WO2022141944A1 - Standard strains of campylobacter jejuni containing specific molecular targets, examination therefor, and application thereof - Google Patents

Abstract

The present invention provides three strains of Campylobacter jejuni, the preservation number of the strain 346-1C being GDMCC 60857, the preservation number of the strain 3853-1A being GDMCC 60858, and the preservation number of the strain 542-1A being GDMCC 60859. And also provided are specific molecular targets respectively used for examining three specific strains of Campylobacter jejuni, the molecular targets comprising nucleotide sequences as shown in SEQ ID NO: 1-3. Each strain has a standard microscopic morphology and physiological and biochemical properties of Campylobacter jejuni thallus, and can be used for examining the accuracy of a color development plate for Campylobacter jejuni and standard strains examined by related reagents. The present invention also provides a lyoprotectant for Campylobacter jejuni.

Description

Standard strain of Campylobacter jejuni containing specific molecular targets and its detection and application technical field

The invention belongs to the technical field of microorganism detection, and particularly relates to the detection of a specific molecular target of Campylobacter jejuni and a standard bacteria containing the target.

Background technique

Diarrhoeal disease is a major public health problem worldwide, with more than 2 million deaths due to infectious diarrhea each year, and up to 10 million deaths annually in developing countries due to diarrheal diseases. It has caused huge economic losses and also posed an important threat to people's lives and health. Campylobacter jejuni is a zoonotic pathogen that can cause a variety of diseases in humans and animals, and is a food-borne pathogen that is considered a major cause of bacterial diarrhea in humans worldwide. Campylobacter jejuni is widely found in meat and meat products, especially in poultry, and is a common and important food-borne pathogen in my country. The pathogenic mechanism of Campylobacter jejuni and its pathogenic factors include four aspects: adhesion, invasion, toxin production and molecular mimicry mechanism. Molecular mimicking mechanism can cause the most serious complication—Guillain-Barre syndrome. Campylobacter jejuni can cause disease through the production of cytotoxic enterotoxins, cytotoxins, and cytotoxic distending toxins. Understanding the epidemic characteristics and genetic evolution of the bacteria is the basis for effective prevention and control of food-borne diseases caused by the bacteria, and whether the appropriate standard strains are used determines the reliability of the research results. At present, the standard strains of Campylobacter jejuni are the main source The American Type Culture Collection (ATCC) in the United States and the my country Medical Microbiology Center (CMCC) do not preserve Campylobacter jejuni.

After May 1, 2015, the inspection of Campylobacter jejuni in food in my country mainly implements the national standard GB/T4789.9-2014 "National Food Safety Standard for Food Microbiological Inspection of Campylobacter jejuni". The identification of Campylobacter jejuni mainly adopts traditional biochemical identification methods. Mainly through oxidase, catalase, hippurate hydrolysis and indole acetate hydrolysis tests, and then confirm whether it is Campylobacter jejuni. At present, my country still lacks representative food isolates as standard control strains. Most of the standard strains currently used are derived from ATCC. The standard strains of ATCC are generally isolated from clinics, and they are not pathogenic with food-borne Campylobacter jejuni strains. The abilities and genotypes are different, and they are different from the strains isolated in China in terms of traits, which cannot represent the characteristics of all C. jejuni, let alone the characteristics of C. jejuni in China, especially the food-borne C. jejuni. No strains of Campylobacter can be retrieved on the Chinese CMCC website. There is a shortage of standard strains from China, and even more from food sources. Therefore, it is very important to construct a representative standard strain of Campylobacter jejuni in China. Necessary.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned technical problems, the present invention provides 3 strains of food isolated strains of Campylobacter jejuni in China, the strains have typical physiological and biochemical characteristics of Campylobacter jejuni, and can better reflect the genetic background of the bacteria in China. Make up for the vacancy in the availability of standard strains of non-foodborne Campylobacter jejuni in China. Campylobacter jejuni strain 346-1C is a pigeon meat sample isolated from Wanshouting Farmers Market, Hangzhou, China. Chicken samples from Hualian Nanmen Square Store, preservation number: GDMCC 60858, classified name: Campylobacterjejuni; Campylobacter jejuni strain 542-1A is a pigeon meat sample isolated from Yaowangshan Farmers Market in Lhasa, China, and its preservation number is: GDMCC 60859 , classified as: Campylobacter jejuni. The Campylobacter jejuni strains 346-1C, 3853-1A, and 542-1A isolated and obtained above are all preserved in the Guangdong Provincial Microbial Culture Collection Center, address: 5th Floor, Laboratory Building, Provincial Institute of Microbiology, No. 100, Xianlie Middle Road, Guangzhou, China, preservation date: October 27, 2019.

The technical scheme adopted in the present invention is:

The present invention provides specific molecular targets for detecting Campylobacter jejuni, the molecular targets are:

(a) any one or several nucleotide sequences shown in SEQ ID NOs: 1 to 3; or,

(b) The nucleotide sequence in (a) is substituted, deleted or added by one or several nucleotides and has more than 90% homology with the nucleotide in (a). When three new strains of Campylobacter jejuni were discovered, it was also found that these three strains each contained a specific molecular target, which could distinguish them from other strains and have strong specificity.

The present invention provides primers for detecting the specific molecular target, and the PCR primers for the amplification of the nucleotide sequence shown in SEQ ID NO: 1 include: the upstream primer shown in SEQ ID NO: 4 and the primers shown in SEQ ID NO: 4 Downstream primers shown in ID NO: 5; PCR primers for amplification of the nucleotide sequence shown in SEQ ID NO: 2 include: upstream primers shown in SEQ ID NO: 6 and upstream primers shown in SEQ ID NO: 7 PCR primers for amplification of the nucleotide sequence shown in SEQ ID NO:3 include the upstream primer shown in SEQ ID NO:8 and the downstream primer shown in SEQ ID NO:9. The inventors designed primers for the above-mentioned specific molecular targets, which can specifically amplify the above-mentioned fragments, and thus can be used as specific primers for detecting the above-mentioned molecular targets.

The present invention also provides a Campylobacter jejuni, which is (a), (b) or (c):

(a) strain 346-1C contains the nucleotide sequence shown in SEQ ID NO: 1;

(b) strain 3853-1A contains the nucleotide sequence shown in SEQ ID NO:2;

(c) Strain 542-1A contains the nucleotide sequence shown in SEQ ID NO:3.

Preferably, the strain 346-1C further comprises the following virulence genes: cdtB, cdtC, ciaB, pldA, flig, dnaJ, racR, cadF, cdtA, docA, imaA, rpon and ceuE; and is resistant to the following antibiotics Properties: Cefoperazone, ciprofloxacin, tetracycline, nalidixic acid and vancomycin.

Preferably, the strain 3853-1A further comprises the following virulence genes: cdtB, cdtC, ciaB, pldA, flig, dnaJ, racR, cadF, cdtA, docA, imaA, rpon, flaA, wlaN and ceuE; and for the following Antibiotics are resistant: cefoperazone, ciprofloxacin, tetracycline, nalidixic acid, and vancomycin.

Preferably, the strain 542-1A further comprises the following virulence genes: cdtB, cdtC, ciaB, pldA, flig, dnaJ, racR, cadF, cdtA, docA, imaA, rpon, flaA and ceuE; and has the following antibiotics Resistance: cefoperazone, clindamycin, amoxicillin, ciprofloxacin, tetracycline, streptomycin, kanamycin, ampicillin, nalidixic acid, and vancomycin.

Preferably, the deposit number of the strain 346-1C is: GDMCC 60857; the deposit number of the strain 3853-1A is: GDMCC 60858; the deposit number of the strain 542-1A is GDMCC 60859.

The invention also provides the application of the Campylobacter jejuni in the study of antibiotic resistance of the Campylobacter jejuni. Preferably, the antibiotic resistance of the strain-1 is resistance to cefoperazone, ciprofloxacin, tetracycline, nalidixic acid and vancomycin; the antibiotic resistance of the strain-2 is resistance to Resistance to cefoperazone, ciprofloxacin, tetracycline, nalidixic acid, and vancomycin; the antibiotic resistance of strain-3 was cefoperazone, clindamycin, amoxicillin, ciprofloxacin Resistance to floxacin, tetracycline, streptomycin, kanamycin, ampicillin, nalidixic acid, and vancomycin.

Preferably, the antibiotics include any one or more of the following antibiotics: cefoperazone, ciprofloxacin, tetracycline, nalidixic acid, vancomycin, streptomycin, kanamycin, ampicillin, Linomycin or and amoxicillin.

The invention also provides the application of the said Campylobacter jejuni in improving the accuracy of testing the color plate of Campylobacter jejuni.

The invention also provides a freeze-drying protective agent for preparing quantitative quality control bacteria of Campylobacter jejuni, comprising the following components in parts by weight: 2-7 parts of fetal bovine serum, 5-20 parts of skimmed milk powder, 0.2-20 parts of potassium lactate 5 parts, 1-4 parts of inositol, 0.1-2 parts of L-cysteine hydrochloride.

Preferably, the following components are included in parts by weight: 5 parts of fetal bovine serum, 20 parts of skim milk powder, 5 parts of potassium lactate, 2 parts of inositol, and 1 part of L-cysteine hydrochloride.

The reason why the above-mentioned components are selected in the present invention is that inositol has strong hydrophilicity. During the freezing or drying process, it can form hydrogen bonds with the phosphate group in the phospholipid of the bacterial cell membrane or with the polar group of the bacterial protein. , to protect the integrity of cell membrane and protein structure and function, skim milk powder can be wrapped in the outer layer of bacteria cells to protect bacteria. Fetal bovine serum has a stabilizing effect on bacterial suspension, potassium lactate and L-cysteine hydrochloride reduce the activity of cellular oxidase during freeze-drying process and long-term storage, and prevent the oxidative deterioration of freeze-dried products.

The beneficial effects of the present invention are: the Campylobacter jejuni GDMCC 60857, GDMCC 60858, and GDMCC 60859 of the present invention have the standard microscopic morphology and physiological and biochemical characteristics of Campylobacter jejuni, and can be used to test the accuracy of the color plate of Campylobacter jejuni , and standard strains verified by relevant reagents. These strains are native food source strains in China, with clear and reliable sources and clear genetic backgrounds. It can be seen from the results of the API Campy reaction that the strain is Campylobacter jejuni, but from the typical value T, it can be seen that its characters are quite different from the standard strain selected by API, and it is a strain with the genetic background and typical characters in China, so these The strain can be used as a specific reference strain for the detection of Campylobacter jejuni in China. GDMCC 60857 and GDMCC 60858 have specific molecular target sites, which can be directly identified by PCR, and are fast, cheap, and easy to operate. GDMCC 60859 has a large resistance island, which can also be identified by PCR or sequencing.

In addition, for the Campylobacter jejuni of the present invention, the inventor provides a freeze-drying protective agent for Campylobacter jejuni. The protective agent has the following advantages: good shape, beautiful appearance, good water solubility, and can be completely dissolved within 1-2 seconds ; The survival rate of freeze-drying can reach more than 40%; it can be stored at -20 ℃ for at least one year, which can ensure that its order of magnitude does not change, and can be used for long-term storage of quality control strains.

Description of drawings

Figure 1 shows the colony morphology of Campylobacter jejuni on the skirrow blood plate and the modified CCD plate, and its morphology after Gram staining;

Figure 2 is a schematic diagram of the specificity of the target site in Campylobacter jejuni (a: No. 346-5 and 346-7 in the figure can amplify the target band, other numbers of Campylobacter jejuni strains have no target band, No. 346- 5 and 346-7 are the strains (strain-1) of the deposit number GDMCC 60857; b: the number 24 (+) in the figure can amplify the target band, and the other numbers of Campylobacter jejuni strains have no target band, and the number 24 is The strain of deposit number GDMCC 60858 (strain-2); c: "+" in the figure can amplify the target band, other numbers of Campylobacter jejuni strains have no purpose band, and the number "+" is the strain of deposit number GDMCC 60859 (strain-3)

Fig. 3 is a schematic diagram of the specificity of the Campylobacter jejuni strain of the present invention's deposit number GDMCC 60857 compared with other species, and the figure shows other genera (including Bacillus thuringiensis, Bacillus cereus, Escherichia coli, Yersinia enterocolitis) Bacillus, Staphylococcus aureus, Listeria monocytogenes, Cronobacter, Vibrio parahaemolyticus and Salmonella) did not amplify the target size band (279bp), while the Campylobacter jejuni of the present invention has the target band. bring.

Figure 4 is a schematic diagram showing the specificity of the Campylobacter jejuni strain with the deposit number GDMCC 60857 of the present invention compared with the standard strains preserved in other institutions, and the figure shows other genera (Listeria monocytogenes [LM19115, LM54002, LM54004], Staphylococcus aureus [Sa25923], Pseudomonas aeruginosa [Pa9027, Pa27853, Pa10104], Salmonella [sal150335, Sal1402], Escherichia coli [Ec25922], Yersinia enterocolitica [Ye5402, JC2, Y802, C009 ], Bacillus cereus [Bc2068] and Campylobacter jejuni [Cj33291]) did not amplify the target band, while the Campylobacter jejuni of the present invention had the target band.

Figure 5 is a schematic diagram of changes in the storage period of Campylobacter jejuni quantitative quality control bacteria.

Detailed ways

In order to show the technical solutions, objects and advantages of the present invention more concisely and clearly, the technical solutions of the present invention are described in detail below with reference to specific embodiments and accompanying drawings.

Example 1 Isolation, identification and cultivation of standard strains of Campylobacter jejuni

Take 25g (mL) sample (50g for fruits, vegetables or aquatic products) and add it to a homogeneous bag with 225mL Bolton broth (if it is a homogeneous bag without a filter, it can be filtered with sterile gauze). Homogenize the filtrate for 1 to 2 minutes with a hit homogenizer, filter it through a filter screen or sterile gauze, and culture the filtrate. Under microaerophilic conditions, incubate at 36 °C ± 1 °C for 4 h, and shake at a speed of 100 r/min if conditions permit. If necessary, measure the pH value of the enrichment solution and adjust it to 7.4±0.2, and continue to culture at 42℃±1℃ for 24h～48h. The 24h enrichment solution, the 48h enrichment solution and the corresponding 1:50 dilution were streaked on Skirrow blood agar and mCCDA agar plates, and incubated at 42℃±1℃ for 24h～48h under microaerophilic conditions. Alternatively, the Campylobacter jejuni chromogenic plate can be used as a supplement. Observe the colony morphology on the agar plates cultured for 24h and 48h, and determine the suspicious colonies on the chromogenic medium of Campylobacter jejuni according to the instructions. Pick 5 (if less than 5, pick all) or more suspicious colonies, inoculate them on Columbia blood agar plates, and incubate them at 42°C ± 1°C for 24h-48h under microaerophilic conditions. Identify according to GB4789.9-2014. The target colonies were resuspended with Brucella broth from the blood plate, fresh glycerol broth (final concentration of 25%) was added, and 0.025% FBP was added, and stored in a -80°C refrigerator. The purified strains can be identified by morphological characteristics, physiology, biochemistry and molecular biology.

Example 2 Identification of physiological and biochemical characteristics of standard strains of Campylobacter jejuni

(1) Growth status of selective medium

Suspicious colonies on mCCDA agar plates are usually pale gray, metallic, moist, flat, and tend to spread out. Type 1 suspicious colonies on Skirrow blood agar plates are gray, flat, moist and shiny, and tend to spread outward along the inoculation line; Type 2 suspicious colonies are usually scattered and raised single colonies with neat and shiny edges. The results are shown in Figure 1.

(2) Microscopic examination

Staining microscopy: smear suspicious colonies, carry out Gram staining, and observe the morphology by microscopy. All Campylobacter jejuni were gram-negative, stained red, slightly curved and comma-shaped, without spores and capsules, and with flagella. The results are shown in Figure 1.

(3) API Campy identification

The colonies of Campylobacter jejuni were scraped from the skirrow plate, and the cell suspension with appropriate turbidity was prepared with normal saline, and identified using the API Campy biochemical identification reagent strip. The identification results are shown in Table 1:

Table 1: API Campy identification results

From the results of the API Campy reaction in Table 1, it can be seen that the strains screened above are Campylobacter jejuni, but from the typical value T, it can be seen that its characters are quite different from the standard strains selected by API, and they have the genetic background and typical characters in China. Therefore, these strains can be used as specific reference strains for the detection of Campylobacter jejuni in China.

Example 3 Identification of virulence factor carrying characteristics of standard strains of Campylobacter jejuni

The virulence genes carried by the strains were identified by PCR. The primers used were synthesized by Shanghai Sangon Biological Co., Ltd. (see Table 2 for primer sequences).

The PCR amplification system (25 μL) contained: 2×Ferment PCRmix, 12.5 μL; 0.4 μM upstream and downstream primers; ddH ₂ O, 8.5 μL and genomic DNA, 2 μL. 8-10 μL of PCR products were loaded on 2.0% agarose gel for electrophoresis separation (120V, 40min), using 2000pb DNA Marker.

Confirmed by PCR and gel electrophoresis, the virulence factors carried by the strain GDMCC 60857 are:

cdtB-cdtC-ciaB-pldA-flig-dnaJ-racR-cadF-cdtA-docA-imaA-rpon-ceuE;

The virulence factors carried by GDMCC 60859 are: cdtB-cdtC-ciaB-pldA-flig-dnaJ-racR-cadF-cdtA-docA-imaA-rpon-flaA-ceuE;

The virulence factors carried by GDMCC 60858 are:

cdtB-cdtC-ciaB-pldA-flig-dnaJ-racR-cadF-cdtA-docA-imaA-rpon-flaA-wlaN-ceuE.

Table 2. Virulence-related gene primers

Example 4 Identification of drug susceptibility of standard strains of Campylobacter jejuni

After culturing Campylobacter jejuni in Brucella broth for about 40 hours, adjust the OD ₆₀₀ to 0.1-0.3, draw 100 μL of the bacterial solution, spread it on the MH plate containing 5% sheep blood, and put the antibiotic paper after the bacterial solution is dry. The sheet was attached to the surface of the medium and incubated at 42°C for 24h. The size of the inhibition zone was measured with a vernier caliper, accurate to 0.01 mm. The selected antibiotics are as follows: cefoperazone, erythromycin, clindamycin, gentamicin, streptomycin, kanamycin, ciprofloxacin, nalidixic acid, amoxicillin, ampicillin, tetracycline, Vancomycin. Escherichia coli ATCC25922 and ATCC25922 were used as quality control strains. The drug resistance spectrum of Campylobacter jejuni GDMCC 60857 is CFP (cefoperazone)-CIP (ciprofloxacin)-TE (tetracycline)-NA (nalidixic acid)-VA (vancomycin); the drug resistance spectrum of GDMCC 60858 It is CFP (cefoperazone)-CIP (ciprofloxacin)-TE (tetracycline)-NA (nalidixic acid)-VA (vancomycin); the drug resistance spectrum of GDMCC 60859 is CFP (cefoperazone)-DA (Clindamycin)-AML(Amoxicillin)-CIP(Ciprofloxacin)-TE(Tetracycline)-S(Streptomycin)-K(Kanamycin)-AMP(Ampicillin)-NA( nalidixic acid)-VA (vancomycin).

Example 5 Multi-locus sequence (MLST) typing analysis of standard strains of Campylobacter jejuni

Using the experimental protocol described on the official website of MLST, the 7 pairs of primers used were synthesized by Shanghai Sangon Biological Co., Ltd. (see Table 3 for primer sequences). The reagent for multilocus sequence typing is Takara's

HS DNA Polymerase, total 50 μL of PCR reaction system: 10 μL of 5×PrimeSTAR Buffer (Mg2+plus), 4 μL of dNTP Mixture, 1 μL of each primer, 1 μL of template, 1 μL of PrimeSTAR HS DNA Polymerase (2.5U/μl), supplemented with ultrapure water together. PCR reaction conditions: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 2 min; annealing at 50°C for 1 min; extension at 72°C for 1 min; a total of 35 cycles; final extension at 72°C for 10 min. After PCR, gel electrophoresis was performed to confirm that the size of the product was correct, and the product was handed over to Thermo Fisher Scientific (China) Co., Ltd. for bidirectional sequencing. The sequencing results were matched with the MLST database, and the allele values of the seven housekeeping gene loci were obtained respectively, and the corresponding allele profiles were formed to determine their sequence types. The typing results are shown in Table 4.

Table 3: Sequences of primers for MLST typing of Campylobacter jejuni

Table 4: MLST typing results

The above seven housekeeping genes were used for MLST typing, and the ST type results are shown in Table 4, among which ST9558 and 9560 are both new types of ST.

Example 6 Characteristic sequence analysis of standard strains of Campylobacter jejuni

The strain-specific non-essential genes were obtained mainly based on the pan-genome analysis results of Campylobacter jejuni. A total of 74 strains of Campylobacter jejuni (including GDMCC 60857, GDMCC 60858, and GDMCC 60859 strains) were selected for pan-genome analysis. The pan-genome was analyzed by the GF method in the prokaryotic Pan-Genomics Analysis Pipeline (PGAP), and the analysis results were processed by the local Perl script to obtain the core gene and non-core gene information of all strains.

Extract the non-core gene protein sequences specific to Campylobacter jejuni, and compare them with the total protein library of Campylobacter jejuni and the NCBI non-redundant protein database (NR) through local Blast, and remove the sequences that can be mapped to the nr library, and the remaining sequences is the specific sequence. The specific sequence of GDMCC 60857_00919 is shown in SEQ ID NO: 1, and the specific sequence of GDMCC 60858_02046 is shown in SEQ ID NO: 2. Primer 5 was used to design primers for these specific sequences, and primers were synthesized by Shanghai Shenggong (see Table 5 for primer sequences), and the reaction conditions and systems were shown in Table 6 and Table 7. The specificity of the unique gene was tested by PCR amplification, including the specificity of the molecular sequence in Campylobacter jejuni, the specificity in other strains, and the specificity in strains preserved by other institutions. The specific PCR results are shown in Figures 2-4.

A relatively special drug resistance gene island was found in strain GDMCC 60859. The drug resistance island carries 9 kinds of drug resistance-related genes. This drug resistance island was first discovered in Campylobacter jejuni, and its specific information is shown in the specific sequence SEQ ID NO:3 .

Table 5: Primers specific to target sites

Table 6. PCR reaction system for specific tags

Note: a Mix used is Thermofisher K8081

Table 7 PCR reaction program for molecular identification of specific molecular tags

The present invention also provides a lyophilized protective agent for quantitative preservation of Campylobacter jejuni. The components of the lyophilized protective agent in different embodiments and comparative examples are shown in Table 8:

Table 8: Components of each group of lyoprotectants

In the comparative example, the weight part of skim milk powder was increased to make up the total amount due to its lack of components.

The freeze-dried survival rate of the freeze-dried bacterial species (GDMCC 60857) of the protective agents of Examples 7 to 9 and Comparative Examples 1 to 3 will be used, and the specific method is as follows:

After the strains are recovered, they are inserted into the culture medium and cultivated to the late logarithmic stage to the early stage of the stable stage. Select an appropriate amount of bacteria and add them to the protective agents of Examples 7-9 and Comparative Examples 1-3, mix them evenly, and distribute them into vials. , and sample for dilution count, which is the bacterial content A0 before freeze-drying. Transfer the half-stoppered vial into the freeze dryer for pre-freezing, the pre-freezing temperature is -40°C, the time is 3 hours, the main drying is turned on, the time is 20-25 hours, and then the analysis and drying stage is entered, and the time is 6 -8 hours, end drying, press the plug under vacuum and remove from the freeze dryer, carry out automatic capping, ensure the complete vacuum state of the sample, and store at -20 ℃ low temperature. Get the sample after freeze-drying and carry out dilution count, and the count result is the bacterial content A after freeze-drying, and the freeze-drying survival rate is the percentage of A and A0, and the results are shown in Table 9:

Table 9: Comparison of lyophilized survival rates of lyoprotectants with different compositions

As can be seen from Table 9, the lyophilized protective agent of Example 9 is the best embodiment of the present invention, therefore, taking Example 9 as a comparison object, the lyophilized protective agents of Comparative Examples 1 to 3 have been prepared, and the results are as shown in Table 9 As shown, since Comparative Examples 1 to 3 lacked one of the components of the lyophilized protective agent of the present invention, their protective effects were all worse than those of the examples, thus illustrating the component lactic acid in the lyophilized protective agent of the present invention. There is a synergistic effect among potassium, inositol and L-cysteine hydrochloride, and the effect of the protective agent of the present invention cannot be achieved without any of them.

The freeze-drying stability will be compared with the freeze-dried strains of the protective agents in Examples 7-9 and Comparative Examples 1-3, and the specific methods are as follows:

According to the preparation method of Example 7, different protective agents were used to prepare quantitative quality control bacteria, and they were stored at -20°C, and 3 bottles were extracted every month to check the bacteria content according to the aforementioned counting method. In order to better compare the effect of each protective agent in the long-term storage process, when preparing quantitative quality control bacteria, the number of viable bacteria before freeze-drying was calculated according to the freeze-drying survival rate of each protective agent, so that after each protective agent was freeze-dried, The bacterial content is about 3000cfu/bottle. The changes in bacterial content of the quantitative quality control bacteria prepared by each protective agent after 12 months of storage are shown in Figure 5 below.

As can be seen from accompanying drawing 5, after 12 months of storage, the bacterial content protected by the lyophilized protective agents of Examples 7 to 9 has no significant change, while the bacterial content protected by the lyophilized protective agents of Comparative Examples 1 to 3 has no significant change after 12 months of storage. The bacterial content decreased significantly after 2 months, and the bacterial content was close to 0 after 12 months.

The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as a limitation on the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims (10)

1. A specific molecular target for detecting Campylobacter jejuni, characterized in that the molecular target is:

   (a) any one or several nucleotide sequences shown in SEQ ID NOs: 1 to 3; or,

   (b) The nucleotide sequence in (a) is substituted, deleted or added by one or several nucleotides and has more than 90% homology with the nucleotide in (a).
2. The primer for detecting the specific molecular target of claim 1, wherein the PCR primer for amplification of the nucleotide sequence as shown in SEQ ID NO:1 comprises: upstream as shown in SEQ ID NO:4 Primers and downstream primers as shown in SEQ ID NO:5; PCR primers for amplification of the nucleotide sequence as shown in SEQ ID NO:2 include: upstream primers as shown in SEQ ID NO:6 and as shown in SEQ ID Downstream primer set forth in NO:7; PCR primers for amplification of the nucleotide sequence set forth in SEQ ID NO:3 include the upstream primer set forth in SEQ ID NO:8 and the upstream primer set forth in SEQ ID NO:9 downstream primers.
3. A kind of Campylobacter jejuni (Campylobacter jejuni) is characterized in that, is (a), (b) or (c):

   (a) strain 346-1C contains the nucleotide sequence shown in SEQ ID NO: 1;

   (b) strain 3853-1A contains the nucleotide sequence shown in SEQ ID NO:2;

   (c) Strain 542-1A contains the nucleotide sequence shown in SEQ ID NO:3.
4. The Campylobacter jejuni of claim 3, wherein the strain 346-1C further comprises the following virulence genes: cdtB, cdtC, ciaB, pldA, flig, dnaJ, racR, cadF, cdtA, docA, imaA , rpon, and ceuE; and resistant to the following antibiotics: cefoperazone, ciprofloxacin, tetracycline, nalidixic acid, and vancomycin.
5. The Campylobacter jejuni of claim 3, wherein the strain 3853-1A further comprises the following virulence genes: cdtB, cdtC, ciaB, pldA, flig, dnaJ, racR, cadF, cdtA, docA, imaA , rpon, flaA, wlaN, and ceuE; and resistant to the following antibiotics: cefoperazone, ciprofloxacin, tetracycline, nalidixic acid, and vancomycin.
6. The Campylobacter jejuni of claim 3, wherein the strain 542-1A further comprises the following virulence genes: cdtB, cdtC, ciaB, pldA, flig, dnaJ, racR, cadF, cdtA, docA, imaA , rpon, flaA, and ceuE; and are resistant to the following antibiotics: cefoperazone, clindamycin, amoxicillin, ciprofloxacin, tetracycline, streptomycin, kanamycin, ampicillin, nelidine acid and vancomycin.
7. Campylobacter jejuni as claimed in claim 3, characterized in that the deposit number of the strain 346-1C is: GDMCC 60857; the deposit number of the strain 3853-1A is: GDMCC 60858; The deposit number is GDMCC 60859.
8. The use of Campylobacter jejuni as claimed in claim 3 in antibiotic resistance of Campylobacter jejuni. Preferably, the antibiotic resistance of the strain 346-1C is resistance to cefoperazone, ciprofloxacin, tetracycline, nalidixic acid and vancomycin; the antibiotic resistance of the strain 3853-1A resistance to cefoperazone, ciprofloxacin, tetracycline, nalidixic acid and vancomycin; antibiotic resistance of the strain 542-1A to cefoperazone, clindamycin, amoxicillin , ciprofloxacin, tetracycline, streptomycin, kanamycin, ampicillin, nalidixic acid and vancomycin resistance.
9. The application of the Campylobacter jejuni according to claim 3 in improving the accuracy of testing the color plate of Campylobacter jejuni.
10. A freeze-drying protective agent for Campylobacter jejuni, characterized in that it comprises the following components by weight: 2-7 parts of fetal bovine serum, 5-20 parts of skim milk powder, 0.2-5 parts of potassium lactate, and 1-4 parts of inositol , 0.1-2 parts of L-cysteine hydrochloride.

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