阅读说明：本技术 一种用于血培养报阳样品预处理的试剂盒及方法 (Kit and method for pretreatment of blood culture positive reporting sample ) 是由 徐健 韩霄 徐腾 朱鹏飞 籍月彤 于 2020-05-26 设计创作，主要内容包括：本发明提供了一种用于血培养报阳样品预处理的试剂盒及方法,所述试剂盒包括保护液、聚沉液；所述保护液含有蛋白胨,牛肉浸粉、牛心浸出粉或牛脑浸出粉中的一种,可溶性淀粉,氯化钠,水；所述聚沉液含有阳离子聚合物,水。所述试剂盒能够实现报阳血培养瓶内微生物与活性炭的有效分离,获得包含微生物的澄清菌液,继而进行培养或其他检测。该试剂盒操作方法简便、耗时短、微生物亲和性高,在临床上具有广泛的应用前景。(The invention provides a kit and a method for pretreating a blood culture newspaper positive sample, wherein the kit comprises a protective solution and a coagulation solution; the protective solution contains one of peptone, beef extract powder, beef heart extract powder or beef brain extract powder, soluble starch, sodium chloride and water; the coagulation liquid contains cationic polymer and water. The kit can realize effective separation of microorganisms and active carbon in the yang blood culture bottle, obtain clarified bacterial liquid containing the microorganisms, and then culture or other detection is carried out. The kit has the advantages of simple and convenient operation method, short time consumption and high microbial affinity, and has wide application prospect clinically.)

1. A kit for pretreating a blood culture newspaper positive sample is characterized by comprising a protective solution and a coagulation solution; the protective solution contains one of peptone, beef extract powder, beef heart extract powder or beef brain extract powder, soluble starch, sodium chloride and water; the coagulation liquid contains cationic polymer and water.

2. The kit of claim 1, wherein the peptone is tryptone, and,Peptone or soybean peptone.

3. The kit for pretreating a blood culture newspaper sample as recited in claim 1, wherein the protective solution comprises 1.00-8.00% (mass fraction) of peptone, 4.00-10.00% (mass fraction) of one of beef extract powder, beef heart extract powder or beef brain extract powder, 0.50-0.80% (mass fraction) of soluble starch, 0.50-1.00% (mass fraction) of sodium chloride, and the balance of water.

4. The kit for pretreatment of blood culture positive samples according to claim 1, wherein the coagulation solution contains 0.67-300 ‰ (volume fraction) of cationic polymer, and the balance is water.

5. The kit of claim 1, wherein the cationic polymer is one of polyhexamethylene dimethyl ammonium bromide, polyhydroxyethyl cellulose ether quaternary ammonium salt, cationic polyacrylamide, polydiallyldimethyl ammonium chloride, cationic starch, or saturated aluminum potassium sulfate solution.

6. The kit for pretreatment of the blood culture newspaper sample as recited in any one of claims 1 to 5, wherein the kit further comprises a centrifuge tube.

7. A pretreatment method for a blood culture positive sample is characterized by comprising the following steps:

s1: establishing an activated carbon coagulation system, which comprises uniformly mixing a blood culture positive sample, a protective solution in the kit according to any one of claims 1 to 5 and a coagulation solution;

s2: the coagulation of the activated carbon and the taking out of the microorganism sample specifically comprise the steps of centrifuging the activated carbon coagulation system established in the step S1, and collecting the supernatant containing the microorganism sample.

8. The method according to claim 7, wherein the step S1 of establishing an activated carbon coagulation system comprises: s11, putting a blood culture positive sample accounting for 70-95% of the total volume into a clean centrifuge tube; s12, adding a protective solution accounting for 5-30% of the total volume; s13 adding the coagulation liquid according to the volume of the protective liquid and the volume ratio of 5:1-50: 1.

9. The method for pretreating the blood culture newspaper positive sample according to claim 8, wherein in the step S12, the protective solution is added and then the mixture is inverted and mixed for 3 times; adding the coagulation liquid, and then, uniformly mixing for 15s by vortex.

10. The method as claimed in claim 7, wherein in another preferred embodiment, in the step S2, the centrifugation time is 10 seconds, and the centrifugation speed is 1000-2000 rpm.

Technical Field

The invention relates to the technical field of pretreatment of detection of clinical pathogenic microorganisms, in particular to a kit and a method for pretreatment of a blood culture newspaper positive sample.

Background

Bloodstream infections (BSIs) are infections caused by the invasion of blood by various pathogenic microorganisms (including bacteria, fungi, etc.), and are a serious systemic infectious disease including bacteremia and septicemia. The fatality rate of blood stream infection of many developed countries in Europe and America is as high as 12% -20%, and is one of the main causes of death of patients. More than 50% of cases occur in the elderly population aged at 65 years old, and the incidence of infection is increased in elderly patients due to hypoimmunity, basic diseases, malnutrition, atypical blood stream infection, etc. In recent years, the basic medical construction of China has been greatly developed, and invasive and traumatic diagnosis and treatment means such as mechanical ventilation, venous catheter indwelling, endoscope and the like are widely applied, but become common infection routes of blood stream infection. In addition, the phenomenon of unreasonable use of broad-spectrum antibiotics, corticosteroids, immunosuppressants and the like still exists, and the incidence rate and the fatality rate of bloodstream infection in China tend to rise year by year and the drug resistance is gradually enhanced.

Blood culture is the gold standard for diagnosis of blood stream infection, and accurate and rapid blood culture results can better guide clinical scientific and reasonable medication and improve the survival rate and cure rate of patients. However, the abuse of antibiotics severely affects the true positive detection rate of blood cultures. In order to ensure the real positive detection rate of blood culture, a blood culture bottle containing a neutralizer or an adsorbent is usually selected clinically, and the currently more commonly used adsorbent is activated carbon and resin. There are many reports at home and abroad about the comparison of the two types of blood culture bottles in the aspects of antibiotic adsorption performance, positive alarm time, positive detection rate and the like, and the general conclusion is that the two types of blood culture bottles are respectively superior and inferior, and the combined use has more advantages than the single use of the one type of blood culture bottle.

After blood culture positive reporting, subsequent detection such as strain identification, drug sensitivity detection and the like is required, and the currently clinically adopted method comprises novel detection methods such as matrix-assisted laser desorption ionization time-of-flight mass spectrometry, PCR amplification and probe hybridization, flow cytometry, single cell Raman spectrum detection and the like besides the traditional culture identification method. The resin-adsorbed blood culture bottle can be used in combination with conventional or above-mentioned novel detection methods after simple treatment. However, the activated carbon adsorption blood culture bottle has limited application because microorganisms and activated carbon are difficult to separate after reporting yang.

Disclosure of Invention

Aims to solve the key problem that in the prior art, the application of an activated carbon adsorption blood culture bottle is limited because microorganisms and activated carbon are difficult to separate after yang reporting. The invention provides a kit for pretreating a blood culture positive sample of a clinical activated carbon adsorption blood culture bottle. The kit can cause the active carbon to spontaneously aggregate through changing the solution environment of the yang-reporting blood culture bottle, thereby realizing the separation of microorganisms (including gram-negative bacteria, gram-positive bacteria, fungi and the like) and the active carbon, and obtaining a microorganism sample which can be used for identifying the strains of the blood-cultured yang-reporting sample and detecting drug sensitivity.

The sample processed by the kit can be subjected to subsequent operations such as culture, mass spectrometry detection, DNA extraction, single cell Raman detection and the like.

In order to achieve the above object, the present invention provides, in one aspect, a kit for pretreating a blood culture newspaper positive sample, the kit comprising a protective solution and a coagulation solution;

the protective solution contains one of peptone, beef extract powder, beef heart extract powder or beef brain extract powder, soluble starch, sodium chloride and water;

the coagulation liquid contains cationic polymer and water.

The peptone is tryptone,Peptone or soybean peptone.

In another preferred example, the protective solution contains 1.00-8.00% (mass fraction) of peptone, 4.00-10.00% (mass fraction) of one of beef extract powder, beef heart extract powder or beef brain extract powder, 0.50-0.80% (mass fraction) of soluble starch, 0.50-1.00% (mass fraction) of sodium chloride, and the balance of water.

In another preferred example, the protective solution contains 1.00-8.00% (mass fraction) of peptone, 4.00-10.00% (mass fraction) of one of beef extract powder, beef heart extract powder or beef brain extract powder, 0.50-0.80% (mass fraction) of soluble starch, 0.50-1.00% (mass fraction) of sodium chloride, 0.25-0.50% (mass fraction) of disodium hydrogen phosphate, and the balance of water.

The coagulation liquid contains cationic polymer and water.

The coagulation liquid contains 0.67-300 per mill (volume fraction) of cationic polymer, and the balance of water.

In another preferred embodiment, the coagulation liquid contains 0.67-300 per mill (volume fraction) of cationic polymer, 0.5-1.0 percent (mass fraction) of sodium chloride, and the balance of water.

In another preferred example, the cationic polymer is one of polyhexamethylene dimethyl ammonium bromide, polyhydroxyethyl cellulose ether quaternary ammonium salt, cationic polyacrylamide, polydiallyldimethyl ammonium chloride, cationic starch or saturated aluminum potassium sulfate solution.

The protective solution comprises the following components in percentage by weight:

the coagulation liquid comprises the following components in percentage by weight:

in another preferred example, the kit further comprises a centrifuge tube.

In another preferred example, the centrifuge tube is a 1.5mL centrifuge tube.

In another preferred embodiment, the kit has a shelf life of at least 1 year.

The invention also provides a pretreatment method of the blood culture positive reporting sample, which comprises the following steps:

s1: establishing an activated carbon coagulation system, which comprises uniformly mixing a blood culture sunrise sample, a protective solution and a coagulation solution;

s2: the coagulation of the activated carbon and the taking out of the microorganism sample specifically comprise the steps of centrifuging the activated carbon coagulation system established in the step S1, and collecting the supernatant containing the microorganism sample.

In another preferred example, the microorganism sample obtained in step S2 may be cultured continuously for traditional strain identification and drug sensitive detection, or may be subjected to DNA extraction or incubation according to the subsequent detection requirement.

In another preferred example, the microorganism sample obtained in step S2 is frozen and fixed according to the microorganism freezing operation if it is not used immediately.

In another preferred example, the step S1 is to establish an activated carbon coagulation system, which specifically includes: s11, putting a blood culture positive sample accounting for 70-95% of the total volume into a clean centrifuge tube; s12, adding a protective solution accounting for 5-30% of the total volume; s13 adding the coagulation liquid according to the volume of the protective liquid and the volume ratio of 5:1-50: 1.

The volume content of each component of the activated carbon coagulation system is as follows:

in another preferred example, in step S12, the mixture is added with the protective solution and then inverted and mixed for 3 times.

In another preferred example, in the step S12, the coagulation liquid is added and then vortexed and mixed for 15 seconds.

In another preferred embodiment, the total volume of the activated carbon coagulation system does not exceed 2 mL.

In another preferred example, in the step S2, the centrifugation time is 10 seconds, and the centrifugation rotation speed is 1000-.

In another preferred example, in the step S2, the activated carbon coagulation system is left standing at room temperature for 15 minutes before the centrifugal treatment.

The invention has the beneficial effects that:

(1) the kit can realize effective separation of microorganisms and active carbon in the yang blood culture bottle, obtain clarified bacterial liquid containing the microorganisms, and then culture or other detection is carried out.

(2) The kit has the advantages of simple and convenient operation method, short time consumption and high microbial affinity, and has wide application prospect clinically.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It is obvious that the drawings in the following description are only some of the embodiments described in the present application, and that other drawings can be derived from these drawings by a person skilled in the art without inventive effort.

FIG. 1 shows the results of the Raman spectroscopy PCA analysis after the treatment of a bacillus coli blood culture positive sample;

FIG. 2 shows the results of the Raman spectroscopy PCA analysis after the enterococcus faecalis blood culture positive sample treatment;

FIG. 3 shows the result of the Raman spectroscopy PCA analysis after the treatment of the blood culture and positive report sample of Candida albicans.

Detailed Description

In order to make those skilled in the art better understand the technical solutions in the present application, the present invention will be further described with reference to the following examples, and it is obvious that the described examples are only a part of the examples of the present application, and not all examples. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

Example 1

Table 1 is an example of a kit component,

TABLE 1 kit composition

The components of the protective solution and the coagulation solution are shown in tables 2 and 3:

TABLE 2 protective solution Components

The coagulation liquid is:

table 3 coagulation fluid composition:

table 4 shows the volume distribution of the components of the activated carbon coagulation system prepared in the blood culture positive sample pretreatment method.

TABLE 4 preparation of activated carbon coagulation system

Remarking: the preparation system is suitable for 1 pretreatment.

Example 2

By utilizing the kit, a gram-negative bacteria blood culture positive sample which is cultured by using an activated carbon adsorption blood culture bottle and represented by escherichia coli is pretreated, and a bacterial Raman spectrum detection is carried out after the treatment to evaluate the pretreatment effect, wherein the kit specifically comprises the following steps:

1. preparing colibacillus blood culture positive sample

The culture bottle for culturing escherichia coli blood and reporting positive is taken down from the culture instrument, inverted and mixed evenly for 5 times, and 2mL of positive reporting sample is extracted by a syringe for standby.

2. Establishing an activated carbon coagulation system

The addition volumes of the components of the operation are shown in Table 5:

TABLE 5 preparation of activated carbon coagulation system for gram-negative bacteria blood culture positive samples represented by E.coli

The specific operation steps are as follows:

(1) taking 750 mu L of escherichia coli blood culture positive-reporting sample to a 1.5mL clean centrifuge tube;

(2) adding 250 μ L of protective solution, and mixing by reversing for 3 times;

(3) add 8. mu.L of the coagulation liquid and vortex for 15 s.

3. Coagulation of activated carbon and removal of microbial samples

And (3) standing the system obtained in the step (2) at room temperature for 15min, centrifuging the system by using a palm centrifuge (1000rpm for 10s), and collecting the supernatant containing the escherichia coli to be detected.

4. Raman detection sampling and sheet making

Taking 800 mu L to 1.5mL of the pretreated supernatant as an experimental group, taking 800 mu L to 1.5mL of a bacterial liquid which is purely cultured by using an Escherichia coli BHI culture medium (being a culture medium component in a blood culture bottle) of the same strain as a positive control group, and taking 800 mu L to 1.5mL of an Escherichia coli blood culture positive sample which is not pretreated as a negative control group. The three groups of samples were centrifuged (12000rpm, 2min) to remove the supernatant, 500. mu.L of ddH2O was added to each group of samples to resuspend, the centrifugation was continued to remove the supernatant, the washing was repeated three times, and 10. mu.L of ddH2O was added to each group of centrifuge tubes to resuspend and mix well to dilute the precipitated samples.

10 μ L of the diluent was spotted onto a clean detection chip (calcium fluoride slide), three samples were spotted in parallel on each sample, and the samples were air-dried in a biosafety cabinet.

5. Raman detection

Detecting a sample by adopting a single cell Raman fast detector, wherein the detection parameters are as follows: the diameter of a pinhole is 125 mu m, the grating is 600, the lens of an objective lens is 100x, the exposure time is 6s, the laser wavelength is 532nm, and the laser intensity is 180 mW.

6. Data processing and result analysis

The main component analysis is carried out according to the Raman spectrum of the bacteria obtained by the single cell Raman fast detector, and the experimental result is shown in figure 1: the main component analysis of the experimental group sample and the positive control group sample has no significant difference, and the main component analysis of the negative control group sample and the positive control group sample has significant difference. The results show that after the gram-negative bacteria blood culture positive (activated carbon culture bottle) sample represented by escherichia coli is pretreated, the interference of activated carbon and other impurities is effectively removed under the condition of ensuring the normal state of bacteria. (the strain number Escherichia coli group is a negative control group, the blood culture-Escherichia coli group is a positive control group, and the pretreatment-blood culture-Escherichia coli group is an experimental group)

Example 3

By utilizing the kit, a gram positive bacteria blood culture positive sample which is cultured by using an activated carbon adsorption blood culture bottle and represented by enterococcus faecalis is pretreated, and a bacterial Raman spectrum detection is carried out after the treatment to evaluate the pretreatment effect, wherein the kit specifically comprises the following steps:

1. preparing enterococcus faecalis blood culture positive sample

Taking the enterococcus faecalis blood culture positive report culture bottle off the culture instrument, reversing and mixing uniformly for 5 times, and drawing 2mL positive report samples for later use by using a syringe.

2. Establishing an activated carbon coagulation system

The addition volumes of the components of the operation are shown in Table 6:

TABLE 6 preparation of activated carbon coagulation system for gram-positive bacteria blood culture positive sample represented by enterococcus faecalis

The specific operation steps are as follows:

(1) taking 900 mu L of enterococcus faecalis blood culture positive sample in a 1.5mL clean centrifuge tube;

(2) adding 100 μ L of protective solution, and mixing by reversing for 3 times;

(3) add 12. mu.L of the coagulation liquid and vortex for 15 s.

3. Coagulation of activated carbon and removal of microbial samples

And (3) standing the system obtained in the step (2) at room temperature for 15min, centrifuging the system by using a palm centrifuge (1000rpm for 10s), and collecting the supernatant containing the enterococcus faecalis to be detected.

4. Raman detection sampling and sheet making

Taking 800 mu L to 1.5mL of the pretreated supernatant as an experimental group, taking 800 mu L to 1.5mL of a bacterial liquid which is purely cultured by using a enterococcus faecalis BHI culture medium (which is a culture medium component in a blood culture bottle) of the same species as a positive control group, and taking 800 mu L to 1.5mL of an enterococcus faecalis blood culture positive sample which is not pretreated as a negative control group in the same bottle of the centrifugal tube. Three groups of samples were centrifuged (12000rpm, 2min) to remove the supernatant and 500. mu.L of ddH was added₂O resuspending, continuing to centrifuge to remove supernatant, repeating washing three times, and finally adding 10. mu.L of ddH into each centrifuge tube₂Resuspend and mix well to dilute the precipitated sample.

10 μ L of the diluent was spotted onto a clean detection chip (calcium fluoride slide), three samples were spotted in parallel on each sample, and the samples were air-dried in a biosafety cabinet.

5. Raman detection

Detecting a sample by adopting a single cell Raman fast detector, wherein the detection parameters are as follows: the diameter of the pinhole is 125 mu m, the grating is 600, the lens of the objective lens is 100x, the exposure time is 3s, the laser wavelength is 532nm, the laser intensity is 100mW, and the EMCCD is 150 times.

6. Data processing and result analysis

The main component analysis is carried out according to the Raman spectrum of the bacteria obtained by the single cell Raman fast detector, and the experimental result is shown in figure 2: the main component analysis of the experimental group sample and the positive control group sample has no significant difference, and the main component analysis of the negative control group sample and the positive control group sample has significant difference. The results show that after pretreatment of gram-negative bacteria blood culture positive (activated carbon culture bottle) samples represented by enterococcus faecalis, the interference of activated carbon and other impurities is effectively removed under the condition of ensuring the normal state of bacteria. (the strain number enterococcus faecalis group is a negative control group, the blood culture _ enterococcus faecalis group is a positive control group, and the pretreatment _ blood culture _ enterococcus faecalis group is an experimental group).

Example 4

By utilizing the kit, a fungus blood culture positive sample which is cultured by using an activated carbon adsorption blood culture bottle and represented by candida albicans is pretreated, and after treatment, the bacterial Raman spectrum detection is carried out to evaluate the pretreatment effect, and the kit specifically comprises the following steps:

1. preparing a white candida blood culture positive sample

Taking the white candida blood culture positive reporting culture bottle off the culture instrument, reversing and mixing uniformly for 5 times, and extracting 3mL positive reporting samples for later use by using a syringe.

2. Establishing an activated carbon coagulation system

The addition volumes of the components of the operation are shown in Table 7:

TABLE 7 preparation of fungal blood culture positive sample activated carbon coagulation system represented by Candida albicans

The specific operation steps are as follows:

(1) taking 1.90mL of a candida albicans blood culture positive sample into a 1.5mL clean centrifuge tube;

(2) adding 100 μ L of protective solution, and mixing by reversing for 3 times;

(3) add 24. mu.L of the coagulation liquid and vortex for 15 s.

3. Coagulation of activated carbon and removal of microbial samples

And (3) standing the system obtained in the step (2) at room temperature for 15min, centrifuging the system by using a palm centrifuge (1000rpm for 10s), and collecting supernatant containing the candida albicans to be detected.

4. Raman detection sampling and sheet making

Taking 1.6mL to 2.0mL of the pretreated supernatant as an experimental group, taking 1.6mL to 2.0mL of the same bacterial liquid which is purely cultured by using a Candida albicans BHI culture medium (being a culture medium component in a blood culture bottle) as a positive control group, and taking 1.6mL to 2.0mL of the same bottle of Candida albicans blood culture positive sample which is not pretreated as a negative control group. Three groups of samples were centrifuged (12000rpm, 2min) to remove the supernatant and 800. mu.L of ddH was added₂Resuspending, centrifuging to remove supernatant, washing for three times, and centrifugingAdd 10. mu.L of ddH to each tube₂Resuspend and mix well to dilute the precipitated sample.

10 μ L of the diluent was spotted onto a clean detection chip (calcium fluoride slide), three samples were spotted in parallel on each sample, and the samples were air-dried in a biosafety cabinet.

5. Raman detection

Detecting a sample by adopting a single cell Raman fast detector, wherein the detection parameters are as follows: the diameter of the pinhole is 125 mu m, the grating is 600, the objective lens is 100x, the exposure time is 2s, the laser wavelength is 532nm, the laser intensity is 100mW, and the EMCCD is 50 times.

6. Data processing and result analysis

The main component analysis is carried out according to the Raman spectrum of the bacteria obtained by the single cell Raman fast detector, and the experimental result is shown in figure 3: the main component analysis of the experimental group sample and the positive control group sample has no significant difference, and the main component analysis of the negative control group sample and the positive control group sample has significant difference. The results show that after the gram-negative bacteria blood culture positive (activated carbon culture bottle) sample represented by candida albicans is pretreated, the interference of activated carbon and other impurities is effectively removed under the condition of ensuring the normal state of bacteria. (strain number Candida albicans group is negative control group, blood culture _ Candida albicans group is positive control group, pretreatment _ blood culture _ Candida albicans group is experimental group).

The three experimental results show that the pretreatment kit for the blood culture and positive reporting (activated carbon adsorption bottle) sample can simply, conveniently, quickly and nondestructively realize the effective separation of microorganisms (including gram-negative bacteria, gram-positive bacteria and fungi) and activated carbon, and a microorganism sample which can be used for the identification of the strain of the blood culture and positive reporting sample and the drug sensitivity detection is obtained.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.