CN112575054B - Rapid synchronous multiple detection method and kit for total number of listeria monocytogenes and bacteria - Google Patents

Abstract

The invention provides a method and a detection kit for rapid and simultaneous multiple detection of Listeria monocytogenes and the total number of bacteria in a sample. The total number of bacteria is labeled with a red fluorescent probe that can label all bacteria, distinguishing bacteria in the sample from other background particles; at the same time, using a green fluorescent probe that is cross-linked by a chemical group for Listeria monocytogenes antibodies, distinguishing monocytogenes and non-Listeria monocytogenes; the flow cytometry analyzer simultaneously counts the red and green fluorescent signals to realize the simultaneous detection of Listeria monocytogenes and the total number of bacteria. The method can simultaneously detect Listeria monocytogenes and the total number of bacteria, is simple and convenient to operate, and takes a short time. Most samples can be detected within 0.5 hours.

Description

Rapid simultaneous multiple detection method and kit for Listeria monocytogenes and total bacterial count

Technical field:

The invention belongs to the field of food microbiological detection, and in particular relates to rapid synchronous multiple detection and a kit for two indicators of Listeria monocytogenes and the total number of bacteria in food samples.

Background technique:

Listeria monocytogenes is a zoonotic pathogen that can cause listeriosis in humans and animals. After infection, the main manifestations are sepsis, meningitis and mononucleosis. The total number of bacterial colonies usually refers to the total number of bacteria, which is used to determine the degree of food contamination by bacteria and the hygienic quality. It marks the quality of food hygiene to a certain extent.

The traditional culture method is time-consuming and labor-intensive for the measurement of Listeria monocytogenes, such as the national standard GB 4789.30-2016 "National Food Safety Standard Food Microbiology Examination of Listeria monocytogenes", this method requires pre-enrichment, enrichment , cultivation, biochemical identification and a series of steps took more than 72 hours.

The plate count method of the total number of bacteria also has many shortcomings, such as the national standard GB 4789.2-2016 "Determination of the total number of colonies", this method requires a series of steps such as sampling, dilution, pouring into plates, and culturing, which takes 48 hours.

These two methods require a lot of manual operation, time-consuming and labor-intensive, and the detection of the two indicators of Listeria monocytogenes and the total number of bacteria must be carried out separately.

Although many new technologies are currently used for the rapid detection of L. monocytogenes or the total number of bacteria, these technologies are difficult to be used for multiple detection of L. monocytogenes and the total number of bacteria. For example, the existing biochip method cannot identify all types of bacteria; the PCR method can only realize multiple detection of different types of bacteria, but cannot realize the detection of the total number of bacteria.

Flow cytometry technology has the potential to realize multiple detection of L. monocytogenes and the total number of bacteria indicators. However, due to many technical difficulties in the development of L. monocytogenes-specific fluorescent probes, there has been no relevant report.

In summary, rapid detection, simultaneous multiple detection, and simultaneous detection of Listeria monocytogenes and the total number of bacteria are the actual needs and technical difficulties in the field of food microbiological testing.

Invention content:

The first object of the present invention is to provide a rapid synchronous multiple detection method for Listeria monocytogenes and the total number of bacteria, especially to meet the following requirements: 1, must be able to distinguish bacteria and impurities; 2, be able to quantitatively measure the total number of bacteria; 3 1. It can specifically identify and quantify Listeria monocytogenes from the total bacteria; 4. Get the test results quickly and synchronously.

The second object of the present invention is to provide a detection kit capable of achieving the above object.

The method has two technical key points: one is how to accurately distinguish bacteria and non-bacteria particles; the other is how to specifically identify Listeria monocytogenes.

For the problems referred to above, the inventor has carried out the following work:

1. Development of fluorescent probes that can identify total bacteria

Due to the wide variety of bacteria, Escherichia coli was selected as the representative strain of Gram-negative bacteria, Staphylococcus aureus and Listeria monocytogenes were used as representative strains of Gram-positive bacteria, and Bacillus subtilis was used as the representative strain of spore-forming bacteria . It is planned to develop and optimize fluorescent probes that can identify total bacteria through the study of four representative strains.

2. Preparation of specific antibodies against Listeria monocytogenes

The antibody of Listeria monocytogenes coupled with a green fluorescent probe is used to specifically fluorescently label the Listeria monocytogenes, and the fluorescence of the Listeria monocytogenes is analyzed using a flow cytometry analyzer.

Screening for Listeria monocytogenes antibodies for flow cytometry is difficult:

First, Listeria monocytogenes serotypes include 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4b, 4ab, 4c, 4d, 4e and "7" 13 serotypes. The serotypes of pathogenic strains are generally 1/2b, 1/2c, 3a, 3b, 3c, 4a, 1/2a and 4b. Among them, more than 95% of L. monocytogenes strains isolated from food, animal and human samples belonged to serotypes 1/2a, 1/2b, 1/2c and 4b. Therefore, applicable Listeria monocytogenes antibodies need to be able to specifically recognize all strains of the above four serotypes.

Second, common antibodies on the market are mainly used in experiments such as ELISA, IHC-Fr, and WB. In these experiments, the antigen-antibody reaction is mainly performed on a stationary phase, such as a reaction plate. In flow cytometry, the reaction of antigen and antibody is carried out in the mobile phase. Therefore, flow cytometry requires antibodies with good affinity.

The present invention has prepared multiple batches of antibodies against Listeria monocytogenes (Example 1), and finally screened out the antibodies against Listeria monocytogenes with high affinity and good specificity, which are suitable for flow analyzers , named as Ab-1 (Example 3, 4), is now preserved in the China Institute of Metrology.

The screening of the antibody goes through three steps:

1. Preliminarily judge whether the antibody can react with Listeria monocytogenes in the mobile phase, and preliminarily evaluate the specificity of the antibody (Example 2).

2. Analyze the affinity between the Listeria monocytogenes antibody and the antigen, and screen the Listeria monocytogenes antibody with high affinity (Example 3).

3. Select 12 strains of Listeria monocytogenes (including four serotypes 1/2a, 1/2b, 1/2c, and 4b) and 24 other strains, and use a flow cytometer to verify whether the antibody can specifically recognize Listeria monocytogenes, whether Listeria monocytogenes can be distinguished from other miscellaneous bacteria (embodiment 4).

The present invention confirms that the technical indicator applicable to the Listeria monocytogenes antibody of the object of the present invention is:

1. The immunogen used to prepare the antibody is a mixture of specific membrane proteins of 4 serotypes of Listeria monocytogenes strains, and the 4 serotypes are 1/2a, 1/2b, 1/2c and 4b respectively;

2. The antibody can react with Listeria monocytogenes in the mobile phase;

3. After the antibody is cross-linked with a green fluorescent probe, it can fluorescently label Listeria monocytogenes, and the fluorescence can be observed by a fluorescent microscope and a flow analyzer;

4. The affinity KD of the antibody to the specific bacterial protein of Listeria monocytogenes should be less than 1×10 ^-9 ;

5. The antibody can recognize Listeria monocytogenes strains with serotypes 1/2a, 1/2b, 1/2c and 4b.

Antibodies that meet the above requirements can be used in the method of the present invention.

Accordingly, the present invention firstly establishes a method for fast and synchronous multiple detection of Listeria monocytogenes and the total number of bacteria using flow analysis technology, which is characterized by: 1) distinguishing bacteria and other background particles in the sample: using the method that can mark all The red fluorescent probe of the bacteria marks the total number of bacteria; 2) Distinguish between Listeria monocytogenes and non-Listeria monocytogenes in the sample: use the green fluorescent probe to cross-link the Listeria monocytogenes antibody through chemical groups, Specific labeling of Listeria monocytogenes in the sample; 3) Simultaneously counting the red and green fluorescent signals generated by 1) and 2) by a flow cytometer to realize simultaneous quantitative detection of Listeria monocytogenes and the total number of bacteria.

The technical indicators of the Listeria monocytogenes antibody are as follows: a) The immunogen used to prepare the antibody is a mixture of specific membrane proteins of 4 serotypes of Listeria monocytogenes strains, and the 4 serotypes are respectively 1 /2a, 1/2b, 1/2c and 4b; b) the antibody can react with Listeria monocytogenes in the mobile phase; c) after the antibody is cross-linked with a green fluorescent probe, it can react to Listeria monocytogenes Listeria is fluorescently labeled, and the fluorescence can be observed by a fluorescence microscope and a flow analyzer; d) the affinity KD of the antibody to the specific bacterial protein of Listeria monocytogenes should be less than 1×10 ^-9 ; e) The antibodies are capable of recognizing Listeria monocytogenes strains of serotypes 1/2a, 1/2b, 1/2c and 4b.

The determination method of Listeria monocytogenes and the total number of bacteria of the method is as follows: for an event produced by a particle, if only red fluorescence is detected, it is judged as bacteria, but not Listeria monocytogenes; If there are two kinds of green fluorescence, it is determined to be Listeria monocytogenes; if no fluorescence is detected, it is determined to be non-bacterial impurity particles.

The counting by the flow analyzer is to gate through the scattered light channel of the flow analyzer, and detect the red and green fluorescent signals through the dual fluorescent channels, thereby counting Listeria monocytogenes and the total number of bacteria; wherein, The forward angle scattered light channel has a gate between 500nm and 2500nm. The gating method is as follows: use a flow analyzer to measure 500nm standard microspheres and 2500nm standard microspheres, and perform gating according to the signal position of the microspheres on the histogram of the forward angle scattering light channel, with the lower limit at 500nm , the upper limit is at 2500nm.

The fluorescence emission spectrum of the red fluorescent probe is 601nm-640nm; the fluorescence emission spectrum of the green fluorescent probe is 501nm-540nm.

The technical indicators and detection parameters of the flow analyzer are: fluorescence sensitivity<10MESF, scattered light sensitivity<50nm, fluorescence resolution RSD<3%, scattered light resolution<3%; analysis speed is 1-30μL/min, The detection time is 15-300s.

In the detection method, the sample to be tested needs to be purified before being tested; the purification method is to add the sample to be tested into the suspension in water, then filter and collect the filtrate, centrifuge the filtrate and discard the upper liquid, and keep the sediment at the bottom , and then add PBS to resuspend to obtain a purified sample bacterial suspension.

The present invention clearly discloses the method of the present invention with a large number of experiments, see embodiment for details. in:

Example 1 is the preparation of Listeria monocytogenes antibody.

Example 2 is the preliminary screening of Listeria monocytogenes antibodies.

Embodiment 3 is the affinity analysis of antigen antibody

Example 4 is the specificity evaluation of antibodies.

Examples 5-8 are evaluations of the detection method established in this patent.

Example 9 is the components of the rapid detection kit for Listeria monocytogenes and the total number of bacteria.

Below is the specific operation of an actual detection of the present invention:

1. Purification of samples to be tested:

Add 25mL or 25g of the sample to be tested into 225mL of deionized water, filter the sample to be tested with a filter membrane with a pore size of 2.5 μm to 15 μm, and retain the filtrate. The filtrate was centrifuged at 5000×g for 5 min. Discard the upper liquid and keep the bottom pellet. Add 2.5 mL of PBS to resuspend the pellet and transfer to a new centrifuge tube to obtain a purified sample bacterial suspension.

2. Add the green fluorescent probe cross-linked Listeria monocytogenes antibody (Gr-Ab) with a final concentration of 1 μg/mL and the red fluorescent probe (Rd) with a concentration of 1 μg/mL to the purified bacterial suspension, mix well Afterwards, incubate in the dark for 5-15 minutes.

3. Flow cytometer detection: the excitation light is set to 450-500nm, the analysis speed is 1-30μL/min, the detection time is 15-300s, and the gate of the forward angle scattering light channel is between 500nm and 2500nm. The concentration of L. monocytogenes and the total number of bacteria were calculated by analyzing the events within the circle gate in the dual fluorescent channel.

The inventor has verified the effect of this detection method:

A mixed bacterial solution of Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, and Bacillus subtilis was prepared, and a 10-fold serial dilution of the mixed bacterial solution was obtained. Use the Listeria monocytogenes plate count method and the total number of bacteria pour plate method to detect the 10-fold serial dilution of the mixed bacterial solution, and use this method to detect the total number of Listeria monocytogenes and the total number of bacteria in the mixed bacterial solution. Comparing the detection results of the plate counting method and the flow cytometry method: for the detection of Listeria monocytogenes, this method has a good linear relationship with the results obtained by the plate counting method, indicating that this method has good accuracy, and the lower limit of detection of the method is 76CFU/mL; For the detection of the total number of bacteria, this method has a good linear relationship with the results obtained by the plate count method, indicating that this method has good accuracy, and the lower limit of detection of the method is 225CFU/mL.

According to the detection method established by the above-mentioned present invention, the present invention also provides a test kit for rapid and simultaneous multiple detection of Listeria monocytogenes and the total number of bacteria in a sample, which is characterized in that: 1. The red fluorescence of all bacteria in the sample can be marked Probe; 2. Using a green fluorescent probe to cross-link specific Listeria monocytogenes antibodies through chemical groups, it can distinguish Listeria monocytogenes and non-Listeria monocytogenes in the sample; 3. Calibration microspheres ( 500nm and 2500nm).

The technical indicators of the Listeria monocytogenes antibody are as follows: a) The immunogen used to prepare the antibody is a mixture of specific membrane proteins of 4 serotypes of Listeria monocytogenes strains, and the 4 serotypes are respectively 1 /2a, 1/2b, 1/2c and 4b; b) the antibody can react with Listeria monocytogenes in the mobile phase; c) after the antibody is cross-linked with a green fluorescent probe, it can react to Listeria monocytogenes Listeria is fluorescently labeled, and the fluorescence can be observed by a fluorescence microscope and a flow analyzer; d) the affinity KD of the antibody to the specific bacterial protein of Listeria monocytogenes should be less than 1×10 ^-9 ; e) The antibodies are capable of recognizing Listeria monocytogenes strains of serotypes 1/2a, 1/2b, 1/2c and 4b.

The fluorescence emission spectrum of the red fluorescent probe is 601nm-640nm; the fluorescence emission spectrum of the green fluorescent probe is 501nm-540nm.

The kit also contains a filter membrane with a pore size of 2.5 μm to 15 μm.

In an example of the present invention, the kit includes the following components:

1. A red fluorescent probe that can label all the bacteria in the sample, and its fluorescence emission spectrum is between 601nm and 640nm;

2. Use the green fluorescent probe to crosslink the specific Listeria monocytogenes antibody through chemical groups, which can distinguish Listeria monocytogenes and non-Listeria monocytogenes in the sample; wherein, the fluorescence emission of the green fluorescent probe The spectrum is between 501nm and 540nm;

3. Calibration microspheres (500nm and 2500nm).

4. Filter membrane (2.7μm～15μm pore size).

The operating method of carrying out Listeria monocytogenes detection with kit of the present invention is characterized in that:

1. Purification of samples to be tested:

Add 25mL or 25g of the sample to be tested into 225mL of deionized water, filter the sample to be tested with a filter membrane with a pore size of 2.5 μm to 15 μm, and keep the filtrate. The filtrate was centrifuged at 5000×g for 5 min. Discard the upper liquid and keep the bottom pellet. Add 2.5 mL of PBS to resuspend the pellet and transfer to a new centrifuge tube to obtain a purified sample bacterial suspension.

2. Add Gr-Ab with a final concentration of 1 μg/mL and Rd with a final concentration of 1 μg/mL to the purified bacterial suspension, mix well and incubate in the dark for 5-15 minutes.

3. Flow cytometer detection: the excitation light is set to 450-500nm, the analysis speed is 1-30μL/min, the detection time is 15-300s, and the gate of the forward angle scattering light channel is between 500nm and 2500nm. The concentration of L. monocytogenes and the total number of bacteria were calculated by analyzing the events within the circle gate in the dual fluorescent channel.

The present invention has the following innovations and advantages:

1. Simultaneous detection of Listeria monocytogenes and the total number of bacteria

By using a green fluorescent probe cross-linked Listeria monocytogenes antibody and a red fluorescent probe for dual fluorescent labeling, the simultaneous accurate and quantitative detection of Listeria monocytogenes and the total number of bacteria can be performed.

A red fluorescent probe stains all bacteria and specifically labels Listeria monocytogenes with an immunofluorescence antibody with green fluorescence. If a bacterium is marked both green and red, it is L. monocytogenes; if a bacterium is marked only red, it is a bacterium, but not L. monocytogenes.

2. The operation is simple and convenient, and the time-consuming is short. Most samples can be detected within 0.5h.

3. The flow analyzer calibration microspheres are attached to the kit, which can ensure the accuracy and accuracy of the test results to the greatest extent.

Description of drawings

Fig. 1 is the electrophoresis result of Listeria monocytogenes antibody in embodiment 1;

Fig. 2 is the FITC-labeled Listeria monocytogenes observed under a fluorescence microscope in Example 2;

Fig. 3 is the result of the Listeria monocytogenes detection double fluorescent label in embodiment 6;

Fig. 4 is the result of the Listeria monocytogenes and miscellaneous bacteria detected by the flow analyzer in embodiment 6 in different concentration ratios;

Fig. 5 is the linear relationship between the method and the plate count method for measuring Listeria monocytogenes results in Example 7.

Fig. 6 is the linear relationship between this method and the plate count method measuring the total number of bacteria results in Example 8.

detailed description

Below in conjunction with specific example, further set forth the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Unless defined otherwise, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The experimental reagent consumables used in the following examples are conventional biochemical reagents unless otherwise specified. For the experimental methods without specific conditions indicated in the following examples, generally follow the conditions in conventional conditions, or follow the conditions suggested by the manufacturer. The bacterial strains involved in the examples belong to the prior art, and those skilled in the art can easily obtain them from open commercial channels.

Approximate language used in the following examples applies to quantitative tenders, indicating that some variation in quantities is permissible without altering essential functionality. Accordingly, values modified by language such as "about", "approximately" and the like are not limited to the exact value itself. In some cases, the language of approximation may relate to the precision of the measuring instrument.

Example 1 Preparation of Listeria monocytogenes antibody

1. Materials and methods

1. A total of 2 experimental rabbits, with an initial weight of 2.2-2.5kg. Immunization method: multiple intradermal injections on the back. Immunogen: Listeria monocytogenes specific membrane protein. Process: A total of three immunizations were carried out with an interval of half a month. Then carry out the potency test. Then the fourth immunization was carried out, and the serum was collected after the titer test was suitable, and the antibody was purified. Two Listeria monocytogenes antibodies were obtained.

2. Use the ELISA endpoint method to detect the titer of the obtained 2 Listeria monocytogenes antibodies. Antibody concentration was detected by SDS-PAGE method.

2. Experimental results

Two Listeria monocytogenes were preliminarily prepared, antibody 1 titer 1:1×10 ⁶ , antibody 2 titer 1:2×10 ⁶ . The electrophoresis results are shown in Figure 1.

Table 1 Antibody Titer Detection

3. Experimental conclusion

Antibody 2 is the antibody prepared from the Listeria monocytogenes antibody, numbered Ab-1.

According to the same procedure, a total of 6 antibodies (Ab-1 to Ab-6) were prepared.

Example 2 Preliminary screening of Listeria monocytogenes antibody

1. Materials and methods

1. Use the FITC labeling kit to label the prepared 6 Listeria monocytogenes antibodies (Ab-1 to Ab-6) with fluorescein FITC.

2. Four standard strains of Listeria monocytogenes, numbered respectively: ATCC15313 (1/2a serotype), ATCC BAA-751 (1/2b serotype)), ATCC19112 (1/2c serotype), ATCC19115 (4b serotype) type). The bacterial solutions of the above-mentioned Listeria monocytogenes at a concentration of approximately 1×10 ⁷ cells/mL were prepared respectively.

3. Add FITC-labeled antibodies (Ab-1 to Ab-6) at a concentration of 1 μg/mL to the above-mentioned four strains of Listeria monocytogenes, and incubate for 15 minutes in the dark. They were detected by fluorescence microscope and flow cytometer respectively.

2. Experimental results

When labeled with Ab-1, Ab-3, and Ab-4, green fluorescence was detected in all four strains of Listeria monocytogenes (Fig. 2). When labeled with Ab-2, no green fluorescence was detected in the 4 strains of Listeria monocytogenes, indicating that the antibody Ab-2 could not react with the antigen in the mobile phase. When labeled with Ab-5 and Ab-6, only part of Listeria monocytogenes detected green fluorescence, indicating that antibodies Ab-5 and Ab-6 could not specifically recognize the four serotypes of Listeria monocytogenes strains.

3. Experimental conclusion

Successfully screened three Listeria monocytogenes antibodies (Ab-1, Ab-3, Ab-4), which can react with Listeria monocytogenes in the mobile phase, and can identify 4 serotypes of monocytogenes after preliminary verification Listeria monocytogenes strains.

Table 2 Evaluation of Listeria monocytogenes antibodies

Example 3 Affinity analysis of Listeria monocytogenes antibody and Listeria monocytogenes membrane protein

1. Materials and methods

1. Four standard strains of Listeria monocytogenes, numbered respectively: ATCC15313 (1/2a serotype), ATCCBAA-751 (1/2b serotype)), ATCC19112 (1/2c serotype), ATCC19115 (4b serotype) type).

2. The 3 Listeria monocytogenes antibodies (Ab-1, Ab-3, Ab-4) obtained by preliminary screening and the 4 Listeria monocytogenes antibodies purchased on the market (numbering Ab-7 to Ab-10 ) were diluted to the same initial concentration, and a 2-fold serial dilution was performed respectively.

3. Prepare the bacterial solution of the above-mentioned Listeria monocytogenes at a concentration of about 1×10 ⁷ cells/mL, lyse the cell membrane after heating to death, and then use the antibody of Listeria monocytogenes to carry out fishing reaction to capture Listeria monocytogenes Bacteria-specific membrane proteins.

4. Linking L. monocytogenes-specific membrane proteins to the carboxy-chip using the EDC/NHS reaction. The carboxyl chip was loaded into an analytical interaction analyzer.

5. For a Listeria monocytogenes antibody, put 2 serially diluted antibodies on the molecular interaction instrument, collect data, and use TraceDrawer to fit a series of reaction curves, and calculate the antibody and membrane protein Affinity KD.

2. Experimental results

The affinities of 7 Listeria monocytogenes antibodies and membrane proteins were analyzed using an analytical interaction analyzer, and the results are shown in Table 1. The results showed that the affinity between antibody Ab-1 and Listeria monocytogenes membrane protein was the best, which was 2.15×10 ^-10 .

3. Experimental conclusion

The present invention screens out the Listeria monocytogenes antibody Ab-1 with the best affinity.

Table 3 Affinities of different Listeria monocytogenes antibodies and antigens

Embodiment 4 Listeria monocytogenes antibody Ab-1 specificity evaluation

1. Materials and methods

1. 12 strains of Listeria monocytogenes (Table 4), and 24 strains of Listeria monocytogenes (Table 5). Rejuvenate and amplify all the above strains, and dilute to an appropriate concentration (approximately 10 ⁶ CFU/mL).

2. Add the green fluorescent-labeled Listeria monocytogenes antibody Ab-1 at a concentration of 1 μg/mL to the above bacterial suspension, and incubate in the dark for 5-15 minutes. Then it was detected by a flow analyzer.

2. Experimental results

The Listeria monocytogenes antibody Ab-1 was reacted with the bacterial suspensions of 36 different strains, and then the specificity was detected by a flow cytometer. The results are shown in Table 4 and Table 5: 12 Listeria monocytogenes The green fluorescence was detected in all bacteria, but the green fluorescence was not detected in the 24 non-Listeria monocytogenes.

Table 4 The method specificity experimental results (12 Listeria monocytogenes strains)

Table 5 The method-specific experimental results (24 non-monocyte Listeria strains)

3. Experimental conclusion

The Listeria monocytogenes antibody Ab-1 can be used in flow cytometry analysis technology, has good specificity, and can accurately identify and distinguish Listeria monocytogenes.

Embodiment 5 method measures the universality of total number of bacteria

1. Materials and methods

1. Standard bacterial strains of different genera, a total of 16 strains (Table 6).

2. Membrane-permeable nucleic acid fluorescent material SYTO 62.

3. Prepare the bacterial suspensions of the above-mentioned 16 bacterial strains respectively, and adjust their McFarland turbidity values to 0.5 McF. The bacterial fluids were stained with SYTO 62 (final concentration: 1 μg/mL), and the reaction time was 15 min.

5. Use a flow cytometer to detect the above-mentioned stained sample.

2. Experimental results

The results showed that red fluorescence was detected by the flow cytometer in 16 fluorescently labeled bacterial liquids. For the 16 non-fluorescence-labeled bacterial solutions, no red fluorescence was detected by the flow cytometer. It indicated that SYTO 62 could stain the 16 strains in the experiment.

Table 6 Fluorescence labeling of different bacterial strains

3. Experimental conclusion

This method can identify all kinds of bacteria and is universal.

Example 6 Detection of different concentration ratios of Listeria monocytogenes and miscellaneous bacteria samples

1. Materials and methods

1. Prepare a Listeria monocytogenes solution with a concentration of about 1×10 ⁷ cells/mL and a miscellaneous bacteria solution (including Escherichia coli, Staphylococcus aureus, and Bacillus subtilis) at a concentration of about 1×10 ⁷ cells/mL.

2. Mix the Listeria monocytogenes suspension and the miscellaneous bacteria suspension in different volume ratios (0/10, 1/9, 5/5, 9/1, 10/0). Samples were then assayed by flow cytometry.

2. Experimental results

Listeria monocytogenes without fluorescent labeling is shown in Figure 3A without any fluorescence; Listeria monocytogenes labeled with green fluorescent antibody is shown in Figure 3B and emits green fluorescence; labeled with green fluorescent antibody and nucleic acid fluorescein The L. monocytogenes, as shown in Figure 3C, fluoresce both green and red.

Listeria monocytogenes and miscellaneous bacteria in different concentration ratios were mixed and detected by flow cytometry, and the results are shown in Figure 4. The results showed that the proportion of Listeria monocytogenes and miscellaneous bacteria detected by flow cytometry was close to the actual value.

3. Experimental conclusion

The detection method of the invention can accurately and quantitatively detect the Listeria monocytogenes and the total number of bacteria in the liquid.

Embodiment 7 The comparison of the method of the present invention and the plate count method detecting Listeria monocytogenes

1. Materials and methods

1. Centrifuge the cultured Listeria monocytogenes suspension at 12,000×g for 5 minutes, discard the supernatant, and resuspend the bacteria sludge in PBS.

2. Carry out 10-fold serial dilutions to the Listeria monocytogenes bacterial suspension with sterilized pure water, and adopt detection method of the present invention and plate count method to carry out quantitative detection to sample, analyze the linearity and the sensitivity of this method.

2. Experimental results

When the concentration of Listeria monocytogenes was between 10 ² and 10 ⁸ CFU/mL, the detection results of the plate counting method and the flow cytometry method were close, and the linearity between them was good (R ² =0.9994) (Figure 5).

3. Experimental conclusion

The detection method of the invention can accurately and sensitively detect the Listeria monocytogenes quantitatively, and has good linearity with the plate counting method.

Embodiment 8 The comparison of the method of the present invention and the total number of bacteria detected by plate counting method

1. Materials and methods

1. Escherichia coli standard strain, No. CMCC 44102; Staphylococcus aureus standard strain, No. ATCC6538P; Bacillus subtilis standard strain, No. ATCC 6633.

2. Serially dilute the cultured three representative bacterial strains by 10 times respectively. Quantitative detection was carried out by flow cytometry and the plate count method in the national standard GB4789.2 "Determination of the total number of colonies in food microbiological examination of food safety national standard". Each concentration was repeated three times by flow cytometry and plate count method.

2. Experimental results

Figure 6 shows the linear relationship between the flow cytometry results and the plate count results of the three bacterial strains. The results showed that when the concentration of bacteria was 10 ³ -10 ⁸ CFU/mL, the results of flow cytometry were basically consistent with the plate counting method, and the linearity was good.

3. Experimental conclusion

The flow cytometric analysis technique in this method has good accuracy in detecting the total number of bacteria, and the detection limit is 225 CFU/mL.

Example 9 Listeria monocytogenes and the total number of bacteria rapid detection kit

The kit contains:

A red fluorescent probe that can label all bacteria in a sample;

Antibody against Listeria monocytogenes cross-linked with green fluorescent probe;

Filter membrane (2.5μm～15μm pore size);

Calibration microspheres (500nm and 2500nm).

Claims (9)

1. A method for rapid synchronous multiple detection of Listeria monocytogenes and total bacterial counts in a non-diagnostic purpose, characterized as follows: 1) Distinguish between bacteria and other background particles in the sample: use a red fluorescent probe that can label all bacteria to mark the total number of bacteria; 2) Differentiate between Listeria monocytogenes and non-Listeria monocytogenes in the sample: use the green fluorescent probe to cross-link the Listeria monocytogenes antibody through chemical groups to specifically label the Listeria monocytogenes in the sample; 3) Simultaneously count the red and green fluorescent signals generated by 1) and 2) through the flow analyzer to realize the simultaneous quantitative detection of Listeria monocytogenes and the total number of bacteria; In the above 2), the immunogen for preparing the antibody is a mixture of specific membrane proteins of 4 serotypes of Listeria monocytogenes strains, and the 4 serotypes are 1/2a, 1/2b, 1/2 2c and 4b; and, the prepared antibodies are screened to select antibodies that meet the following a-d technical indicators, which are antibodies that can simultaneously detect Listeria monocytogenes and the total number of bacteria: a said antibody can react with Listeria monocytogenes in mobile phase; b After the antibody is cross-linked with a green fluorescent probe, it can fluorescently label Listeria monocytogenes, and the fluorescence can be observed by a fluorescent microscope and a flow analyzer; c The affinity KD of the antibody to the specific bacterial protein of Listeria monocytogenes should be less than 1 × 10-9; d said antibody is capable of recognizing Listeria monocytogenes strains with serotypes 1/2a, 1/2b, 1/2c and 4b; In the above 3), the determination method of Listeria monocytogenes and the total number of bacteria is as follows: For an event produced by a particle, if only red fluorescence is detected, it is judged as bacteria, but not Listeria monocytogenes; if red fluorescence is detected at the same time and green fluorescence, it is determined to be Listeria monocytogenes; if no fluorescence is detected, it is determined to be non-bacterial impurity particles. 2. The method according to claim 1, said counting by flow analyzer is to carry out gate by the scattered light channel of flow analyzer, and claim 1 detects said red and green fluorescent signals by double fluorescence channels, Thereby counting Listeria monocytogenes and the total number of bacteria; wherein, the gate of the forward angle scattered light channel is between 500 nm and 2500 nm. 3. the method described in claim 2, described gate, its method is: use flow analyzer to measure 500 nm standard microsphere and 2500 nm standard microsphere, on the histogram of forward angle scattered light channel, according to The signal position of the microspheres was gated with a lower limit at 500 nm and an upper limit at 2500 nm. 4. The method of claim 1, wherein the fluorescence emission spectrum of the red fluorescent probe is between 601 nm and 640 nm; the fluorescence emission spectrum of the green fluorescent probe is between 501 nm and 540 nm. 5. The method according to claim 1, the technical index and detection parameters of the flow analyzer are: fluorescence sensitivity＜10MESF, scattered light sensitivity＜50 nm, fluorescence resolution RSD＜3%, scattered light resolution＜3 %; the analysis speed is 1-30 μL/min, and the detection time is 15-300 s. 6. The method according to any one of claims 1 to 5, the sample to be tested needs to be purified before being detected; the purification method is to add the sample to be tested to the suspension in water, then filter and collect the filtrate, centrifuge the filtrate and discard Remove the upper liquid, keep the bottom precipitate, and then add PBS to resuspend to obtain the purified sample bacterial suspension. 7. A Listeria monocytogenes antibody for rapid simultaneous multiple detection of Listeria monocytogenes and the total number of bacteria in a sample, the technical indicators are as follows: a The immunogen used to prepare the antibody is a mixture of specific membrane proteins of 4 serotypes of Listeria monocytogenes strains, the 4 serotypes being 1/2a, 1/2b, 1/2c and 4b respectively; b said antibody can react with Listeria monocytogenes in mobile phase; c. After the antibody is cross-linked with a green fluorescent probe, it can fluorescently label Listeria monocytogenes, and the fluorescence can be observed by a fluorescent microscope and a flow analyzer; d The affinity KD of the antibody to the specific bacterial protein of Listeria monocytogenes should be less than 1 × 10-9; e The antibody is capable of recognizing Listeria monocytogenes strains of serotypes 1/2a, 1/2b, 1/2c and 4b. 8. A test kit for rapid synchronous multiple detection of Listeria monocytogenes and the total number of bacteria in a sample, characterized in that it has: A red fluorescent probe that can label all bacteria in a sample; Specific Listeria monocytogenes antibody cross-linked by a green fluorescent probe through a chemical group; 500 nm and 2500 nm calibration beads; The Listeria monocytogenes antibody is as described in claim 7. 9. The kit according to claim 8, further comprising a filter membrane with a pore size of 2.5 μm to 15 μm; and the fluorescence emission spectrum of the red fluorescent probe is at 601 nm to 640 nm; the fluorescence emission spectrum of the green fluorescent probe is The spectrum is between 501nm and 540nm.

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