WO2021120805A1 - Preparation of serum diagnostic kit of listeria monocytogenes serotype 4h and use thereof - Google Patents

Abstract

Provided are Listeria monocytogenes XYSN and the use thereof, wherein the strain is deposited with the deposit number CCTCC NO: M 2019765 and is isolated and preserved from the brains of sheep that have been infected with listeriosis. The Listeria monocytogenes XYSN can be used to prepare a polyclonal antibody for identifying or immunofluorescently labeling the Listeria monocytogenes serotype 4h.

Description

Preparation and application of a 4h serological diagnostic kit for Listeria monocytogenes serotype Technical field

The present invention relates to the field of biotechnology, in particular to a Listeria monocytogenes XYSN and its use.

Background technique

Listeria monocytogenes (Listeria monocytogenes, Lm) is a gram-positive facultative intracellular parasitic bacteria that is extremely tolerant to the environment and can infect mammals, birds, fish, and crustaceans Among them, ruminants are susceptible hosts, and the mortality rate after infection is 20%-100%. Animal-derived Lm is transmitted to humans through contaminated farms and through food processing. It is an important cause of human listeriosis. It can cross the intestinal barrier, placental barrier, and blood-brain barrier to cause meningitis, sepsis, abortion, etc. Suffering from listeriosis is mainly the elderly, newborns, pregnant women and people with low immunity, and the mortality rate is as high as 20%-30%, which has important public health significance.

So far, the food-borne pathogen Listeria monocytogenes consists of 14 serotypes (1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e). , 4h, 7), of which serotype 4h is a newly discovered serotype in my country. Studies have shown that this serotype of Listeria monocytogenes has caused an outbreak of animal listeriosis and has super virulence. , Posed a great threat to public health security. Therefore, the establishment of a fast and effective method for the detection and identification of serotype 4h Listeria monocytogenes is very important for the monitoring, prevention and control of Listeria. At present, the existing serological diagnostic kits cannot effectively identify Listeria monocytogenes of serotype 4h, so it is particularly necessary to prepare a diagnostic kit for Listeria monocytogenes of serotype 4h.

Summary of the invention

In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a Listeria monocytogenes XYSN and its use to solve the lack of effective identification of the serotype 4h Listeria monocytogenes in the prior art. Special bacteria means.

In order to achieve the above objectives and other related objectives, the present invention provides a Listeria monocytogenes XYSN, and the collection number of the Listeria monocytogenes XYSN is: CCTCC NO: M 2019765.

The microorganism XYSN used in the present invention was isolated and preserved by the inventor from the brains of sheep with outbreaks of listeriosis.

The Listeria monocytogenes XYSN strain of the present invention was identified by morphological characteristics, and the identification results were as follows:

Morphological characteristics: This genus of bacteria is a gram-positive bacillus. Observed under the microscope, it is short rod-shaped, with blunt or spherical ends at both ends, and the size is (0.4～0.5)μm×(0.5～2.0)μm. Aerobic or facultative anaerobic, no spores, no capsules, exercise at 20-25℃, no exercise at 37℃, catalase positive. The (G+C) mol% of DNA is 36 to 37.95.

The main physiological and biochemical characteristics of the XYSN strain: the bacteria can decompose glucose, maltose, esculin, lactose, mannose, trehalose, but cannot decompose rhamnose, mannitol, xylose, MR-VP test is positive, phosphatidyl phospholipase The C test was positive, the phosphatase test was negative, and the urease test was negative.

After morphological identification and 16S rRNA amplification sequence development tree analysis, it was determined that the strain was Listeria monocytogenes. The strain was named according to the international naming rules: genus name + species name + plant name, genus name, species name , The plant names are Listeria, monocytogenes and XYSN, respectively, named Listeria monocytogenes XYSN, and the deposit number is: CCTCC NO: M 2019765. The nucleotide sequence of Listeria monocytogenes XYSN isolated in the present invention is shown in SEQ ID NO.1.

The Listeria monocytogenes XYSN of the present invention has been registered and deposited in the China Type Culture Collection (CCTCC) on September 27, 2019, and the deposit number is CCTCC NO: M 2019765.

Another aspect of the present invention provides the use of the above-mentioned Listeria monocytogenes XYSN for preparing polyclonal antibodies.

Another aspect of the present invention provides a polyclonal antibody prepared by using Listeria monocytogenes XYSN as an immunogen.

Further, the object immunized with the immunogen is a rabbit. For example, the New Zealand White Rabbit.

Another aspect of the present invention provides the use of the above-mentioned polyclonal antibody for identifying or immunofluorescence labeling of Listeria monocytogenes of serotype 4h.

Another aspect of the present invention provides a method for identifying Listeria monocytogenes of serotype 4h, which includes the steps of: picking freshly cultured Listeria monocytogenes and mixing the above-mentioned polyclonal antibody, and observing agglutination Reaction, if agglutination occurs, it means that the serotype of Listeria monocytogenes is 4h.

Another aspect of the present invention provides a method for immunofluorescence labeling of Listeria monocytogenes of serotype 4h, which comprises the steps of: using the polyclonal antibody to label Listeria monocytogenes of serotype 4h.

The above-mentioned polyclonal antibody can be used as a primary antibody to bind to Listeria monocytogenes of serotype 4h, and then use a fluorescent secondary antibody to bind to the above-mentioned polyclonal antibody to label the serotype 4h of Listeria monocytogenes bacteria.

Another aspect of the present invention provides a method for preparing the above-mentioned polyclonal antibody, which is obtained by immunizing animals with Listeria monocytogenes XYSN.

Specifically, the method includes the following steps:

(1) Preparation of immunogen using Listeria monocytogenes XYSN;

(2) Immunize animals;

(3) Collect blood to obtain serum containing polyclonal antibodies.

Further, the method for preparing the immunogen in the step (1) may be to collect Listeria monocytogenes XYSN cultured in a 37°C incubator for 24 hours, wash twice with PBS, adjust the appropriate bacterial concentration, and use a boiling water bath. Boil for 1 hour to destroy the H antigen of the bacteria, add phenol to a final concentration of 0.5%, and finally ultrasonically break.

Further, the animal in the step (2) is selected from rabbits, rats, guinea pigs, chickens, pigs, sheep and the like.

Further, the method of immunization in step (2) is to use antigens to contact animals for multiple times. For example, the following operation can be used: ear vein immunization of adult New Zealand white rabbits, the first immunization dose is 0.5mL (0.5×10 ⁹ CFU), interval Immunization for 4 days, a total of 5 times, each time an increase of 0.5mL immunogen.

Further, the step (3) before blood sampling also includes the determination of the antibody titer of the animal serum to ensure that the polyclonal antibody in the serum reaches an effective dose, for example, an ELISA method can be used.

Further, the step (3) includes post-treatment after blood collection, and the post-treatment method is standing and centrifugation, and the supernatant is serum containing polyclonal antibodies.

Another aspect of the present invention provides a kit containing the above-mentioned polyclonal antibody.

Another aspect of the present invention provides the use of the above kit for identification or immunofluorescence labeling of Listeria monocytogenes of serotype 4h.

As mentioned above, the Listeria monocytogenes XYSN of the present invention and its application have the following beneficial effects:

Using the polyclonal antibody in this application to detect Listeria monocytogenes of serotype 4h has high sensitivity and good specificity, and can effectively identify and fluorescently label Listeria monocytogenes of serotype 4h.

The preservation information of the strain of the present invention is as follows:

Strain name: Listeria monocytogenes XYSN;

The deposit number is: CCTCC NO: M 2019765;

Date of preservation: September 27, 2019;

Name of the depository: China Center for Type Culture Collection;

Abbreviation of depository institution: CCTCC;

Depository address: School of Life Sciences, Wuhan University, Luojiashan Street, Wuchang, Wuhan.

Description of the drawings

Figure 1 Test results of antibody titer of rabbit polyclonal antibodies:

The serum dilution ratios were 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, 1:102400, 1:204800, 1:409600.

Figure 2 Glass plate agglutination identification results:

1-3. Listeria monocytogenes serotype 4h strain; 4-15. Serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, Listeria monocytogenes of 4d and 7; 16. Listeria escherichia; 17. Listeria innocuous; 18. Listeria serratia; 19. Listeria spp; 20. Listeria weseri Bacteria; 21. Listeria monocytogenes XYSN of serotype 4h (PBS).

Figure 3 Fluorescent labeling results of 4h rabbit polyclonal antibody serum:

1-3. Listeria monocytogenes serotype 4h strain; 4-15. Serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, Listeria monocytogenes of 4d and 7; 16. Listeria escherichia; 17. Listeria innocuous; 18. Listeria serratia; 19. Listeria spp; 20. Listeria weseri bacteria.

Detailed ways

The following describes the implementation of the present invention through specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the examples of the present invention are used to describe specific specific embodiments. It is not intended to limit the scope of protection of the present invention; in the specification and claims of the present invention, unless the context clearly indicates otherwise, the singular forms "a", "one" and "this" include plural forms.

When numerical ranges are given in the examples, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, those skilled in the art can also use the methods, equipment, and materials described in the embodiments of the present invention based on their grasp of the prior art and the description of the present invention. Any methods, equipment and materials that are similar or equivalent to the prior art are used to implement the present invention.

Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in the technical field. Conventional technology. These technologies have been fully explained in the existing literature. For details, see Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel, etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987 and periodic updates; the series Methods IN ENZYMOLOGY, Academic Press, San Diego; Wolfe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, Vol. PMWassarman and APWolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (PBBecker, ed.) Humana Press, Totowa, 1999, etc.

Materials and their sources:

BHI medium (BactoTM Brain Heart Infusion) was purchased from BD; goat anti-rabbit IgG was purchased from Bioworld; goat anti-rabbit (Alexa 488) was purchased from Abcam; DAPI was purchased from Biyuntian; TMB was purchased from Solarbio.

Example 1: Isolation and extraction of Listeria monocytogenes XYSN and detection of 16S rRNA

Listeria monocytogenes XYSN was isolated and preserved by the inventors from the brains of sheep with listeriosis outbreaks. The 16S rDNA of XYSN is the gene sequence corresponding to the 16S rRNA on the bacterial chromosome, and its sequence is as SEQ ID NO. 1:

Example 2: Preparation and immunization of immunogen

Pick the serotype of Listeria monocytogenes XYSN for 4 hours and place it on the BHI solid plate with 3-zone lines, and place it in a 37°C incubator for 24 hours. Collect the bacteria with PBS, wash 2 times at 8000rpm, 2min, adjust the bacterial concentration (OD ₆₀₀ ＝0.8), boil in a boiling water bath for 1 hour, destroy the H antigen of the bacteria, add phenol to a final concentration of 0.5%, and finally ultrasonically disrupt (amplitude 50 , Time 10min). Adult New Zealand white rabbits were immunized through ear vein. The first immunization dose was 0.5 mL (0.5×10 ⁹ CFU), and immunization was performed every 4 days for a total of five times, each time 0.5 mL of immunogen was added, and PBS was used as a negative control.

Example 3: Detection of serum antibody titer

1. Treat the slats: add 100μL of 5% glutaraldehyde solution (25% glutaraldehyde 5mL+0.1M NaHCO ₃ 95mL) to each well of the ELISA slats, and act in a humidified chamber at 37°C for 2 hours;

2. After taking it out, wash with SW 3 times, 5 minutes each time, and pat dry the ELISA plate;

3. Whole bacteria coating: adjust the XYSN concentration to 1×10 ⁸ CFU/mL, 50μL/well coating, and place in a 56°C oven to dry for 20-24 hours;

4. The next day, add 100μL of pre-cooled anhydrous methanol to each well, let it work at room temperature for about 15 minutes, and wash with SW 3 times, 5 minutes each time;

5. Sealing: add 5% skimmed milk solution to each hole for sealing, 200μL/well, water bath at 37°C for 2 hours, after taking it out, wash with PBST 3 times, each time for 5 minutes;

6. Dilute the XYSN poly-antiserum sample 200 times with PBS, mix well and add 200μL to the first hole of the ELISA plate, mix well, then aspirate 100μL of liquid and carefully move it to the next well (100μL of PBS has been added), Dilute continuously in this way until the last well is evenly mixed and then draw 100μL and discard it. At the same time, set up a negative control (not immunized) and a blank control (with PBS instead of serum samples), and bathe in water at 37°C for 2 hours;

7. Wash with PBST 5 times, 5min each time. Add HRP-goat anti-rabbit IgG enzyme-labeled secondary antibody diluted 1:8000 with PBST containing 2% BSA, 100 μL/well, and water bath at 37°C for 1 hour;

8. Wash 7 times with PBST, 5min each time, try to pat dry the remaining liquid;

9. Add TMB color developing solution, 100μL/well, 37℃ dark water bath for 10-15min;

10. Add 2M H ₂ SO _{4 to} stop the reaction, 50 μL/well, and detect the OD ₄₅₀ value with a microplate reader.

The results of serum antibody titer determination are shown in Figure 1. When the serum is diluted to 1:409600, its P/N=11.10, which is greater than 2.1, indicating that the rabbit polyclonal antibody titer at this time is greater than 1:409600, which meets the expected requirements .

Example 4: Mass preparation of 4h rabbit polyantiserum

Blood was collected from the heart, left standing overnight at 4°C, and centrifuged at 6000 rpm at 4°C for 10 minutes the next day. The supernatant was taken to be the rabbit polyantiserum for 4 hours, aliquoted, and stored at -70°C.

Example 5: Glass plate agglutination and identification of Listeria monocytogenes

Table 1 involves a total of 20 strains of bacteria, including 3 strains of Listeria monocytogenes serotype 4h, 12 strains of Listeria monocytogenes of other serotypes, Listeria escherichia, Listeria innocuous, and Strains There are 1 strains of Listeria spp., Listeria spp. and Listeria spp.

Table 1 Test strains

Note: +agglutination; -not agglutination

Dilute the 4h rabbit polyclonal antibody serum by 50 times with PBS, take 5μL on a glass slide, pick out fresh Listeria monocytogenes of different serotypes and polyantibody serum and mix them (see Table 1 for strains), observe the agglutination reaction.

The results are shown in Table 1 and Figure 2. The 3 strains of Listeria monocytogenes of serotype 4h all had an obvious agglutination reaction with the 4h rabbit polyantiserum, and the other serotypes of Listeria monocytogenes and Ehrlich Listeria, Listeria innocuous, Listeria serovar, Listeria spp. and Listeria weseri are not agglutinating with the 4h rabbit poly-antiserum. PBS replaces the rabbit poly-antiserum and serotype 4h The XYSN reaction of Listeria monocytogenes was used as a negative control, indicating that the prepared 4h rabbit polyclonal antiserum can be used as a serological diagnostic kit to identify Listeria monocytogenes serotype 4h.

Example 5: Fluorescence identification of Listeria monocytogenes with 4h rabbit polyclonal antibody serum

1. Collect Listeria monocytogenes of different serotypes with PBS (see Table 1 for strains), wash twice with PBS, 9000rpm, 3min;

2. Fix with 4% paraformaldehyde for 20min, wash twice with PBS, 9000rpm, 3min;

3. PBS containing 5% BSA was blocked in a 37°C water bath for 2 hours, washed twice with PBS, 9000rpm, 3min;

4. Dilute the 4h rabbit poly-antiserum 800 times with 5% BSA-containing PBS, incubate in a 37°C water bath for 2 hours, wash 5 times with PBS, 5 minutes each time;

5. Add goat anti-rabbit (Alexa 488), incubate in a 37°C water bath for 2 hours, wash with PBS 5 times, 5 minutes each time;

6. DAPI marked the nucleus in a 37℃ water bath for 20 minutes, washed 4 times with PBS, 5 minutes each time;

7. Take 10μl and mix with a drop of glycerin, mount the slide with nail polish, and observe with laser confocal.

The results are shown in Figure 3. The 4h rabbit polyclonal antiserum can well label the 3 strains of Listeria monocytogenes of serotype 4h. Obvious green fluorescence was observed on the surface of the bacteria. The monocytes of the other serotypes Listeria hyperplasia, Listeria escherichia, Listeria innocuous, Listeria sederia, Listeria spp. and Listeria spp. all do not see green fluorescence on the surface of the bacteria. The results show that the prepared The 4h rabbit polyclonal antiserum can successfully label Listeria monocytogenes serotype 4h.

The above examples are intended to illustrate the disclosed embodiments of the present invention, and should not be construed as limiting the present invention. In addition, the various modifications listed herein and the changes in the method and composition of the invention are obvious to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications that are obvious to those skilled in the art to obtain the invention as described above should be included in the scope of the present invention.

Claims (12)

1. A Listeria monocytogenes XYSN is characterized in that: the collection number of the Listeria monocytogenes XYSN is: CCTCC NO: M 2019765.
2. The Listeria monocytogenes XYSN according to claim 1, wherein the nucleotide sequence of the Listeria monocytogenes XYSN is shown in SEQ ID NO.1.
3. Use of the Listeria monocytogenes XYSN according to any one of claims 1 to 2 for preparing polyclonal antibodies.
4. A polyclonal antibody, characterized in that: the polyclonal antibody is prepared by using Listeria monocytogenes XYSN as an immunogen.
5. The use of the polyclonal antibody according to claim 4 for identifying or immunofluorescently labeling Listeria monocytogenes of serotype 4h.
6. A method for identifying Listeria monocytogenes of serotype 4h, comprising the step of mixing an object to be identified with the polyclonal antibody of claim 4.
7. A method for immunofluorescence labeling of Listeria monocytogenes of serotype 4h, comprising the step of labeling Listeria monocytogenes of serotype 4h with the polyclonal antibody of claim 4.
8. The method for preparing polyclonal antibodies according to claim 4, which is obtained by immunizing animals with Listeria monocytogenes XYSN.
9. The preparation method according to claim 8, wherein the method comprises the following steps:

   (1) Use Listeria monocytogenes XYSN to prepare immunogen to immunize animals;

   (2) Collect blood to obtain serum containing polyclonal antibodies.
10. The preparation method according to claim 9, wherein the animal is selected from rabbits, mice, chickens, pigs or sheep.
11. A kit, characterized in that: the kit contains the polyclonal antibody according to claim 4.
12. The kit according to claim 11 is used to identify or immunofluorescently label Listeria monocytogenes of serotype 4h.

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