阅读说明：本技术 一种基于数字pcr技术检测椰毒假单胞菌的试剂盒及其应用 (Kit for detecting pseudomonas cocoi based on digital PCR technology and application thereof ) 是由 杨佳怡 高运华 王志栋 吴枭 于 2021-01-07 设计创作，主要内容包括：本发明公开一种用于PCR检测椰毒假单胞菌的引物和探针组合,其上游引物序列为：TTGTCTGGCGGTAGAAACC,下游引物序列为：下游引物：CGAGTCAACTGACCCGAAC,探针序列为：TCGATCCCGTCTGCCTCCACCAATCT。还包括上述引物和探针组合的检测试剂盒。本发明提供的优化设计的引物探针更适用于椰毒假单胞菌的定量检测,且同时适用于荧光定量PCR的检测体系,两种方法表现出良好的线性相关性,引物探针的优化设计扩展了该试剂盒的应用范围,方便检测者根据具体的实验条件进行应用。(The invention discloses a primer and probe combination for PCR detection of pseudomonas cocoanut, wherein the upstream primer sequence is as follows: TTGTCTGGCGGTAGAAACC, the sequence of the downstream primer is: a downstream primer: CGAGTCAACTGACCCGAAC, the probe sequence is: TCGATCCCGTCTGCCTCCACCAATCT are provided. Also comprises a detection kit combining the primer and the probe. The primer probe with optimized design provided by the invention is more suitable for quantitative detection of pseudomonas cocoanut and is also suitable for a fluorescent quantitative PCR detection system, the two methods show good linear correlation, the application range of the kit is expanded by the optimized design of the primer probe, and a detector can conveniently apply the primer probe according to specific experimental conditions.)

1. A primer and probe combination for PCR detection of Pseudomonas cocoanut is characterized in that,

an upstream primer: TTGTCTGGCGGTAGAAACC

A downstream primer: CGAGTCAACTGACCCGAAC

And (3) probe: TCGATCCCGTCTGCCTCCACCAATCT are provided.

2. The primer and probe combination of claim 1, for use in digital PCR or fluorescent PCR.

3. The primer and probe combination of claim 2, wherein the probes are as follows:

FAM-TCGATCCCGTCTGCCTCCACCAATCT-BHQ1。

4. a kit for detecting Pseudomonas cocoi, comprising the primer and probe combination according to any one of claims 1 to 3.

5. The kit according to claim 4, further comprising a negative control, a positive control, wherein the negative control is genomic DNA of an Aspergillus strain.

6. A method for detecting Pseudomonas cocoanut in food, which is characterized in that the kit of claims 4 to 5 is used for detecting Pseudomonas cocoanut in food to be detected by digital PCR or fluorescence PCR.

7. The detection method as claimed in claim 6, wherein after the DNA is extracted from the sample of the food to be detected, the digital PCR or the fluorescent quantitative PCR detection is directly carried out by using the primer probe in the kit, and preferably, the positive control and the negative control in the kit are added in the detection process for quality control.

8. The detection method according to claim 6 or 7, wherein the working concentrations of the primer and the probe in the reaction system of PCR are respectively 700nM and 300nM, preferably 600nM and 400 nM.

9. The method of claim 6 or 7, wherein the annealing temperature is 56 ℃ under the PCR reaction conditions in the assay.

10. The detection method according to claim 6 or 7, which is a digital PCR, wherein the reaction procedure is as follows:

Technical Field

The invention belongs to the fields of genetic engineering and food detection, and particularly relates to a kit for detecting pseudomonas cocoanut based on a digital PCR technology and application thereof.

Background

The pseudomonas cocoanut belongs to gram-negative bacteria, is facultative and anaerobic, is easy to grow on the surface of food, is easy to pollute fermented cereal products (fermented corn flour, waxy corn dumpling flour, fermented waxy millet and the like), potato products (potato vermicelli, sweet potato starch and the like), deteriorated tremella, agaric and the like. Food poisoning caused by pseudomonas cocoanut has extremely high lethality rate, and poses serious threat to health. The fatality rate of food poisoning caused by the bacteria is as high as 40-100%. At present, the method of initial emetic, catharsis and later-stage plasma replacement is mainly adopted to treat poisoned patients, and no specific treatment means or medicine exists. The growth temperature of the pseudomonas cocoanut is 37 ℃, the toxin production temperature is 26 ℃, wherein the toxic events are frequently generated in summer and autumn, and high-generation areas of China comprise Guangxi, Yunnan, Guizhou and the like. The sour soup poisoning event happens in 2020, and high-concentration rice ferment acid is found after the corn flour sample is detected, and the food poisoning caused by the pseudomonas cocoanut is primarily determined.

The toxic effects of pseudomonas cocoi are mainly due to two toxins produced: since both the mirostrobin and the toxoflavin are fatty acid micromolecular substances, researchers develop methods based on liquid chromatography and chromatography-mass spectrometry to detect the mirostrobin and the toxoflavin, but the methods need expensive instruments, are difficult to detect and operate, and are not beneficial to rapid field detection. The detection of the pseudomonas cocoanut is also paid more attention, and the classical plate culture method is simple in operation, long in time consumption and large in error range. GB/T4789.29-2003 'test of food hygiene microbiology for testing coconut toxin pseudomonad leavened rice flour subspecies' is the main basis for detecting coconut toxin pseudomonad, but the method for culturing bacteria on a flat plate is long in time consumption, has various influence factors, and is not beneficial to rapid detection under emergency. In recent years, the detection method based on the PCR amplification technology has received more and more attention, and researchers detect the nucleic acid of the pseudomonas cocoanut through a loop-mediated isothermal amplification (LAMP) method and a fluorescent quantitative PCR method, so that the detection efficiency is improved. However, the quantitative results of the existing nucleic acid detection methods all depend on standard curves, and the accuracy is to be improved (the research on rapid detection of the pseudomonas cocoanut by using the loop-mediated isothermal amplification technology [ J ]. food industry science and technology, 2013,34(03):321 + 324; Linjie, Fangchenyu, Lujinfang, Yidanhan, Zhaoqiqi, Wangjiasheng ] research on real-time fluorescence PCR detection of the pseudomonas cocoanut zymomonas rice subspecies in food [ J ]. food safety and quality detection science, 2020,11(11):3538 + 3544). The principle of fluorescence quantitative PCR is to measure the fluorescence signal accumulated during PCR amplification, which depends on a standard curve during quantitative detection. In general, the existing nucleic acid detection method for pseudomonas cocoi has obvious limitations: 1. the method mainly adopts semi-quantitative analysis, and can not realize 'absolute quantification' independent of external standards; 2. lack of quality control of the detection process. Both of these problems may affect the accuracy of the detection. In order to improve the reliability of the detection result of pseudomonas cocoanut, it is urgently needed to develop a detection method capable of carrying out absolute quantification, and add appropriate positive and negative controls to further form a complete detection kit, which is convenient for detection researchers to use. No research report for detecting the pseudomonas cocoanut based on the digital PCR technology exists, and the detection accuracy of the existing method needs to be further improved.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a complete kit for detecting pseudomonas cocoanut, which comprises a positive control, a negative control and a primer probe.

The invention firstly provides a primer and probe combination for PCR detection of Pseudomonas cocoanut, which is characterized in that,

an upstream primer: TTGTCTGGCGGTAGAAACC

A downstream primer: CGAGTCAACTGACCCGAAC

And (3) probe: FAM-TCGATCCCGTCTGCCTCCACCAATCT-BHQ 1.

Preferably, it is used for digital PCR or fluorescence PCR. More specifically, the probes are as follows:

FAM-TCGATCCCGTCTGCCTCCACCAATCT-BHQ1。

the invention designs a primer and a probe aiming at a 16S-23S rRNA sequence with high evolutionary conservation in the genome of pseudomonas cocoanut, and optimizes a reaction system. Compared with primers and probe sequences generated by common primer design software, the primer probe optimally designed by the method is more suitable for quantitative detection of pseudomonas cocoanut and is also suitable for a detection system of fluorescence quantitative PCR (polymerase chain reaction), the two methods show good linear correlation, the application range of the kit is expanded by the optimal design of the primer probe, and a detector can conveniently apply the primer probe according to specific experimental conditions. The method based on the digital PCR technology is beneficial to improving the detection accuracy, and compared with the existing qualitative detection method, the establishment of the method has important significance for preventing food poisoning caused by pseudomonas cocoi.

The invention further provides a kit for detecting pseudomonas cocoanut, which is characterized by comprising the primer and probe combination. Further, the kit also comprises a negative control and a positive control, wherein the negative control is the genome DNA of the aspergillus flavus strain.

In order to provide a reference for quality control in the measurement process, genome reference substances of positive control and negative control determined by sources are added, and the extracted and purified pseudomonas cocoanut genome is used as the positive control in the kit; in the process of selecting the negative control, the aspergillus flavus is considered to be a saprophytic fungus through analysis and research, the aspergillus flavus is commonly found in improperly stored grains and grain products and can also cause pollution to milk products, edible oil and the like, and the metabolite aflatoxin of the aspergillus flavus is classified as a class 1 carcinogen by the world health organization. The aspergillus flavus and the pseudomonas cocovorans can pollute food-related products and have strong pathogenicity, and the confusion of the detection of the aspergillus flavus and the pseudomonas cocovorans is not beneficial to scientific prevention and diagnosis. The positive control and the negative control have higher purity and are convenient to take and use, and the positive control and the negative control are measured simultaneously with a sample to be detected, thereby being beneficial to improving the effectiveness and the accuracy of detection.

At present, most of the existing detection methods for pseudomonas cocoanut stay in the laboratory research stage, and a complete detection kit containing a primer, a probe, a positive control reference substance and a negative control reference substance is not formed, so that the detection work is inconvenient. The digital PCR can carry out quantitative detection on nucleic acid without depending on a standard curve, the tracing of the quantity value to a natural unit is ensured, and the detection method based on the digital PCR technology is the basis of the development of standard substances in the later period and is beneficial to improving the comparability and the consistency of different detection methods. And positive and negative control genome reference substances are added into the test reagent box, so that quality control can be provided for the detection process, possible interference of aspergillus flavus pollution can be eliminated in the practical application process, and scientific prevention of food poisoning events is facilitated. That is to say, the genome of the pseudomonas cocovorans is used as a positive control, the genome of the aspergillus flavus is used as a negative control, the possible confusion problem of the pseudomonas cocovorans and the aspergillus flavus is avoided, reference is provided for quality control in the detection process, the accuracy of nucleic acid detection of the pseudomonas cocovorans is improved, and the pseudomonas cocovorans and the aspergillus flavus can be widely applied to detection of potential contaminated food.

Drawings

FIG. 1 shows the effect of primer probes generated by software applied to a digital PCR system.

FIG. 2 is a diagram showing the effect of the optimized primer probe applied to a digital PCR system.

FIG. 3 digital PCR system annealing temperature optimization.

FIG. 4 working concentration optimization of primers and probes in digital PCR reaction system.

FIG. 5 shows the result of the numerical PCR method in terms of the measurement range.

The digital PCR method of fig. 6 shows good linearity.

FIG. 7 is the linear correlation between the fluorescent quantitative PCR of Pseudomonas cocoanut and the digital PCR detection method.

FIG. 8 is a schematic view of the construction of a Pseudomonas cocoanut detection kit.

FIG. 9 shows the results of detection of actual samples using the kit.

Detailed description of the preferred embodiments

The invention is further illustrated, but not limited, by the following specific examples.

EXAMPLE 1 design of primers and probes

The applicability of primers and probes is very important because the amplification needs to be carried out based on the whole genome of the pseudomonas cocoi during the detection process. The invention designs a primer and a probe of a digital PCR system aiming at a 16S-23S rRNA sequence (GenBank gene number: EF552059) highly conserved in the evolution process of a genome of pseudomonas cocoanut. The results of primer probes automatically designed using primer5.0 software are as follows. Wherein the 5 'end of the probe is connected with FAM, and the 3' end is connected with BHQ 1.

TABLE 1 primer probe sequence for detecting Pseudomonas coco by automatic design of digital PCR

Name (R)	Sequence of
		Upstream primer	GTCTTTGTCATTGGCGATT
Downstream primer	ATACAATCACAACCCGGAT
		Probe needle	FAM-ACGCCCATCTCAATAGACGCTT-BHQ1

The amplification experiment was performed using a primer probe automatically designed by software, and the results are shown in FIG. 1. It can be observed that in the reaction system, the positive and negative droplet units are difficult to distinguish, which affects the accuracy of quantitative detection.

Based on this, the present inventors optimized primers and probes. The primers and probes were designed by optimizing the GC content and the length of the amplification product, and the sequence information of the optimally designed primers and probes as shown in table 1 was obtained.

Table 2 primer probe sequence for detecting Pseudomonas cocoi by optimized digital PCR

Name (R)	Sequence of
		Upstream primer	TTGTCTGGCGGTAGAAACC
Downstream primer	CGAGTCAACTGACCCGAAC
		Probe needle	FAM-TCGATCCCGTCTGCCTCCACCAATCT-BHQ1

Using genome DNA of pseudomonas cocoi as a template, measuring the concentration of the DNA by Nanodrop, calculating the copy number concentration by combining the relative molecular weight of the genome, calculating the concentration of the template DNA in the reaction to be 4000 copies/mu L by dilution, and preparing a digital PCR reaction system according to the components in the following table:

the temperature cycling process of the digital PCR reaction is shown in the following table:

after the PCR amplification is completed, reading is carried out, and the result is shown in FIG. 2, so that an amplification effect graph is obtained, in the reaction system, the positive droplet unit and the negative droplet unit can be well distinguished, and the positive reaction signals are relatively concentrated. The applicability of the primer and the probe is an important basis for continuously optimizing a digital PCR reaction system subsequently.

EXAMPLE 2 optimization of annealing temperature

This example optimizes the annealing temperature of the PCR reaction. The annealing temperature was set at a gradient of 54 deg.C, 56 deg.C, 58 deg.C, 60 deg.C and 62 deg.C.

Using genome DNA of pseudomonas cocoi as a template, measuring the concentration of the DNA by Nanodrop, calculating the copy number concentration by combining the relative molecular weight of the genome, calculating the concentration of the template DNA in the reaction to be 4000 copies/mu L by dilution, and preparing a digital PCR reaction system according to the components in the following table:

the temperature cycling process of the digital PCR reaction is shown in the following table:

after the PCR amplification is completed, reading is performed, as shown in fig. 3, and the annealing temperature of the reaction system is finally determined to be 56 ℃ by taking the most significant difference between the positive and negative signals as the determination criterion.

EXAMPLE 3 optimization of working concentrations of primers and probes

This example is done to determine the optimal working concentration of primers and probes. Orthogonal assays were performed with primer concentration gradients (200nM, 400nM, 600nM, 800nM) and probe concentration gradients (200nM, 300nM, 400nM), respectively.

Using genome DNA of pseudomonas cocoi as a template, measuring the concentration of the DNA by Nanodrop, calculating the copy number concentration by combining the relative molecular weight of the genome, calculating the concentration of the template DNA in the reaction to be 4000 copies/mu L by dilution, and preparing a digital PCR reaction system according to the components in the following table:

the temperature cycling process of the digital PCR reaction is shown in the following table:

after the PCR amplification is completed, the reading is performed, as shown in fig. 4, and the working concentrations of the finally selected primers and probes are 600nM and 400nM, respectively, with the most significant difference between the positive and negative signals as the determination criterion.

Example 4 digital PCR reaction measurement Range determination

The measurement range of the digital PCR is 1-10⁵The copies/. mu.L, using the genome DNA of Pseudomonas coco as the template, measuring the concentration of DNA by Nanodrop, calculating the copy number concentration according to the relative molecular weight of the genome, and performing the quantitative analysis of digital PCR after the DNA template is diluted in a gradient manner. The digital PCR reaction system was formulated according to the composition in the following table:

the temperature cycling process of the digital PCR reaction is shown in the following table:

after the PCR amplification is finished, reading is carried out, the dilution times are calculated in the process of gradient dilution through a gravimetric method, the copy number concentration calculated by the DNA concentration and the quantitative detection result of the digital PCR are counted in the following table, wherein the quantitative result of the digital PCR is the average value of three repeated experiments. In FIG. 5, NTC represents a blank control without a template, which is a result of the determination of the numerical PCR within the measurement range.

Next, linear regression analysis was performed, and as shown in FIG. 6, the digital PCR method showed good linearity (R) in its measurement range²＝0.998)

Example 5 comparison of digital PCR reaction with fluorescent quantitative PCR

It is to be noted that, by designing an optimized primer probe, which is also applicable to the fluorescent quantitative PCR system, the measurement of the fluorescent quantitative PCR was performed using the above-mentioned DNA template diluted in a gradient, and the results of the quantitative detection of the fluorescent quantitative PCR and the digital PCR were counted in the following table, and the quantitative results in the table are the average values of three repeated experiments (refer to the reaction system and the reaction program in example 4)

Under the concentration of 10 copies/mu L, the fluorescent quantitative PCR has no effective reading, which indicates that the digital PCR detection method has higher sensitivity and is more suitable for detecting trace samples, and linear correlation analysis is carried out in the common measurement range of the two methods. As shown in FIG. 7, both methods of fluorescent quantitative PCR and digital PCR showed good linear correlation (R)²＝0.999)。

Considering that the instrument used in the digital PCR reaction is expensive and complex to operate, and the application range of the fluorescent quantitative PCR is wider, the experimental result proves that the kit is simultaneously suitable for the digital PCR and the fluorescent quantitative PCR detection method, and provides greater convenience for use.

Example 6 quality control during measurement

In order to provide a reference for quality control in the measurement process, genome reference substances of positive control and negative control determined by sources are added, and the extracted and purified pseudomonas cocoanut genome is used as the positive control in the kit; in the process of selecting the negative control, through analytical research, the aspergillus flavus is considered as a saprophytic fungus, is commonly found in improperly stored grains and food products, and can also pollute milk products, edible oil and the like, and the metabolite aflatoxin of the aspergillus flavus is classified as a type 1 carcinogen by the world health organization. The aspergillus flavus and the pseudomonas cocovorans can pollute food-related products and have strong pathogenicity, and the confusion of the detection of the aspergillus flavus and the pseudomonas cocovorans is not beneficial to scientific prevention and diagnosis. The positive and negative controls have higher purity and are convenient to take and use, and the positive and negative controls are measured simultaneously with a sample to be detected, so that the effectiveness and the accuracy of detection are improved, and fig. 8 is a schematic diagram of the composition of the detection kit.

Using a liquid medium to culture Pseudomonas cocoanut bacteria solutions of different durations as detection samples, the detection kit of the present invention was used to perform digital PCR detection in the following manner (see the reaction system and procedure in example 4)

The experimental results are shown in fig. 9 and the following table, wherein sample 1 is a overnight-cultured and obviously turbid pseudomonas natans liquid, sample 2 is a pseudomonas natans liquid which is not obviously turbid after 6 hours of culture, and the quantitative results in the table are the average values of three repeated experiments.

	Digital PCR fixed value (copies/. mu.L)
		Positive control	4220
Sample 1	168
		Sample 2	27.67
Negative control	0

The experimental result shows that the kit can be used for detecting a reagent sample with lower concentration (without obviously turbid bacterial liquid), the accurate copy number concentration is provided without depending on a standard curve, and meanwhile, the positive control and the negative control in the kit can play a role in quality control. The kit is suitable for quantitative detection of pseudomonas cocoanut and has important significance for preventing food poisoning.

Sequence listing

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