阅读说明：本技术 用于低浓度李斯特菌属的检测和计数的方法 (Methods for detection and enumeration of low concentrations of listeria ) 是由 T·谢尔达尔 T·马西森·法伦格 A·弗格里 I·科菲西奥·索努·库乔 于 2019-12-20 设计创作，主要内容包括：本文件涉及一种用于检测和/或计数样本中的李斯特菌,例如单核细胞增生李斯特菌的方法,所述方法包括在包含鼠李糖、一种或多种抗生素、pH颜色指示剂和LiCl的培养基中培养可能含有李斯特菌的样本。(This document relates to a method for detecting and/or enumerating listeria, such as listeria monocytogenes, in a sample, the method comprising culturing the sample, which may contain listeria, in a medium comprising rhamnose, one or more antibiotics, a pH color indicator, and LiCl.)

1. A method for detecting listeria rhamnosus, such as listeria monocytogenes, in a sample, the method comprising or consisting of the steps of:

i) preparing a suspension of a sample that may contain listeria in a first medium comprising rhamnose, one or more antibiotics, a pH color indicator, and LiCl;

ii) incubating the suspension under conditions that allow growth of listeria; and

iii) identifying a positive sample.

2. The method of claim 1, further comprising counting listeria rhamnosus, said method comprising the steps of ia) transferring the suspension prepared in step i) to a multi-well tray, and step iv) performed after step iii) calculating the concentration of the listeria in the sample, e.g. by using the most probable method.

3. The method of claim 1 or 2, further comprising the step of confirming the presence of listeria monocytogenes, e.g. by using molecular methods, e.g. by plating the positive sample identified in step iii) on a second growth medium, e.g. ALOA medium, by polymerase chain reaction or in situ hybridization, ELISA, VITEC and/or API, which is performed after step iii) in claim 1 or step iv) in claim 2.

4. The method of any one of the preceding claims, wherein the sample possibly containing listeria rhamnosus is obtained by dividing the sample into small pieces, e.g. by homogenization, slicing and/or chopping, before performing step i) and/or in the first culture medium of step i).

5. The method of any one of the preceding claims, wherein the antibiotic is one or more of nalidixic acid, ceftazidime, polymyxin B sulfate, cycloheximide, amphotericin B, preferably a combination of two or more of any of the antibiotics.

6. The method of any one of the preceding claims, wherein the pH color indicator is phenol red.

7. The method according to any of the preceding claims, wherein the LiCl is present in an amount of about 5-17g/l, such as about 7-13g/l, such as about 10 g/l.

8. The method of any one of the preceding claims, wherein the rhamnose is present in an amount of about 5-17g/l, such as about 7-13g/l, such as about 10 g/l.

9. The method according to any of the preceding claims, wherein the sample is a food sample, an environmental sample, or a sample from an animal, such as a human, such as a tissue sample or a stool sample.

10. The method of any of the preceding claims, wherein the food sample is raw or processed meat, poultry or fish product, vegetable or ready-to-eat food product.

11. The method according to any one of claims 1-9, wherein the environmental sample is a water sample, a soil sample or a food industry environmental sample, such as a surface swab sample.

12. The method of any of the preceding claims, wherein the method is performed in a closed system.

13. A culture medium for growing and/or detecting listeria rhamnosus, such as listeria monocytogenes, comprising:

e) rhamnose at a concentration of about 5-15g/l, such as about 7-13g/l, for example about 10 g/l;

f) one or more antibiotics, such as nalidixic acid, ceftazidime, polymyxin B sulfate, cycloheximide, amphotericin B, preferably at least two of these antibiotics;

g) pH color indicators, such as phenol red; and

h) LiCl at a concentration of about 5g/l to about 15g/l, such as about 7g/l to about 13g/l, such as about 10 g/l.

14. A kit for detecting and/or enumerating listeria rhamnosus, e.g. listeria monocytogenes, in a sample, the kit comprising:

a) a container comprising a culture medium comprising or consisting of rhamnose, one or more antibiotics, a pH color indicator and LiCl;

b) a container or multi-well tray for culturing a sample that may contain listeria; and

c) optionally a color scale for identifying positive samples; and

d) optional instructions for use.

15. A computer-implemented calculator for displaying a prediction of growth of listeria, such as listeria monocytogenes, based on detection and enumeration of colony forming units in a sample by the method of any one of claims 1-12, the calculator comprising:

c) an input device, such as a keyboard or a microphone;

b) an output device, such as a display, a computer or mobile phone screen or a speaker;

d) software that may be downloaded or stored, such as a smartphone or an application on a web page;

wherein the software accepts input of any number of colony forming units present in the sample ranging from 0.04cfu/g to 1 cfu/g.

Technical Field

This document relates to methods and means for detecting and/or enumerating Listeria (Listeria), such as Listeria monocytogenes (Listeria monocytogenes), in a sample, wherein the methods comprise culturing the sample, possibly containing Listeria, in a selective growth medium, thereby allowing for more rapid and sensitive detection of Listeria.

Background

Listeria monocytogenes is a food-borne pathogen that causes thousands of listeriosis cases each year. In europe alone, about 1500 cases are reported each year (EFSA 2018). Although there are fewer cases of listeriosis than salmonellosis and campylobacteriosis, the mortality rate of listeriosis is higher.

Listeria monocytogenes is a pathogenic bacterial species that causes the disease listeriosis. It is a gram-positive facultative anaerobe that can survive in the presence or absence of oxygen. It can grow and multiply in host cells and is one of the most virulent food-borne pathogens. Listeriosis infection in high risk individuals can be fatal.

Listeria monocytogenes (l.monocytogenes) is an ubiquitous bacterium, meaning that it is present in many different niches such as soil, water, food production environments, animals and humans. It is easy to form a depot in a food production facility even in a facility having a high hygienic standard and implementing a HACCP system. EFSA has reported an prevalence of 0.5-6% of listeria monocytogenes in a particular class of ready-to-eat foods. Prevalence rates are highest in fish and seafood products (EFSA 2018).

Listeria monocytogenes has the ability to grow at temperatures as low as 0 ℃ or even as low as-1.5 ℃, which allows for proliferation at typical refrigeration temperatures, which thereby greatly increases its ability to evade human food control.

The most likely to become listeria infected people, especially the elderly, people with compromised immune systems, and pregnant women (fetuses). Even a small amount of bacteria, as low as 100-. Healthy humans are not normally affected by listeria monocytogenes, but high doses can result in mild symptoms that are generally harmless. During the last decades, norway recorded 20-50 cases of listeriosis per year. This is not much different from other parts of europe when measured by the number of residents.

The consequences of listeriosis are very severe, where they can lead to death and very high social costs. Listeria monocytogenes can infect the brain, spinal cord membrane, and/or the blood stream of a host.

The combination of the following facts has led to a strong interest in listeria monocytogenes in legislation and by customers, including norwegian fish buyers: listeriosis is a very serious disease for humans; listeria in food occurs relatively frequently; and listeria monocytogenes in food can be kept below pathogenic numbers by relatively simple means.

Listeria can be analyzed qualitatively or quantitatively. ISO methods 11290-1 (qualitative) and 11290-2 (quantitative) are standard methods in legislation. However, the detection limits of these methods are 1cfu/25g and 10cfu/g, respectively, which is not sufficiently detailed for the analysis of process steps in which contamination may occur, such as during fish slaughter and willow cutting before the product is sent to market. The fisherman trade has thus described the limitations of the ISO method: "if the method is used to measure blood pressure, the result will be that there is a blood pressure below 1000. Such information is not fine enough to be useful. In the same way, there is a need for a method for measuring the concentration of listeria in the range between 0.04cfu/g and 10 cfu/g.

Listeria is capable of growing in food during refrigeration. How high a concentration is reached when the consumer consumes food will depend on the product conditions, storage conditions, processing conditions and the vulnerability of the consuming consumer. In some products, such as heat-treated meat intended for cooling use in salad or fish for sushi or sashimi, if the initial concentration is higher than 2cfu/g, the concentration may reach a level constituting a food health risk to healthy consumers under reasonably foreseeable conditions, but for fragile consumers the initial concentration needs to be lower than 0.2 cfu/g. In other products the concentration may be higher, but in most cases an acceptable concentration from a food safety point of view is between 0.2cfu/g and 5 cfu/g. The ISO method does not distinguish between these concentrations because levels below 10cfu/g cannot be counted unless many plates are applied, samples are filtered or samples of higher concentrations in the broth are obtained by other means.

The ISO method has recently been revised (2017). The changes compared to the previous version (1997) are the following:

to ISO 112902: the main changes in 1998 are as follows.

The counts of listeria monocytogenes have been modified, as listed below.

Primary suspension with buffered peptone water, semi-frenzer broth with or without supplements, and all applicable diluents mentioned in ISO 6887 (all parts).

The recovery step was deleted.

The microscopic aspects for validation, catalase and CAMP tests are optional.

Includes new performance characteristics.

In addition, the listeria count is already included in the range, and the title changes accordingly.

In these revisions, counts of listeria monocytogenes concentrations below 10cfu/g were not addressed in the revisions, although the correlation is known.

Qualitative ISO method 11290-1 includes a step in which listeria obtains optimal propagation conditions, plating on selective agar medium, followed by a confirmation step to qualitatively determine whether listeria is present in the sample. However, the actual number of bacteria in a sample cannot be determined using this qualitative method, and even samples with high or low numbers of bacteria cannot be distinguished. This qualitative ISQ method takes one week to perform after the sample has been received in the laboratory.

In quantitative ISO method 11290-2, a 10 gram sample was analyzed. One problem with this quantitative determination is that its detection limit is 10cfu/g, which is not sufficient. If the qualitative test is positive, but the quantitative test is negative, it can only be concluded that the product has less than 10cfu/g listeria, but not that only 5cfu or none at all.

The first step in quantitative ISQ method 11290-2 was to plate serial dilutions of the sample into selective growth medium ALOA, and the second step involved validation of rhamnose, xylose, gram bacteria and catalase tests. Lithium chloride was used as the selection pressure in the ALOA medium, and a color indicator was used to indicate putative colonies. Confirmation of the use of rhamnose and xylose is based on the fact that only a few bacteria can utilize these sugars. Thus, the quantitative ISO method comprises the following steps: the samples were incubated on selective agar plate medium for two days and then confirmed. This ISO method takes 5 days to perform after the sample has been received in the laboratory.

Typically, a combination of the two ISO methods described above is used. However, when talking about the problem of listeria in fresh salmon, the combination of these two approaches would not be sufficient, as it is not necessarily required that the fish must be completely free of listeria in order to be considered safe. There may also be considerable variation within a product batch, and the sample taken does not necessarily represent the entire batch.

One problem with the current method is that the amount of sample that can be analyzed is low, which means that if bacteria are unevenly distributed in the sample to be tested, bacteria may be missed and the sample is assessed as listeria negative.

Food legislation in europe has established food safety standards for listeria monocytogenes in ready-to-eat foods. The essence is that the concentration of Listeria monocytogenes should not exceed 100cfu/g at any time during the shelf life (EU regulation 2073/2005). Some countries have zero tolerance for listeria monocytogenes, at least in part because of challenges in low concentration listeria counts, and this option is discussed in several countries. However, 1) the relatively high prevalence of listeria monocytogenes in food, 2) the challenge of removing listeria monocytogenes from production facilities, and 3) high concentrations before the potential for listeriosis increases indicate that zero tolerance is not realistic.

The 100cfu/g concentration is considered a reasonable trade-off between food safety and actual production. This limit is consistently considered sufficient to avoid listeriosis morbidity, provided that the assay is sensitive and accurate enough to detect and count listeria monocytogenes within the desired concentration range, and that the bacteria are distributed sufficiently uniformly throughout the batch so that the sample taken from the batch contains virtually a representative concentration of bacteria.

Unfortunately, none of these conditions are met. Studies of naturally contaminated foods in commercial production have shown that only a few listeria monocytogenes (usually 1 per 100g to 2 per g) are transferred into the product during processing. These bacteria can grow but can remain in the food during storage and form clusters in the food. The sampling plan given in the microbiological standards in EU regulation 2073/2005 stipulates 5-10 samples of 25 grams each, but it is doubtful whether this small sample size can cover variability in concentration. Secondly, sampling of food is mainly done at the process level, which means that the concentration of listeria monocytogenes is well below 10 cfu/g. The detection level of the current reference ISO methods (ISO 11290-1 and 11290-2) was 1cfu/25g in qualitative analysis and 10cfu/g in counts. This means that samples that were positive in the qualitative analysis but negative in the counts were contained in the range of 0.04-9.9 cfu/g. This is a large range, resulting in some food suppliers, customers and authorities already setting the limit of food recovery at the time of testing (0.04 cfu/g-1 cfu/25g), which represents the least risk even though it may lead to food loss. Others set the limit to 10cfu/g even if these concentrations are too high to ensure that the limit of 100cfu/g is not exceeded at the end of the shelf life.

In summary, when sampling must be done at a process level before the product is put on the market, implementation of food safety standards for listeria monocytogenes in ready-to-eat foods is challenging and requires more accurate methods in the range of 0.04-10 cfu/g.

Therefore, there is a need for a faster, even more sensitive assay that can also process larger sample volumes to detect listeria, such as listeria monocytogenes, in food samples as well as swabs used for sampling device surfaces.

It is therefore an object of the present invention to overcome or at least alleviate one or more of the problems described herein.

Disclosure of Invention

This document relates to a method for detecting listeria rhamnosus, such as listeria monocytogenes, in a sample, the method comprising or consisting of the steps of:

i) preparing a suspension of a sample that may contain listeria in a first medium comprising rhamnose, one or more antibiotics, a pH color indicator, and LiCl;

ii) incubating the suspension under conditions that allow growth of listeria; and

iii) identifying a positive sample.

The method may further comprise counting listeria rhamnosus, wherein the method comprises the steps of ia) transferring the suspension prepared in step i) into a multi-well tray, and step iv) performed after step iii) calculating the concentration of the listeria in the sample, e.g. by using the most probable method.

Thus, a method of enumerating listeria rhamnosus, e.g. listeria monocytogenes, comprising or consisting of the steps of:

i) preparing a suspension of a sample that may contain listeria in a first medium comprising rhamnose, one or more antibiotics, a pH color indicator, and LiCl;

ia) transferring the suspension obtained in step i) into a porous plate;

ii) incubating the suspension under conditions that allow growth of listeria;

iii) identifying a positive sample; and

iv) calculating the concentration of the listeria in the sample, e.g., by using a most probable method.

The methods of this document can further include the step of confirming the presence of listeria monocytogenes by distinguishing listeria monocytogenes from other species of listeria, for example, by plating the positive sample identified in step iii) on a second growth medium, such as an ALOA medium, by using molecular methods, such as by polymerase chain reaction or in situ hybridization ELISA (enzyme linked immunosorbent assay), VITEC, or API (analytical signature index). This confirmation step is performed after step iii) in the qualitative method and after step iv) in the quantitative method.

The sample, which may contain listeria rhamnosus, may be processed by dividing said sample into small pieces, e.g. by homogenization, slicing and/or chopping, before performing step i) and/or in the first culture medium of step i).

The antibiotic may be one or more of nalidixic acid, ceftazidime, polymyxin B sulfate, cycloheximide, amphotericin B, preferably a combination of two or more of any of said antibiotics.

The pH color indicator may be phenol red.

LiCl may be present in an amount of about 5-17g/l, such as about 7-13g/l, such as about 10 g/l.

Rhamnose may be present in an amount of about 5-17g/l, such as about 7-13g/l, for example about 10 g/l.

The sample may be a food sample, an environmental sample, or a sample from an animal, such as a human, for example a tissue sample or a stool sample.

The food sample may be raw or processed meat, poultry or fish products, vegetables or ready-to-eat food products.

The environmental sample may be a water sample, a dirt sample or a food industry environmental sample, such as a surface swab sample.

The method may be performed in a closed system.

This document also relates to a culture medium for growing and/or detecting listeria rhamnosus, e.g., listeria monocytogenes, comprising:

a) rhamnose at a concentration of about 5-15g/l, such as about 7-13g/l, for example about 10 g/l;

b) one or more antibiotics, such as nalidixic acid, ceftazidime, polymyxin B sulfate, cycloheximide, amphotericin B, preferably at least two of these antibiotics;

c) pH color indicators, such as phenol red; and

d) LiCl at a concentration of about 5-17g/l, such as about 7-13g/l, such as about 10 g/l.

This document also relates to a kit for detecting and/or enumerating listeria rhamnosus, e.g., listeria monocytogenes, in a sample, the kit comprising:

a) a container comprising a culture medium comprising or consisting of rhamnose, one or more antibiotics, a pH color indicator and LiCl;

b) a container or multi-well tray for culturing a sample that may contain listeria;

c) optionally a color scale for identifying positive samples; and

d) optional instructions for use.

The concentrations of the components of the medium and the concentrations of the components of the medium of the kit are the same as those described herein for the first medium.

The present document also discloses a computer-implemented calculator for displaying a prediction of growth of listeria, such as listeria monocytogenes, based on detection and enumeration of colony forming units in a sample by the methods according to the present document, the calculator comprising:

a) an input device, such as a keyboard or a microphone;

a) an output device, such as a display, a computer or mobile phone screen or a speaker;

b) software that may be downloaded or stored, such as a smartphone or an application on a web page;

wherein the software accepts input of any number of colony forming units present in the sample ranging from 0.04cfu/g to 1 cfu/g.

Other features and advantages of the invention will be apparent from the following detailed description, the accompanying drawings, examples, and the claims.

Definition of

Most Probable Number (MPN) is a method for estimating the concentration of viable microorganisms in a sample by replicating liquid culture growth at x-fold dilutions (e.g. 15x, 10x or 2x dilutions). The most probable number is useful for estimating microbial populations in soil, water, agricultural products, and is particularly useful for samples containing particulate matter that would interfere with the plate count counting method.

Colony forming units (cfu) are units used to estimate the number of viable bacterial or fungal cells in a sample. Viability is defined as the ability to propagate by binary division under controlled conditions. Enumeration using colony forming units requires culturing the microorganisms and enumerating only viable cells. The visual appearance of colonies in cell culture requires significant growth.

"culture (culturing)" and the like mean to grow or maintain living cells in a solid or liquid growth or culture medium designed to support the growth of microorganisms or cells. The growth or culture medium contains nutrients essential and relevant for the intended growth of the respective microorganism or cell.

The ALOA (Listeria agar Medium after Ottajiani and August) medium is a selective chromogenic agar medium for selective and differential isolation of Listeria monocytogenes. The medium is described in the ISO method.

Polymerase Chain Reaction (PCR) is a method of preparing multiple copies of a DNA sequence involving repetitive reactions using a Polymerase.

In situ hybridization ISH is a technique that allows for the precise localization of specific nucleic acid segments within a tissue section. The basic basis of ISH is that if nucleic acids are sufficiently preserved in histological specimens, they can be detected by applying the complementary strand of nucleic acid attached to a reporter molecule.

The term "first medium" refers in the context of this document to a medium comprising rhamnose, one or more antibiotics, a pH color indicator and LiCl. One example of a first medium is "SensiList broth," as disclosed elsewhere herein.

By "rhamnose-fermenting listeria" is meant herein a listeria that is capable of fermenting rhamnose. Examples of such bacteria include listeria monocytogenes, listeria inonotus (l.innocua), and listeria welshimeri (l.welshimeri).

Drawings

Fig. 1 shows an overview of the steps of the method disclosed in this document. The left-hand trajectory is a step when the method is used qualitatively, while the right-hand trajectory shows a step when the method is used quantitatively.

Fig. 2 shows the stacking pattern used when testing the sensitivity of SensiList broth.

Figure 3 shows the sensitivity test results of the method of this document.

Fig. 4 shows the results of stability test of SensiList culture solution.

Fig. 5 shows the results of tests performed in a company with listeria monocytogenes in a production facility. Samples 1, 3, 5 and 6 are production environment samples. Samples No. 3 and 5 turned yellow after incubation. Samples 2 and 4 are 100g salmon samples.

Fig. 6 shows the results of the analysis in example 3.

Figure 7 shows the results from putative positive samples in example 3.

Detailed Description

The present inventors have developed a new method for detecting and/or counting listeria rhamnosus with high sensitivity and at least as accurate as existing methods, which is faster to perform and has lower detection limits than current ISO methods. The methods can detect and count very small amounts of listeria rhamnosus, such as listeria monocytogenes and listeria inonotus, in large samples or pooled samples (e.g., food samples, such as salmon or chicken) as well as in surface swab samples.

The principle of the method of this document is to inoculate a sample, possibly comprising listeria, in a medium for culture (growth) which is selective for listeria rhamnosus, e.g. listeria monocytogenes and listeria lnoko. Both bacteria are able to ferment rhamnose to organic acids, which lowers the pH of the medium. The reaction is detected as a result of a change in color of the culture due to the presence of a pH indicator (e.g., phenol red) in the culture medium. The color change of the culture was sufficient to detect listeria, limited to listeria monocytogenes and listeria inonotus. Confirmation studies can then be performed to distinguish between listeria monocytogenes and listeria lnoko, for example, by growing the bacteria on an ALOA medium selective for listeria monocytogenes. The culture medium also contains LiCl, which allows selection pressure against listeria and antibiotics selected depending on the type of sample, and it is known that bacteria other than listeria are present in such samples.

Accordingly, this document discloses a method for detecting listeria rhamnosus, e.g., listeria monocytogenes, in a sample, the method comprising or consisting of:

i) preparing a suspension of a sample that may contain listeria in a first medium comprising rhamnose, one or more antibiotics, a pH color indicator, and LiCl;

ii) incubating the suspension under conditions that allow growth of listeria; and

iii) identifying a positive sample (i.e., a Listeria-containing sample).

If quantitative analysis of the amount of listeria rhamnosus in a sample is required, the above method comprises the additional step ia) of transferring the suspension obtained in step i) into a multi-well tray, and step iv) of calculating the concentration of said listeria in said sample, e.g. by using the most probable method. Accordingly, a method for detecting and enumerating listeria in a sample comprises or consists of:

i) preparing a suspension of a sample that may contain listeria in a first medium comprising rhamnose, one or more antibiotics, a pH color indicator, and LiCl;

ia) transferring the suspension obtained in step i) into a porous plate;

ii) incubating the suspension under conditions that allow growth of listeria;

iii) identifying positive samples (i.e., listeria-containing samples); and

iv) calculating the concentration of the listeria in the sample, e.g., by using a most probable method.

In order to confirm the presence of listeria monocytogenes in the positive samples identified in the above-described method (i.e. in the samples containing listeria rhamnosus), said method comprises this confirmation as a further step performed after step ii) in the qualitative method and after step iv) in the qualitative method. The confirmation step may be performed, for example, by plating the positive sample on ALOA medium, by using molecular methods such as PCR or in situ hybridization, ELISA, VITEC, and/or API.

The same method steps are basically used in the ISO 11290-1 and ISO 11290-2 methods, but in a different order and the composition of the media used in the different steps is also different, which allows integrating more than one step of the ISO method in the same step of the method, which results in that the selectivity of the method of this document is very similar to that of the ISO method. However, the methods of this document construct higher selectivity in the first step of the analysis compared to the ISO reference method, which in turn leads to fewer putative positive samples and more rapid identification of samples positive for listeria, e.g., listeria monocytogenes.

Using the methods of this document, samples of up to 125 grams can be analyzed at a detection limit of 1 cfu/sample, i.e., a detection limit of 1cfu/125g, and accurately counted at concentrations ranging from 1cfu/25 grams up to about 2000 cfu/g. In the ISO 11290-1 and 11290-2 methods, the sample size is limited to a maximum of 25g in the qualitative method and to a maximum of 10g in the quantitative method. Furthermore, the limit of detection in the ISO method is 10cfu/g unless the method is modified to obtain a more concentrated sample. The selectivity and specificity of the methods of this document are the same as the reference ISO methods used today (ISO 11290-1 and 11290-2), which means that the number of false negatives and false positives in the methods of this document are the same as in the reference methods.

The methods of this document are applicable to analyzing food (e.g., salmon and chicken) and swabs used to collect material from surfaces in production facilities. Furthermore, the method has the benefit that sampling and enumeration can be performed in a production facility, while validation and further characterization, such as whole genome sequencing, can be performed in an external laboratory.

Another advantage of the method of this document is that sampling and sample preparation can be performed by non-professional personnel, thereby ensuring that the food manufacturer or auditor can be responsible for this part themselves. Negative samples can be assessed locally, while putative positive samples can be sent to the laboratory for confirmation.

The methods disclosed herein can be used qualitatively and quantitatively (by adding two method steps as described elsewhere herein) and can be used on pooled samples such that one single analysis can be used to test for compliance with the standards in EU regulation 2073/2005. Pooled samples are generally more likely to give false negatives due to an excessive increase in false positives, but this is not the case if the methods of this document are used quantitatively. In this case, the solution is distributed in many wells, which results in that it is unlikely that listeria monocytogenes and false positives are in the same chamber, as if the entire solution were analyzed at once. Thus, the presence of steps such as steps ia) and iv) described herein is a significant benefit, as it allows to reduce the number of false positives.

The method of this document ensures correct counting even if the sample has been exposed to abusive temperature conditions during transport to the laboratory. This is possible because sample preparation and the start of counting can be performed together with sampling. This is important because abusive temperature conditions between sampling in a facility and sample preparation in an external laboratory often lead to overestimation of listeria concentration in food and thus lead to unnecessary waste, recalls and wrong decisions.

Compared to other rapid listeria detection methods, such as the InSite listeria test (hygienia) Watford UK) and the Path-Chek Hygiene listeria test (Microgen Bioproducts ltd., Camberley, UK), the frequency of false negatives is lower with the method according to this document ((r): u)C.T.Schirmer，Solveig Langsrud，TrondTherese Hagtvedt, Even Heir, 2012, Performance of two general vertical Methods for sampling and detection of Listeria in small-scale chemistry and salmon processing environments, journal of Microbiological Methods, Vol. 91, No. 2, p. 295-: // doi.org/10.1016/j.mimet.2012.08.013.)

The ISO method is given as a standard in legislation and new methods need to be comparable to these methods in terms of specificity and other properties relevant for method validation. For users of analytical methods, the methods need to be cost effective and deliver results quickly. This is particularly important if the results will impact decisions on shipping, recalls, extra cleaning, etc. Therefore, there is a need for an alternative method that meets these criteria with associated selectivity and sensitivity.

The ISO method consists of several steps. The first step is to homogenize the sample in culture broth and then culture in culture broth (qualitative) or on agar plates with specialized growth media (quantitative method). These growth media have been developed and optimized in several steps over the years to obtain

Recovery of stressed cells to avoid false negatives.

The selectivity was increased by removing bacteria that can also grow on the medium and covering the listeria on the agar plate.

The growth medium in the ISO method is suitable for counting down to 10cfu/g and is very suitable for laboratory facilities where open systems (such as agar plates) may be used. This is not the case in industries where lower concentrations are required and where closed analytical systems have to be used. Counting in culture using the MPN (most probable) method is possible, but requires a way to read the difference between positive and negative wells. The culture broth used for selective enrichment in the ISO method does not allow this. For this reason, if low-level counting with the MPN method should be used, additional growth medium is required.

Although the growth medium in the first step of the ISO method is suitable for detecting and enumerating as low as 10cfu/g of listeria monocytogenes, confirmation of putative positive colonies is still required. In one of the confirmation steps, a rhamnose broth is used, since listeria monocytogenes is able to ferment rhamnose and produce an acid, which can be detected as a color change of the pH indicator. In the ISO method, there is no selective pressure in the rhamnose broth (which is used first in the later stages of the ISO method), since only single colonies of isolates that, unlike the bacteria in the food, have been adjusted to grow at the incubation temperature can be tested in this way. The culture broth does not need to stimulate stress recovery.

In the method of this document ("SensiList" method), a modified version of rhamnose broth was used as the main growth medium and combined with the MPN method to allow counting concentrations as low as 0.2 cfu/g. To achieve this, the rhamnose broth is designed and adjusted in iterative steps to obtain:

selectivity of Listeria monocytogenes (avoidance of false positive and false negative bacteria)

Detection limits applicable to quantitation in the range of 0.2-100cfu/g food material

Stress recovery of bacteria

Significant differences between positive and negative results

The system needs to be closed to ensure biosafety.

The price of the culture medium makes the method cost-effective.

More rhamnose-utilizing bacteria than listeria. These bacteria should not be able to grow, at least not to a concentration that would give a false positive signal in the method. Thus, the rhamnose broth used in the initial enrichment step of the method of this document has been added with antibiotics and other selective pressure components like LiCl used in the initial growth medium in the ISO method.

The antibiotic used in the first medium (antibiotics/antibiotics) was selected to reduce or avoid false positive results from other rhamnose fermenting bacteria. Thus, the particular antibiotic used may vary from sample type to sample type. The database and commercial food samples can be searched for microorganisms that can give false positive results. The skilled person can thus easily adapt the antibiotics used to the rhamnose-fermenting bacteria theoretically present in this particular sample type. Thus, the antibiotics used in the first medium are specifically adapted to the type of sample and any rhamnose-fermenting non-listeria that may be present in such a sample type.

The list of antibiotics given in the formulation of the "SensiList" broth in this document applies specifically to fish samples.

Recovery of stressed cells involves the need for the cells to have sufficient nutrients and good conditions to adapt their metabolism to the growth pattern. Listeria is capable of surviving sublethal conditions in a non-growth mode, but this mode cannot be used to detect bacteria. Thus, the components that cause the selective pressure in the culture medium have been tuned to be optimal between the conditions for recovery and selectivity. In addition, low concentrations of abundant media components, such as meat extracts, have been added to the culture broth to supply nutrients for stress recovery. This is especially important for samples that are almost free of organic matter, such as production equipment and water swab samples.

The detection principle of the method of this document is the fermentation of rhamnose to an acid, which changes the color of the pH indicator. In order to obtain a sufficiently high acid yield, the bacteria need to use rhamnose as a source of nutrition and energy, which in turn involves that the amount of other organic substances in the broth (including the sample) must be low, otherwise the bacteria would act as a source of nutrition and energy. Furthermore, the amount of rhamnose must be high enough to allow sufficient bacteria and acid to be produced. The culture broth has been optimized and tested to balance these aspects. It has been found that the amount and concentration of rhamnose and the concentration of the acid ultimately produced are critical for the detection of listeria monocytogenes. The method has been optimized to ensure that all samples, even swab samples from water-rich areas, are correctly detected.

The method of this document is based in principle on the same principles as the ISO methods (ISO 11290-1 and 11290-2), but the order of the method steps is different and modifications are made to the culture medium. By performing these modifications, it was surprisingly found that the sample size can be increased while still maintaining or even increasing the sensitivity of the method, thereby allowing a lower number of bacteria to be detected in the sample compared to the ISO method. Furthermore, the time from sampling to obtaining the results is greatly reduced.

The methods of this document differ from the ISO method in the steps used, the media used in the different steps, and the order of the steps. Table 1 and table 2 below list some similarities between the ISO method and the methods of this document (listeria monocytogenes abbreviated as l.mono).

TABLE 1

TABLE 2

To distinguish between listeria monocytogenes and listeria lnoko and other false positives that may be caused by other rhamnose fermenting bacteria, positive samples (i.e., yellow and orange wells if phenol red is used as a pH indicator) may be plated on the ALOA medium and colonies with characteristic regions indicate a positive result for listeria monocytogenes.

The method of this document is faster than the ISO method; if a classical confirmation test is used, it takes up to two days to obtain a negative sample after going, and three days to obtain a confirmation result. Confirmation using PCR takes only a few hours. For comparison, the first step in the ISO enumeration method (i.e., ISO 11290-2) was plating serial dilutions of the samples into selective growth media, and the second step was validation of rhamnose. The method requires up to 5 ISO test methods (ISO 11290-1, test level 1cfu/25g) with the following steps: the samples were incubated in selective medium for two days and then plated on selective agar, after which time it took one week to confirm after the sample was received in the laboratory. Furthermore, since larger sample sizes can be used, for example, a set of 10 samples, each of 10 grams, can be used in one kit. This also results in lower analysis costs for the food supplier.

Culture medium

The first medium used in steps i) -iii) of the methods of this document (an example of which is the so-called "SensiList broth" herein) is selective for listeria monocytogenes and listeria lnokhei using three methods.

Rhamnose is used as a carbon and energy source in the first culture medium. Only a few bacteria can utilize rhamnose, such as listeria monocytogenes and listeria lnoko. Listeria welshimeri can also ferment rhamnose, but is a very rare listeria compared to listeria inonotus and listeria monocytogenes, and even if this bacterium would grow in a medium containing rhamnose, the specificity of the method of this document is considered to be sufficiently high, at least as high as currently available methods.

In the process of this document, the selectivity for rhamnose has been used in the first step of enriching the bacteria (step ii) in the process of this document.

Rhamnose is present in the first medium at a concentration of at least 5g/l, for example about 5-17g/l, for example about 7-13g/l, for example about 5g/l, about 6g/l, about 7g/l, about 8g/l, about 9g/l, about 10g/l, about 11g/l, about 12g/l, about 13g/l, about 14g/l, about 15g/l, about 16g/l or about 17g/l, usually about 10 g/l. Analytical grade rhamnose is an expensive component and since the volume of rhamnose broth required in the method of this document is high compared to the ISO method, the price of rhamnose is not only crucial for the detection but also for the price of the kit. Thus, the use of significantly cheaper food grade rhamnose in the present method has been tested and it has been found that this quality of rhamnose gives the same results as analytical grade, thereby reducing the price for performing the method.

Lithium chloride (LiCl) was also added to the first medium. This component has been shown to increase the selection pressure in selective enrichment media for listeria (including in frazier broth and ALOA plates, which are used as selective enrichment media and diagnostic agar plate media, respectively, in, for example, the ISO method). Lithium chloride is present in an amount of at least about 5g/l, such as about 5-17g/l, such as about 7-13g/l, such as about 5g/l, about 6g/l, about 7g/l, about 8g/l, about 9g/l, about 10g/l, about 11g/l, about 12g/l, about 13g/l, about 14g/l, about 15g/l, about 16g/l or about 17g/l, typically about 10 g/l.

As explained elsewhere herein, antibiotics are added to minimize the growth of other gram-positive bacteria. Antibiotics suitable for use in the methods of this document include one or more of nalidixic acid, ceftazidime, polymyxin B sulfate, cycloheximide, amphotericin B. Preferably, a combination of two or more of said antibiotics of any of said antibiotics is used, e.g. three, four, even more preferably all five combinations are used. Antibiotics are typically used at their normal concentrations, for example as specified in the SensiList broth formulation below. The requirement of antibiotics depends on the selection pressure required for the sample to be analyzed. Furthermore, the same specificity is expected if the antibiotic used in the first medium is exchanged with other antibiotics having a similar mode of action.

By using the three methods described above, a selective pressure is achieved to minimize bacterial growth other than listeria monocytogenes and listeria inonotus (as well as other rhamnose-fermenting bacteria that may be present in the sample). To be able to distinguish listeria monocytogenes from listeria lnoko from other rhamnose fermenting bacteria, further confirmation is required, for example plating on diagnostic agar (e.g., ALOA agar), performing polymerase chain reaction or in situ hybridization analysis on a particular listeria monocytogenes gene, or by using methods such as ELISA, VITEC, API and/or performing the maldi toff test.

In addition, the first medium may comprise an organic carbon and energy source to initiate rapid growth and stress recovery during the phase. Sodium chloride may be added to obtain preferred bacterial penetration conditions.

The first medium also contains a pH indicator. Fermentation is a process of lowering pH. Thus, when bacteria capable of fermenting rhamnose are present in the sample, these bacteria will ferment the rhamnose present in the first medium, which will lower the pH of the medium. This change in pH is detected by including a pH indicator in the first medium. Thus, a change in color of the culture during incubation of the sample in the first medium indicates the presence of listeria rhamnosus, e.g., listeria monocytogenes and listeria lnokheiner, in the sample. A suitable pH indicator for use in the methods of this document is phenol red, which changes color from red to yellow when listeria is present in the sample, although any other pH indicator that changes color at a pH similar to phenol red can be used.

The first medium may be used as a ready-to-use product or prepared from a single component. The phenol red broth and rhamnose can be sterilized by autoclaving at 121 ℃. The antibiotic was sterile filtered and added after autoclaving.

The first culture medium can be prepared in different ways, for example:

mixing all components into a ready-to-use medium (if the medium is sterilized using autoclaving, this is done before adding antibiotics (which are usually sterile filtered)

Preparing a rhamnose and phenol red broth base as a solution and adding antibiotics shortly before use but after autoclaving. Antibiotics are generally less stable than other components of the growth medium, so delayed addition is a way to extend the shelf life of the medium and minimize the probability of false positives.

Concentrated media (e.g. 5-15x concentrated, e.g. 10x concentrated) with rhamnose and phenol red broth can be formulated and diluted with water before use. This requires sterile filtration of the medium rather than autoclaving due to the caramelization reaction of the medium.

The first culture medium is preferably a liquid.

In all cases, the culture broth can be placed directly into the kit, or separately into a large volume flask or bag.

The first medium comprises or consists of:

a) rhamnose at a concentration of about 5-15g/l, such as about 7-13g/l, for example about 10 g/l;

b) one or more antibiotics, such as nalidixic acid, ceftazidime, polymyxin B sulfate, cycloheximide, amphotericin B, preferably at least two of these antibiotics;

c) pH color indicators, such as phenol red; and

d) LiCl at a concentration of about 5g/l to about 15g/l, such as about 7g/l to about 13g/1, such as about 10 g/l.

An example of a first medium that provides good selective pressure is given below. This medium is denoted herein as "SensiList broth":

SensiList culture fluid formula:

preparation of 1% rhamnose medium:

15g phenol red culture solution foundation

10g rhamnose

10g LiCl

1000ml of distilled water

The components were dissolved in distilled water. Sterilized at 118 ℃ for 15 minutes. Cooling before adding the antibiotic.

Preparing an antibiotic:

10mg/ml nalidixic acid

200000 IE/ml polymyxin B

2mg/ml amphotericin B

2mg/ml ceftazidime

Antibiotics were dissolved in distilled water to the indicated concentrations. Sterilized by sterile filtration.

Complete SensiList broth:

1L 1% rhamnose culture medium

2ml nalidixic acid (10mg/ml)

0.3835ml polymyxin B (200.000IE/ml)

5ml amphotericin B (2mg/ml)

4ml ceftazidime (2mg/ml)

Antibiotics were added to the 1% rhamnose dilution. And (4) stirring uniformly.

pH：7.4±0.2

SensiList broth is particularly useful for the detection and enumeration of listeria in fish samples.

Due to the presence of both rhamnose and LiCl (in combination with antibiotics), the selection pressure for listeria obtained in the first culturing step ii) in the method of the present document is higher than the selection pressure for listeria in the quantitative and qualitative ISO method, which uses two culturing steps to reach the same result.

To confirm the presence/absence of listeria monocytogenes in the samples identified as positive for listeria rhamnosus in step iii) of the methods of this document, the positive samples can be plated on a second growth medium, e.g., an ALOA medium, which distinguishes listeria monocytogenes from listeria inogenes in that the former forms a clear zone of precipitation while the latter does not. The ALOA medium contains a substrate specifically purified against phosphatidylinositol-specific phospholipase C, which allows to distinguish between listeria monocytogenes and listeria lnoko, since the former forms an opaque halo around the colony when metabolizing the substrate. The ALOA medium is preferably a solid medium, such as an agar medium.

Samples and sample preparation

The sample can be any sample used to check for the presence or absence of listeria (e.g., listeria monocytogenes). The sample may be, for example, a food sample, an environmental sample, or a stool sample.

The food sample may be, for example, a raw food sample or a sample of processed meat, poultry or fish products, such as salmon, vegetables or ready-to-eat food products. The environmental sample may be a water sample (e.g. from a blow tank, a thaw tank, wash water, sea water), a soil sample, or a food industry environmental sample, such as a surface swab sample or an equipment surface sample.

Food product samples, typically of size 10-125 grams, can be added to e.g. Buffered Peptone Water (BPW) or physiological saline in a 1: 1 ratio and homogenized in a homogenizer bag by hand or in a homogenizer, e.g. the ratio of homogenized sample to medium is 1: 10, before transfer to the first medium. Alternatively, the sample may be homogenized directly in the first culture medium.

An environmental swab sample, such as cloth (dried or buffered) that may contain listeria, may be added directly to the first medium and left there during incubation. The procedure is otherwise different than for food samples.

Water samples, such as process water, including wash water, cooling water, permeate water, and the like, may be added to the first medium. As for food product samples, a concentrated first culture medium may be used to reduce the detection level, while at the same time limiting the volume and thus the space in the incubator. For example, 11ml of 10 × concentrated first medium can be added to a 100ml water sample to obtain a detection limit of 1cfu/100 ml. The incubation and detection procedures for putative positive samples were the same as for food samples.

Method for detecting and/or counting

As noted above, the methods disclosed herein may be performed qualitatively or quantitatively for all different kinds of samples, depending on the steps included therein.

A method (i.e. a qualitative method) for detecting listeria rhamnosus, e.g. listeria monocytogenes, in a sample, comprising or consisting of the steps of:

iv) preparing a suspension of a sample potentially containing listeria in a first medium comprising rhamnose, one or more antibiotics, a pH color indicator and LiCl;

v) incubating the suspension under conditions that allow growth of listeria; and

vi) identifying a positive sample.

As mentioned above, the method may further comprise counting listeria rhamnosus, wherein the method comprises the steps of ia) transferring the suspension prepared in step i) into a multi-well tray, and step iv) performed after step iii) calculating the concentration of said listeria in said sample, e.g. by using the most probable method.

A method of counting the number of listeria rhamnosus, e.g. listeria monocytogenes (i.e. a quantitative method) comprises or consists of the steps of:

i) preparing a suspension of a sample that may contain listeria in a first medium comprising rhamnose, one or more antibiotics, a pH color indicator, and LiCl;

ia) transferring the suspension obtained in step i) into a porous plate;

ii) incubating the suspension under conditions that allow growth of listeria;

iii) identifying a positive sample; and

iv) calculating the concentration of the listeria in the sample, e.g., by using a most probable method.

The methods of this document can further include the step of confirming the presence of listeria monocytogenes by differentiating listeria monocytogenes from other listeria species, for example, by plating the positive sample identified in step iii) on a second growth medium, such as an ALOA medium, by using molecular methods, such as by polymerase chain reaction or in situ hybridization, ELISA, VITEC, or API. This step is performed after step iii) of the qualitative method and step iv) of the quantitative method.

For both qualitative and quantitative analysis of the presence of listeria in a sample, the first step in the method is to prepare a suspension of the sample, possibly containing listeria, in a first culture medium, as described elsewhere herein.

The sample is typically incubated at a temperature of about 37 ℃, even though temperatures of, for example, between 25 ℃ and 38 ℃, e.g., about 30 ℃ may also be used, and the color of the culture is observed after 1 and 2 days. When phenol red is used as a pH indicator, a change in color, for example, from red to yellow or orange, indicates a putative positive sample. The color can be read as such or the color code can be found by comparison with a color chart.

If a qualitative method is to be performed, it is sufficient to incubate the suspension of the sample, which may contain listeria rhamnosus, to see if there is a color change during the incubation of the sample in the first culture medium (i.e. the suspension of the sample, which may contain listeria rhamnosus, in the first culture medium). If a color change is observed, a listeria rhamnosus, e.g., listeria monocytogenes or listeria inonotus, is present in the sample, and thus the sample is positive for listeria rhamnosus. If no color change is observed, the sample is absent of Listeria rhamnosus, i.e., the sample is negative for Listeria.

For the method to be performed to count the concentration of listeria rhamnosus in the original sample, the suspension of the sample, possibly containing listeria rhamnosus, in a first culture medium is transferred to a multi-well Tray (plate), e.g. a quantification Tray, e.g. a Quanti-Tray 2000 from IDEXX. During incubation under conditions such as those described above, the pores containing listeria rhamnosus will change color due to the presence of the pH indicator in the first medium. The most likely concentration of listeria rhamnosus in the original sample can then be estimated using the MPN (most probable number) method. For example, if a quantification tray is used to estimate the concentration of listeria rhamnosus in a sample, and the total volume of the suspension that may contain the sample of listeria rhamnosus is 100ml, and the amount of sample is 5g, the limit of detection will be 1cfu/5g of sample, which corresponds to an average concentration of 0.2cfu/g of sample. If the concentration of Listeria rhamnosus is high, e.g. 100cfu/g, there will be 500 bacteria in 100ml of solution containing 5 grams of sample. This corresponds to 5 cfu/ml. It is likely that most 1ml wells in the dosing tray will yellow, while up to 50% of 0.1ml wells will yellow. Fermentation of listeria with double concentration of rhamnose, up to 100% of +0.1ml wells will yellow. Above this concentration, all wells will be yellow and reach the upper limit of the effective count. The minimum and maximum limits of counts obtained using 96 1ml wells and 96 0.1ml wells will be in the range of 1cfu/5 grams (i.e. 0.2cfu/g) to 200 cfu/g. The limit of detection can be adjusted if another ratio between the sample and the first culture medium is applied.

The methods of this document allow for the detection and enumeration of very low levels of listeria monocytogenes and listeria rhamnosus in large samples or pooled samples. The sample size can be as high as 100 grams and the level detected is as low as 5cfu/100g of food, i.e. 1cfu/20g of food. Due to the large sample size, a set of e.g. 10 samples, 10 grams per sample, can be used in one kit. This also results in lower analysis costs for the food supplier. Furthermore, allowing for a larger sample size or a larger possible sample size of pooled samples is an important factor in overcoming problems due to maldistribution of listeria in food products.

The analysis performed with the methods of this document can be performed in a production facility because the system can be made closed so that the enriched listeria positive sample cannot leak bacteria back to the production facility or the person handling the sample. Closing the system means that after the sample is added to the first culture medium, whether the method is quantitative or qualitative, the container (e.g. flask, bag, beaker, multi-tray, etc.) is sealed so that bacteria that may grow in the culture medium do not come into contact with the air, the person handling the sample or the surface that contacts the sample. Sample preparation, such as homogenization and cutting, can be accomplished in a closed system. No gas exchange is required because the listeria can be grown in the absence of oxygen. Closing the system may for example be achieved by sealing the sample, e.g. with a lid or a plastic film, e.g. in a way that only allows opening the system with a specific tool, etc. However, confirmation of the presence of listeria monocytogenes in putative positive samples must be performed in a laboratory classified as listeria assay, since the container containing the culture fluid needs to be opened.

Various samples can be shipped to a laboratory to confirm/test for the presence of listeria monocytogenes. If the sample is a putative positive sample, i.e., the color has changed to yellow after incubation of the sample in the first medium, then the temperature should be kept lower during transport to preserve the bacteria. In case the sample is delivered shortly after having been placed in the first culture medium, no cooling is needed, since growth during transport would shorten the detection time after the sample has arrived at the laboratory.

There are several methods available for identifying listeria monocytogenes:

-forming regions on ALOA agar. Listeria monocytogenes forms a region, whereas Listeria inokosa does not.

-PCR assay for the detection of listeria monocytogenes. In fact, the test can be performed before the sample turns yellow, since the number of cells reached before a positive PCR reaction is obtained is so high that the pH is below the level at which the color turns yellow.

In situ hybridization analysis of specific Listeria monocytogenes genes,

any other identification method, such as ELISA, VITEC, API, MaldiToff test and whole genome sequencing.

As is evident from the above, the method of this document allows to obtain putative positive and negative results for both qualitative and quantitative methods within 1 to 2 days and to confirm positive results after at most one day, instead of 5 or 7 days of the ISO method (for quantitative and qualitative methods, respectively). Furthermore, the advantage of the present method is that it does not have to be performed in a laboratory, but can be performed, for example, at the site where the sample is taken, for example, in the industry itself.

Reagent kit

This document also relates to a kit for detecting listeria, e.g., listeria monocytogenes, in a sample, the kit comprising:

a) a container comprising a culture medium comprising or consisting of rhamnose, one or more antibiotics, a pH color indicator and LiCl;

b) a container or multi-well tray for culturing a sample that may contain listeria;

c) optionally a color scale for identifying positive samples; and

d) optional instructions for use.

The concentrations of the components of the medium in the kit are as disclosed elsewhere herein for the first medium. The first medium is preferably a liquid, but it may also be a solid.

Computer-implemented calculator

The present document also relates to a computer-implemented calculator for displaying a prediction of growth of a listeria, such as listeria monocytogenes, based on detection and enumeration of colony forming units in a sample, the calculator comprising:

a) an input device, such as a keyboard or a microphone;

a) an output device, such as a display, a computer or mobile phone screen or a speaker;

b) software that may be downloaded or stored, such as a smartphone or an application on a web page;

using the input device, the number of colony forming units is set to any number found in the sample, in the range of 0.04cfu/g to 10cfu/g, in particular in the range of 0.04cfu/g to 1 cfu/g. The prior art does not allow such details to be provided at a low level.

The calculator may input parameters such as the pH of the sample or the type of sample, e.g. raw salmon, raw fish, sushi, chicken or beef. In addition, there is input of parameters such as temperature and storage time in production, shop and home refrigerators, and room temperature and outdoor refrigeration hours. Based on the inputs, the calculator will calculate from the stored model and then graphically or numerically display the various predictions, e.g., the highest growing days, the most likely growth, the least likely growth, and plot these against legal limits, the likely disease limits of exposed consumers, and the likely disease limits of healthy adults.

The calculator may be implemented as a single application for each food type, or as an integrated calculator offering different choices to the user.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

Experimental part

Experimental part

1Example 1: development and testing of SensiList culture fluid

1.1 Sensilist culture solution

Hereinafter, the content of the culture medium is referred to as "Sensilist culture solution" as described above.

1.2 sensitivity of Sensilist culture solution

The first study of selectivity and sensitivity of SensiList broth was performed using salmon samples taken directly from the production company. The salmon sides were inoculated with 0, 2, 20 or 200cfu/g of Listeria monocytogenes. As inoculum, a single strain or a mixture of 5 strains was used. The samples were used immediately or frozen until use.

The concentrations of LiCl and rhamnose in SensiList cultures were tuned in these studies.

Samples were analyzed using both the SensiList method and the ISO method to compare results. The ISO method is adjusted to allow the detection level to be reduced in two ways. First, a larger volume of sample suspension was plated by using five times more plates. This method is not feasible in everyday work, but is used here to verify the sensitivity of the method herein. Secondly, a 5x concentration of the sample suspension was used by adding less diluent before inoculation onto agar medium as described in ISO method.

Analysis was performed using the methods of this document: salmon samples (product samples) were prepared as described below and placed in SensiList medium. For qualitative samples (presence/absence), the samples and culture broth were placed in 100ml or 500ml flasks. For counting using the MPN (most probable) method, the culture broth containing the sample was placed in sterile glass tubes (1-10ml) or Quantitray 2000 from IDEEXX with 49 large wells and 24 small wells in a total volume of 100 ml. The culture broth was incubated as described below, and the color of the culture broth was observed after one and two days. All wells were further analyzed by transferring the droplets onto an ALOA plate using a sterile needle. Blue-green colonies with typical regions are presumed to be positive for listeria monocytogenes, and blue-green colonies without regions are presumed to be positive for listeria lnoko. Colonies of other colors were negative. Blue-green colonies were confirmed by gram staining and catalase according to ISO 11290-1.

The study was repeated approximately 10 times in triplicate with salmon inoculated with 2, 20 or 200cfu/g Listeria monocytogenes. In some experiments, one listeria positive sample (cube, about 2x 2cm) was put together with 9 negative samples of the same size and stacked in different formats, as shown in fig. 2. The aim was to investigate whether positive samples were detected in the following three sampling procedures: i) analyzing all samples in the stack, ii) analyzing the broth after shaking the samples with the broth and then incubating the broth, and iii) analyzing the water released from the fish.

1.3 SensiList selection of the culture broth

False positive and false negative bacteria were searched by two means:

1. strains were selected for use as positive and negative control strains in listeria diagnostics and in kit tests.

2. Salmon from different suppliers were purchased from stores in oslo and cultured in SensiList broth without antibiotics and LiCl to allow the growth of all bacteria capable of utilizing rhamnose. Tubes that changed the color of the broth from red to yellow during incubation were plated on blood agar and ALOA agar, and the bacteria were isolated. The isolates were used in subsequent experiments to 1) formulate antibiotic compositions in sensiist broth and 2) test the stability of the broth.

1.4 confirmation of putative Positive samples Using PCR

Confirmation using PCR was tested using the following methods:

use ofBlood and tissue kits (Qiagen) extracted DNA from listeria monocytogenes (strain VI 58361, serotype 1/2a) cultured in growth medium broth. Using these growth media: sensiist medium, sensiist medium without LiCl and antibiotics, sensiist medium without antibiotics. In addition, semi-frazier medium and buffered peptone water were included as controls. The listeria monocytogenes colonies were transferred from the agar plates to different media. DNA was extracted using the kit according to procedures from the supplier and DNA concentration was measured using Nanodrop (spectrophotometer from ThermoFischer for measuring DNA concentration).

Use of BioRad iQ-Listeria monocytogenes II kit, isolated DNA was analyzed using qPCR (quantitative PCR) according to the following genetic markers: ORF2819, ORF2110, lmo1118, lmo0737, plcA and prs.

1.5 stability of SensiList culture fluid

Stability of different formulations of SensiList broth was checked as follows:

four formulations of SensiList broth were prepared in sufficiently large amounts to perform multiple analyses during one year of storage. The formulations used were SensiList broth without LiCl and antibiotics, broth with LiCl but without antibiotics, broth with antibiotics added immediately before use, and whole broth. The experiment was repeated with three separate batches of each formulation.

Experiments were performed using a single strain of the group of listeria monocytogenes (8 strains), other listeria (2 strains) and other bacteria that might give false positive results. The latter strain was selected based on its own experiments on salmon (see above), the strain used to test the ISO method and the strains that give false positive results in other listeria monocytogenes kits. The strains are described in more detail below.

A. Listeria monocytogenes strain:

a.2 strains were used to test the ALOA medium in the ISO method.

i. Listeria monocytogenes serotype 4b VI 60847(WDCM00021/ATCC 13932)

Listeria monocytogenes serotype 1/2a, VI 51285(WDCM00109/CCUG 15527)

B. Listeria monocytogenes strains used in experiments with SensiList broth:

a.VI 51503

b.VI 58363(00EB250LM)

c.VI 58365(00EB254LM)

d.VI 59994

e.VI 59998

f.VI 58366

C. listeria inolica: for testing ALOA medium in the ISO method.

a.VI 51284(WDCM00017/CCUG 15531)

D. Listeria monocytogenes: for testing ALOA medium in the ISO method.

a.VI 51040(ATCC 19119)

E. Other strains:

a. for testing of ALOA:

i. enterococcus faecalis (VI 52179).

Escherichia coli VI 51656(WDCM00013/CCUG 17620)

b. Candida albicans VI06652(ATCC10231)

F. In the early version of SensiiList, 18 strains from salmon produced false positive results when no antibiotics were applied

a.5 species of Hafnia alvei: VI 54910, VI 54914, VI 54918, VI 54924 and VI 54927

b.5 Citrobacter species: VI 54915, VI 54916, VI 54928, VI 54931, VI 54940

c.2 Aeromonas species: VI 54922, VI 54926

d.2 species of Enterobacter cloacae: VI 54935 and VI 54941

e.1 Alcaligenes faecalis: VI 54929

f.1 enterococcus faecalis: VI 54946

g.1 species of Bacillus cereus: VI 54948

h.1 indeterminate identifications, which may be of the species Megacoccus maxima: VI 54952

G.2 strains that gave false positives in other kits

a. Bacillus stearothermophilus: VI 61406

b. Enterococcus casseliflavus: VI 61407

H. Strain capable of fermenting rhamnose:

a. lactobacillus rhamnosus: VI 60846

The listeria monocytogenes strains were cultured and cold-adapted at 7 ℃ for 7 days to adapt them to stress conditions representative of food samples. Other strains that may give false positive samples were cultured overnight at 37 ℃ and transferred to BHI medium. Different temperatures were applied to prepare for worst case scenarios: the listeria monocytogenes strain should be given a practical stress to detect false negative results, while the false positive strain should be given the best option to grow in culture during incubation.

Formulations of SensiList broth (0.9ml) were inoculated (0.1ml) into 96-well cassettes (type). The mixture was diluted ten times in trays in 8 steps resulting in one column per strain as shown in tray 1 below. Three trays were required for each medium formulation to test all strains. The box was covered before incubation at 37 ℃ and the colour change was observed after 1 and 2 days of incubation.

Table 3: distribution of strains used in stability tests in 96-well plates (12 columns and 8 rows in each plate). Each column contains 1 strain diluted 10-fold. Row a contains the first 10-fold dilution of the strain, row B contains the second 10-fold dilution of the strain, and so on. The top 5 alphanumeric numbers are the identity of each strain.

1.6 treatment of samples for analysis with the methods of this document

1.6.1 food product samples

10-125 grams of sample was added to Buffered Peptone Water (BPW) in a 1: 1 ratio and homogenized by hand or in a bag or in a homogenizer. 10ml of the homogenized solution were added to 90ml of SensiList broth. The whole sample was incubated at 37 ℃ and the color was observed after 1 and 2 days. A change in the solution from red to yellow indicates a putative positive sample. The color can be read as such or the color code can be found by comparison with a color chart. For example, compounds fromwww.gioco.noA color scale of Y50R indicates a positive result.

If prepared as described above, the test solution contained 5 grams of product. The entire solution was included, which resulted in a detection level of 1cfu/5 g. If another level of detection is required, this level of detection can be obtained by applying another ratio between the sample and the culture liquid. Some examples are given below. If a large sample, e.g. a pooled sample of 5x 25g samples, is required to meet the criterion of absence of microorganisms in 25g measured in 5 samples, the total volume may be limited by adding the culture solution as a concentrated solution. For example, a 6-fold concentration of 125 grams of sample +125ml of BPW +50ml of SensiList medium limits the total volume to 300 ml. This is a high enough liquid fraction to observe color changes, but some foods will require higher dilution factors in the quantitative analysis.

TABLE 4 examples of different broth to sample ratios in view of the desired level of detection.

1.6.2 swabs from production Environment samples

Swabs are various types of cloth, dried or buffered, and can be added directly to sensiist broth and left in the container during incubation. The procedure is otherwise different from that for food products.

1.6.3 Water samples

Process water, including wash water, cooling water, permeate water, etc., may be added to the SensiiList broth. For example, for product samples, concentrated culture solution may be used to reduce the level of detection while limiting the volume and thus space in the incubator. For example, a 100ml water sample can be added to 11ml of 10 XSensiList medium to obtain a detection limit of 1cfu/100 ml. The incubation and detection procedures for putative positive samples were the same as for food samples.

1.6.4 counting method-quantitative analysis

All classes of samples can be performed qualitatively or quantitatively. To count the concentration of listeria in a sample, the liquid fraction of the SensiList broth solution described above is transferred, for example, to a quantification tray, where the broth is separated in 96 small and large wells. If the entire volume (100ml) contains 1 listeria, one of the wells will turn yellow while the other wells will remain red. The most likely concentration can be estimated using the MPN method. In case a 100ml solution contains 5 grams of sample, the limit of detection will be 1cfu/5g of sample, which corresponds to an average concentration of 0.2cfu/g of sample. If the concentration of Listeria is high, e.g., 100cfu/g, then there will be 500 bacteria in 100ml of solution containing 5 grams of sample. This corresponds to 5 cfu/ml. It is then likely that all 1ml wells yellow, while up to 50% of 0.1ml wells yellow. With double concentration of listeria, up to 100% of +0.1ml wells yellow. Above this concentration, all wells will be yellow and reach the upper limit of the effective count. The minimum and maximum limits of counts obtained using 96 1ml wells and 96 0.1ml wells will be in the range of 1cfu/5 grams to 200 cfu/grams.

The limit of detection can be tuned by using another ratio between the sample and the SensiList broth.

1.7 testing of this document with artificially and naturally contaminated specimens

1.7.1 case 1:salmon contaminated artificially and naturally

The released thawed water of the inoculated samples was analyzed using the SensiList method and the modified ISO method. Procedures are as described above, and the study objective is in the results section.

1.7.2 case 2:contaminated heat-treated chicken

The samples were inoculated with low concentrations of listeria monocytogenes and analyzed according to the food sample protocol described above.

1.7.3 case 3:naturally contaminated meat and milk products, processed products from several animal species

Samples were analyzed according to the food sample protocol described above.

1.7.4 case 4: swab for testing a cleaned production surface

The samples were inoculated and analyzed according to the protocol for the environmental samples described above.

1.7.5 case 5:testing in production companies

SensiList broth in the flask was sent to salmon slaughter and willow cutting facilities. A short explanatory note and color map is also provided. The company collected samples according to normal procedures, added the samples to SensiList broth, and incubated the broth at 37 ℃. After incubation, the company takes a photograph and sends the photograph to us in order to discuss the results. They also sent the sample to a local laboratory for confirmation.

1.8 results

1.9 development of the method

1.9.1 accuracy and sensitivity of the method-detection level

For concentrations of 200cfu/g and 20cfu/g, there was a good correlation between the inoculation concentration, the concentration counted with the method of this document and the concentration counted with the ISO method. For salmon at 2cfu/g, only Listeria monocytogenes was detected using the methods of this document. This is expected because the detection level of the ISO method is 10cfu/g when performed according to standard procedures. For all concentrations, a putative positive concentration was confirmed.

The sensitivity of the method was further tested to investigate whether homogenization of the whole salmon bias was required or whether shaking of the sample with SensiList broth was sufficient. One positive sample was mixed with 9 negative samples to obtain accurate inoculation levels of very low concentrations of listeria monocytogenes (0.2-20 cfu/g). The theoretical concentration in the culture solution was 10 times lower because the sample and the culture solution were mixed in a ratio of 1: 2.5. The results are shown in fig. 3.

All inoculated samples, even the lowest concentration of listeria monocytogenes, were tested using the methods of this document. The ISO method only detected the highest concentration (20 cfu/g). After overnight incubation of the broth, all samples were assayed at a concentration of 2cfu/g or higher by ISO method, but only one of the three samples was assayed at a concentration of 0.2cfu/g, even though Listeria monocytogenes was assayed immediately after shaking the salmon side with the broth using the method of this document. The most probable explanation for this observation is that only small volumes (0.1-1.0ml solution) were plated using the ISO method, whereas the whole culture broth was analyzed using the methods of this document.

1.9.2 false positive and false negative bacteria

Similar experiments as described above were performed using different mixtures of listeria strains and salmon samples, but using an adapted version of the ISO method to obtain higher sensitivity of the method (see materials and methods). In all cases there is a good correlation between the methods, but here the methods of this document have also been found to be the most sensitive methods. However, in samples containing listeria inonotus, either alone or in admixture with listeria monocytogenes, the methods of this document give a concentration of putative positives higher than the concentration of confirmed positives compared to the ISO method. This is because listeria inonotus is also able to ferment rhamnose and has similar tolerance to antibiotics and LiCl as listeria monocytogenes. Therefore, a confirmation step is required to separate listeria monocytogenes from listeria inonotus.

Strains 1-18 are false positives obtained from salmon purchased from fish deli shops. Strains were identified by Malditoff as Hafnia alvei, Citrobacter, Aeromonas, Enterobacter cloacae, Alcaligenes faecalis, enterococcus faecalis, Bacillus cereus and Mesococcus (the latter being indeterminate) using the Malditoff technique. Strains 19-24 are Listeria monocytogenes. The conclusion is that the antibiotic tested was sufficient to avoid false positive results.

Stability of 1.9.3 SensiiList culture solution

The test strains were cultured in different SensiList broth preparations for a period of one year. The results of 48 hours incubation of all strains in fresh medium and SensiList medium stored in medium about one year before use are as follows. As expected, all strains of listeria monocytogenes (columns 1-7 of tray a and column 8 of tray C) and listeria lnokhei (tray a, column 8) gave positive results on all preparations. However, other strains only give false positive results in formulations without LiCl and/or without antibiotics. All three batches obtained correct results, indicating that the whole culture broth was stable and accurate, whether the antibiotic was added immediately before use or had been present in the culture broth for one year. The results are shown in FIG. 4.

1.9.4 confirmation of a positive result of a presumption

In all experiments using SensiList broth, the confirmation tests described by plating on ALOA and in the ISO method yielded correct results.

Confirmation by PCR technique does give correct results. Commercial kits for DNA isolation provide sufficient amounts and quality of DNA to perform PCR analysis in all test cases. The qPCR method used is the same as used for determining the gene serogroup, also referred to as molecular serogroup. The results for one of the following strains are listed in the table below. The same gene gave the correct signal for all preparations of SensiList broth. The negative signal (marked 0) is negative because the test strain is serogroup IIa.

Any other PCR method for detecting listeria monocytogenes can be used, but this method is chosen because it allows the identification of both listeria monocytogenes and serogroups. Testing of serogroups is typically performed in a single assay. Here, confirmation and sufficient characterization to examine the association with outbreaks or similarity to other isolates has been obtained in a preliminary analysis of the sample by using it as a confirmation step.

TABLE 5

Example 2: demonstration of the availability of the methods of this document for detecting Listeria in a food sample

2.1 case 1:salmon contaminated artificially and naturally

The above study was performed with salmon. The results were good in all cases.

In addition, studies have been performed on frozen and thawed salmon sides. The released thawed water was sampled using the iso (aloa) method and methods of this document. Two different mixtures of Listeria monocytogenes were used, inoculated at concentrations of 2cfu/g and 20 cfu/g. For the incubation, two temperatures were tested. The results are shown in Table 5 below. In all samples analyzed, the ISO method gave 6 negative results, whereas the method of this document gave only 1 negative result in the inoculated samples. All differences were observed for the lowest vaccination level. The increase in the frozen storage time and the decrease in the count of fish appear to be due to a decrease in viable bacteria during frozen storage.

The conclusion of this experiment is that the methods of this document are suitable for detecting and quantifying low concentrations of listeria from fresh and frozen salmon.

TABLE 6

2.2 case 2: contaminated heat-treated chicken

The heat-treated chicken was inoculated with listeria monocytogenes and analyzed with a lower dilution factor sample using the methods of this document and modified iso (aloa), resulting in a detection level of 1 cfu/g. The aim was to investigate first whether the chicken interfered with the SensiList broth and gave a false positive result, and secondly whether the concentration was correctly estimated. The results are given below.

The method of this document produces in all cases a correct detection, while the modified ISO method gives false negative results. This is likely due to the higher sensitivity of the method of this document.

The conclusion is that heat treating chicken does not interfere with the process of this document.

Furthermore, the methods of this document provide accurate counts down to 0.2cfu/g using a quantification tray and accurate detection down to 1-3 cfu/total sample compared to the modified ISO method, see table 6.

TABLE 7

2.3 case 3: naturally contaminated meat and milk products, processed products from several animal species

Naturally contaminated food samples were obtained from a reference laboratory for listeria monocytogenes in european foods. The sample is frozen and the listeria may have died during storage. However, the samples were used firstly to test the interference of the food matrix with the method of the present document, secondly to test the interference with the normal background flora and thirdly to test the count level.

Also in this case, a modified ISO method is used to obtain a sufficiently low detection level for comparison with the method of this document.

The results are shown in the following table.

In general, there is a good correlation between the modified ISO methods. The method of this document detects listeria monocytogenes in one more sample (chicken-containing noodles) than the ISO method, possibly due to higher sensitivity. The measured concentration was 0.4 cfu/g.

No interference with the test food matrix was observed to result in false positive or false negative results using the methods of this document.

One of the samples contains listeria welshimeri. This gives false positive results when using SensiList and on ALOA. The results show that false positive results can be obtained in both the methods of the invention and the prior art methods for detecting and enumerating listeria. However, listeria welshimeri is a very rare listeria compared to listeria inonotus and listeria monocytogenes, and the specificity of the method of this document is also considered to be sufficiently high, at least as high as currently available methods.

The conclusion from this study is that the methods of this document produce realistic results on naturally contaminated samples. No significant negative interference of the complex food matrix was observed. See table 7.

Table 8.

2.4 case 4: swab for testing a cleaned production surface

Swabs from testing of production equipment surfaces were obtained from three facilities. Such swabs are supplied with a neutralizing solution. This test was performed to investigate whether materials, neutralizing solutions, or residues from cleaning solutions would interfere with the methods of this document, e.g., by introducing growth inhibiting compounds or increasing buffer capacity, both of which could lead to possible false negative results.

Samples obtained from these companies were inoculated with 0, 15 or 1500 cfu/swab of listeria monocytogenes. The swabs were placed in SensiList broth and the solution was transferred to a quantification dish and incubated as described above.

None of the uninoculated swabs gave a putative positive result. For swabs inoculated with 15cfu of listeria monocytogenes, 4-9 of 96 wells were yellowed and confirmed to be positive for listeria monocytogenes. For swabs inoculated with 1500cfu, 58-96 wells yellow. In a few cases, some wells turned from red to orange, but not yellow. The color change was lower than that qualified for a putative positive result.

The conclusion from this study is that the methods of this document result in the correct detection of listeria monocytogenes in swabs for production environment sampling, and that the counts are sufficient.

2.5 cases 5 testing of production samples in salmon production

A salmon processing company drawn samples of process water and tampons from "sluk" and inserted the samples into SensiList broth. The samples were incubated in the company and sent to the local laboratory for validation. The results after the first culture are shown in FIG. 4.

According to the company, the sampling and reading of the color change is easy. Confirmation of the sample indicated that the yellow flask contained listeria inonotus, while the red flask did not contain listeria.

Water and "sluk"/drainage samples were collected with a bibulous (wad). None of these causes any interference.

Salmon sample 100g each.

The conclusion of this study, as shown in fig. 5, is that SensiList is sufficiently user friendly for use in companies. The putative positive results were consistent with the laboratory results.

Example 3: comparison of food-grade rhamnose with analytical-grade rhamnose

Rhamnose is a rare carbohydrate and is relatively expensive. Since the first culture medium according to this document uses this component and has a large volume, this component will have a large impact on the price of the method/kit. To check whether food grade rhamnose could be used instead of analytical grade rhamnose, two versions of SensiList broth were prepared and compared. The comparison was performed in two large trials:

1. the stability test of the culture broth was performed using a single cell culture with the same strain. Qualitative methods were applied.

2. Both naturally contaminated and uncontaminated samples from two fish slaughtering and processing companies were used. Samples were water, wet swabs, dry swabs and fish. Both qualitative and quantitative methods are applied. After culturing in SensiList medium, confirmation was performed using the following three agar media: blood agar, listeria monocytogenes (Rapid L' mono), and ALOA were confirmed.

Results

Testing of a single strain correctly identified both analytical and food grade rhamnose in all cases.

For samples from fish production facilities there was a good correlation between samples, which means that positive and negative results were positive for both food grade rhamnose and analytical grade rhamnose. However, there are some deviations. This is likely due to the very low concentration of listeria monocytogenes, i.e. never more than 6 cfu/total sample. At such low concentrations, if the transfer of listeria into which of the two media is random, this in turn means that only the media containing listeria is likely to give a positive result.

An overview of the results from a company with samples taken over time is given below. Crosses indicate negative samples and Pos indicates detection. All concentrations were very low. Previous revisions of the ISO method used less diluent, so that the count detection level of 2cfu/g was not sufficient to detect listeria in these samples.

Sample positive samples from companies: no more than 3 positive wells: indication < 6 cfu/sample

Table 9 this table is an example of what is used in the industry. The norwegian text in the left-most column shows the position in the production line. The top row shows the date of the sample, where "Pos" represents a positive sample. The rightmost column has comments on the test, such as "swab of 10 fish filets", "meat sample", "water".

The results for three of the samples are given in fig. 6. The left box represents a qualitative method in which 1 listeria monocytogenes can be detected in a sample, in this case 20ml of water. Only the left sample was positive. The same samples were analyzed using a quantitative method (right panel). In this case, 100m1 samples (10ml water and 90ml SensiiList broth) were transferred to the quantification dish. Only one well changed color from red to yellow, which means that the most likely concentration of listeria monocytogenes in water was 1cfu/10 ml. Results were obtained after 24 hours of incubation.

The SensiList method (i.e. the method of this document) is applicable to all samples, but the dilution ratio needs to be taken into account, especially for very wet samples and for fish meat. On the one hand, this is due to the dilution of the SensiList broth, since the pH is related to the amount of acid produced and thus to the concentration of rhamnose. On the other hand, high relative amounts of other carbon and energy sources, such as fish, may result in bacteria growing on these instead of rhamnose, and will not produce sufficient amounts of acid. Both of these challenges can be easily addressed in SensiList broth if a sufficiently high proportion of broth is used.

The results of the putative positive samples are given in figure 7. Two holes are yellow, some are orange, and most holes are red. The material from each well was transferred to three different growth media to test specificity. Only yellow wells hemolyze on blood agar, and a typical region of listeria monocytogenes appears on ALOA agar. Orange and red wells either did not grow or bacteria that are not listeria monocytogenes were grown. Thus, the sensitivity and selectivity of SensiList using rhamnose broth was as good as previously found when using assay grade rhamnose.

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Each of the preferred features described herein can be used in combination with any and all of the other preferred features described herein, unless expressly stated to the contrary.

Reference to the literature

Vitullo et al.，Real-time PCRs assay for serogrouping Listeria monocytogenes and differentiation from other Listeria spp.，Molecular and Cellular Probes，Volume 27，Issue 1，February 2013，Pages 68-70.

C.T.Schirmer，Solveig Langsrud，TrondTherese Hagtvedt，Even Heir，2012.Performance of two commercial rapid methods for sampling and detection of Listeria in small-scalecheese producing and salmon processing environments.Journal of Microbiological Methods，Volume 91，Issue 2，Pages 295-300，ISSN 0167-7012，https：//doi.org/10.1016/j.mimet.2012.08.013。