CN112899379A

CN112899379A - Campylobacter jejuni standard strain containing specific molecular target and detection and application thereof

Info

Publication number: CN112899379A
Application number: CN202011611248.3A
Authority: CN
Inventors: 王涓; 吴清平; 丁郁; 陈谋通; 薛亮; 张菊梅; 曾海燕; 叶青华; 吴诗; 张淑红; 庞锐; 雷涛; 古其会; 张友雄; 韦献虎; 陈惠元; 相欣然; 汪智; 唐胜君; 陈鲁
Original assignee: Guangdong Detection Center of Microbiology of Guangdong Institute of Microbiology; Guangdong Huankai Biotechnology Co Ltd
Current assignee: Guangdong Detection Center of Microbiology of Guangdong Institute of Microbiology; Guangdong Huankai Biotechnology Co Ltd
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2021-06-04
Anticipated expiration: 2040-12-30
Also published as: WO2022141944A1; CN112899379B

Abstract

The invention provides 3 strains of campylobacter jejuni, wherein the strain 346-1C has a deposition number of GDMCC 60857, the strain 3853-1A has a deposition number of GDMCC 60858, and the strain 542-1A has a deposition number of GDMCC 60859; meanwhile, specific molecular targets for detecting 3 specific campylobacter jejuni strains are provided, and the molecular targets comprise nucleotide sequences shown in SEQ ID NO. 1-3. The strain has the microscopic morphology and physiological and biochemical characteristics of the standard campylobacter jejuni thallus, and can be used for detecting the accuracy of a campylobacter jejuni color development plate and the standard strain verified by related reagents. The invention also provides a freeze-drying protective agent which has the advantages of good forming, beautiful appearance and good water solubility, and can be completely dissolved within 1-2 seconds; can be stored for more than one year at the temperature of minus 20 ℃, can ensure that the magnitude of the strain is not changed, and can be used for storing quality control strains for a long time.

Description

Campylobacter jejuni standard strain containing specific molecular target and detection and application thereof

Technical Field

The invention belongs to the technical field of microbial detection, and particularly relates to a target for detecting a campylobacter jejuni specific molecule and a standard bacterium containing the target.

Background

The diarrhea disease is an important public health problem worldwide, the death number of people caused by infectious diarrhea exceeds 200 million every year, and the death number of people caused by the diarrhea disease reaches 1000 million every year in developing countries, thereby causing huge economic loss to the global public health industry and also forming important threat to the life health of people. Campylobacter jejuni is a zoonosis pathogen, can cause various diseases of human and animals, is a food-derived pathogen, and is considered to be a major cause of human bacterial diarrhea worldwide. Campylobacter jejuni is widely existed in meat and meat products, especially poultry meat, and is a common important food-borne pathogenic bacterium in China. The pathogenic mechanism of campylobacter jejuni and its pathogenic factors include adhesion, invasion, toxin production and molecular simulation mechanism, which can cause the most serious complication, guillain-barre syndrome. Campylobacter jejuni can be pathogenic by producing a cytotoxm, a cytotoxin, and a cytolethal swollenin. Understanding the epidemic characteristics and genetic evolution of the campylobacter jejuni is the basis for effectively preventing and controlling food-borne diseases caused by the campylobacter jejuni, and whether a proper standard strain is used determines the reliability of research results, and the campylobacter jejuni is mainly obtained from American Type Culture Collection (ATCC) which is the main source of the standard strain of the campylobacter jejuni at present, and is not stored in China medical microorganism Culture center (CMCC).

After 5 months and 1 day 2015, the inspection of campylobacter jejuni in food in China mainly executes national standards GB/T4789.9-2014 inspection of campylobacter jejuni in national food safety standards. The identification of the campylobacter jejuni mainly adopts a traditional biochemical identification method. Whether the bacteria are campylobacter jejuni or not was confirmed mainly by the oxidase, catalase, hippurate hydrolysis and indoleacetate hydrolysis tests. At present, representative food isolates are still lacking in China as standard control strains, most of the existing standard strains are derived from ATCC, the standard strains of the ATCC are generally obtained by clinical separation, are different from food-borne campylobacter strains in pathogenic capability and genotype, are different from the strains obtained by Chinese separation in properties, cannot represent the characteristics of all campylobacter jejuni, and cannot represent the characteristics of the Chinese campylobacter jejuni, particularly the food-borne campylobacter jejuni. Any strain of campylobacter cannot be searched on the CMCC website in china, and since the standard strain having a native source in china is very scarce in resources and the strain having a food source is very scarce, it is necessary to construct a representative standard strain of campylobacter jejuni in the country.

Disclosure of Invention

In order to solve the technical problems, the invention provides 3 strains of Campylobacter jejuni in China, wherein the strains have typical physiological and biochemical characteristics of Campylobacter jejuni, can better reflect the genetic background of the Campylobacter jejuni in China, and can make up for the vacancy of the Campylobacter jejuni standard strains in China, which are not food-borne. Campylobacter jejuni strain 346-1C is a pigeon meat sample isolated from Tageton farmer market in Hangzhou City, China, with the following preservation numbers: GDMCC 60857, classification name: campyylobacter jejuni; campylobacter jejuni strain 3853-1A is isolated from chicken samples of the Square stores of Nanmen of Beijing Hualian, Yinchuan, China, and the preservation numbers are as follows: GDMCC 60858, classification name: campyylobacter jejuni; the campylobacter jejuni strain 542-1A is a pigeon meat sample separated from farmer market in Wangshan mountain of Lassa, China, and the preservation number is as follows: GDMCC 60859, category name: camphyllobacter jejuni. The isolated Campylobacter jejuni strains 346-1C, 3853-1A and 542-1A are all preserved in Guangdong province microorganism culture Collection with the following addresses: the preservation date of the fifth building of the experimental building of the microbiological institute of 100 province in the first furious Zhonglu city of China: 2019, 10 and 27.

The technical scheme adopted by the invention is as follows:

the invention provides a specific molecular target for detecting campylobacter jejuni, which comprises the following steps:

(a) 1-3 of any one or more nucleotide sequences shown in SEQ ID NO; alternatively, the first and second electrodes may be,

(b) the nucleotide sequence in (a) is subjected to substitution, deletion or addition of one or more nucleotides, and has more than 90% homology with the nucleotides in (a). 3 new campylobacter jejuni strains are found, and the three strains are also found to respectively contain a section of specific molecular target, so that the three strains can be distinguished from other strains and have stronger specificity.

The invention provides a primer for detecting the specific molecular target, and a PCR primer for amplifying the nucleotide sequence shown as SEQ ID NO. 1 comprises: an upstream primer shown as SEQ ID NO. 4 and a downstream primer shown as SEQ ID NO. 5; the PCR primer for the nucleotide sequence amplification shown as SEQ ID NO. 2 comprises: an upstream primer shown as SEQ ID NO. 6 and a downstream primer shown as SEQ ID NO. 7; the PCR primers for the amplification of the nucleotide sequence shown as SEQ ID NO. 3 include an upstream primer shown as SEQ ID NO. 8 and a downstream primer shown as SEQ ID NO. 9. The inventors designed primers for the specific molecular targets and amplified the fragments specifically, and thus the primers were useful as specific primers for detecting the molecular targets.

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

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

(b) strain 3853-1A contains a nucleotide sequence shown as SEQ ID NO. 2;

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

Preferably, said strain 346-1C further comprises the following virulence genes: cdtB, cdtC, ciaB, pldA, flig, dnaJ, racR, cadF, cdtA, docA, imaA, rpon, and ceuE; and resistance to the following antibiotics: 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 resistance to the following antibiotics: 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 resistance to the following antibiotics: cefoperazone, clindamycin, amoxicillin, ciprofloxacin, tetracycline, streptomycin, kanamycin, ampicillin, nalidixic acid and vancomycin.

Preferably, the strain 346-1C has a deposit number of: GDMCC 60857; the preservation number of the strain 3853-1A is as follows: GDMCC 60858; the strain 542-1A has a accession number GDMCC 60859.

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

Preferably, the antibiotic comprises any one or more of the following antibiotics: cefoperazone, ciprofloxacin, tetracycline, nalidixic acid, vancomycin, streptomycin, kanamycin, ampicillin, clindamycin or amoxicillin.

The invention also provides application of the campylobacter jejuni in improving the accuracy of detecting the campylobacter jejuni color development plate.

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

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

The components are selected because inositol has strong hydrophilicity, and can form hydrogen bonds with phosphate groups in thallus cell membrane phospholipid or thallus protein polar groups in the freezing or drying process to protect the integrity of cell membrane and protein structures and functions, and the skim milk powder can wrap the outer layer of thallus cells to protect the thallus. The fetal calf serum has a stabilizing effect on the bacterial suspension, and the potassium lactate and the L-cysteine hydrochloride reduce the activity of cellular oxidase in the freeze-drying process and long-term storage, so that the oxidative deterioration of a freeze-dried product is prevented.

The invention has the beneficial effects that: the campylobacter jejuni GDMCC 60857, GDMCC 60858 and GDMCC 60859 have the microscopic morphology and physiological and biochemical characteristics of standard campylobacter jejuni, and can be used for checking the accuracy of a campylobacter jejuni color development plate and standard strains verified by related reagents. The strains are native food source strains in China, and have clear and reliable sources and clear genetic backgrounds. As can be seen from the result of API Campy reaction, the strain is campylobacter jejuni, but the characteristics of the strain are greatly different from those of the standard strain selected by API, and the strain has genetic background and typical characteristics in China, so that the strain can be used as a specific reference strain for detecting campylobacter jejuni in China. GDMCC 60857 and GDMCC 60858 have specific molecular target sites, can be directly identified by PCR, and have the characteristics of rapidness, low cost, easy operation and the like. GDMCC 60859 has a large drug resistance island and can also be identified by PCR or sequencing.

In addition, aiming at the campylobacter jejuni of the present invention, the inventors provide a campylobacter jejuni freeze-drying protective agent, which has the following advantages: the molding is good, the appearance is beautiful, the water solubility is good, and the solution can be completely dissolved within 1 to 2 seconds; the freeze-drying survival rate can reach more than 40 percent; can be stored for more than one year at the temperature of minus 20 ℃, can ensure that the magnitude of the strain is not changed, and can be used for storing quality control strains for a long time.

Drawings

FIG. 1 is a diagram showing the morphology of colonies of Campylobacter jejuni on a skerrow blood plate and a modified CCD plate, and their morphology observed by gram staining and microscopic examination;

FIG. 2 shows the specificity of the target site in Campylobacter jejuni (a: No. 346-5 and 346-7 amplify the band of interest, and no other Campylobacter jejuni strain (No. 1), No. 346-5 and 346-7 strain (No. GDMCC 60857), b: No. 24(+) amplify the band of interest, No. other Campylobacter jejuni strain (No. 2), No. 24 strain (No. GDMCC 60858), c: "+" amplify the band of interest, No. other Campylobacter jejuni strain (No. 3), No. "+" strain (No. GDMCC 60859)

FIG. 3 is a schematic diagram showing the specificity of Campylobacter jejuni strain with the accession number GDMCC 60857 of the present invention compared with other species, in which the target size band (279bp) is not amplified by other genera (including Bacillus thuringiensis, Bacillus cereus, Enterobacter coli, Yersinia enterocolitica, Staphylococcus aureus, Listeria monocytogenes, Cronobacter saxatilis, Vibrio parahaemolyticus and Salmonella) but by Campylobacter jejuni of the present invention.

FIG. 4 is a schematic diagram showing the specificity of Campylobacter jejuni strain with the accession number GDMCC 60857 of the present invention compared with other standard strains stored in other institutions, wherein 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]) are not amplified to form a target band, and the Campylobacter jejuni strain of the present invention has a target band.

FIG. 5 is a schematic diagram showing the change of Campylobacter jejuni in the storage period of quantitative quality control bacteria.

Detailed Description

In order to more concisely and clearly demonstrate technical solutions, objects and advantages of the present invention, the following detailed description of the technical solutions of the present invention is provided with reference to specific embodiments and accompanying drawings.

Example 1 isolation, identification and culture of Campylobacter jejuni Standard Strain

Adding 25g (mL) of sample (50 g for fruit, vegetable or aquatic product) into a homogenization bag containing 225mL Bolton broth and a filter screen (sterile gauze can be used for filtering if the homogenization bag is not provided with the filter screen), homogenizing for 1 min-2 min by using a beating type homogenizer, filtering by using the filter screen or the sterile gauze, and culturing the filtrate. Culturing at 36 + -1 deg.C for 4h under microaerophilic conditions, and shaking at 100r/min if allowed. If necessary, measuring the pH value of the enrichment fluid, adjusting the pH value to 7.4 +/-0.2, and continuously culturing for 24-48 h at the temperature of 42 +/-1 ℃. And respectively streak-inoculating the 24h enriched liquid, the 48h enriched liquid and the corresponding 1:50 diluent on Skirrow blood agar and mCCDA agar plates, and culturing for 24 h-48 h at 42 +/-1 ℃ under a microaerobic condition. Alternatively, Campylobacter jejuni color development plates may be used as a supplement. The colony morphology on 24h culture and 48h culture agar plates is observed, and suspicious colonies on the campylobacter jejuni chromogenic medium are judged according to instructions. 5 (if less than 5, all) or more suspected colonies are picked and inoculated onto a Columbia blood agar plate and cultured at 42 ℃. + -. 1 ℃ for 24-48 h under microaerobic conditions. The identification is carried out according to GB 4789.9-2014. The target colonies were resuspended in Brookfield broth from blood plates, fresh glycerol broth (25% final concentration) was added, and 0.025% FBP was added and stored in a-80 ℃ freezer. The purified strain can be identified by morphological characteristics, physiological biochemistry, molecular biology and the like

Example 2 identification of physiological and biochemical characteristics of Campylobacter jejuni Standard Strain

(1) Growth status of Selective Medium

Suspect colonies on mCCDA agar plates are usually pale grey, metallic shiny, moist, flat, and prone to diffuse growth. The first type of suspicious colonies on Skirrow blood agar plates are gray, flat, moist, glossy and prone to spread outwards along the inoculation line; the second type of suspicious colonies often appeared as single colonies with scattered bulges, with regular edges and shining, and the results are shown in fig. 1.

(2) Microscopic examination

And (3) dyeing microscopic examination: and smearing the suspicious colonies, performing gram staining, and observing the morphology by microscopic examination. All campylobacter jejuni were gram negative, stained red, slightly bent, comma-shaped, spore-and capsule-free, flagellated, and the results are shown in fig. 1.

(3) API Campy identification

Colonies were scraped from Campylobacter jejuni from skerrow plates, prepared into cell suspensions of appropriate turbidity with physiological saline, and identified using API Campy biochemical identification reagent strips, with the results shown in Table 1:

table 1: API Campy identification results

From the results of API Campy reaction in table 1, it can be seen that the selected strains are campylobacter jejuni, but from the typical value T, the characteristics thereof are greatly different from the standard strains selected by API, and the strains are strains having genetic background and typical characteristics in china, and therefore, the strains can be used as specific reference strains for campylobacter jejuni detection in china.

Example 3 identification of virulence factor-carrying characteristics of a standard strain of Campylobacter jejuni

Identifying virulence genes carried by the strain by a PCR method. The primers used were synthesized by Shanghai Biotechnology Limited (see Table 2 for primer sequences).

The PCR amplification system (25. mu.L) contained: 2 × Ferent PCR mix, 12.5 μ L; 0.4. mu.M of upstream and downstream primers; ddH₂O, 8.5. mu.L and genomic DNA, 2. mu.L. 8-10. mu.L of the PCR product was applied to a 2.0% agarose gel for electrophoretic separation (120V, 40min) using 2000pb DNA Marker.

Through PCR and gel electrophoresis confirmation, the strain GDMCC 60857 carries virulence factors:

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；GDMCC

60858 carry virulence factors:

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

TABLE 2 virulence associated gene primers

Example 4 drug susceptibility characterization of Campylobacter jejuni Standard Strain

After campylobacter jejuni was cultured in Brookfield broth for about 40 hours, OD was adjusted₆₀₀Sucking 100 mu L of bacterial liquid, coating the bacterial liquid on an MH plate containing 5% of sheep blood, sticking an antibiotic paper sheet on the surface of a culture medium after the bacterial liquid is dried, and culturing for 24 hours at 42 ℃. And measuring the size of the bacteriostatic zone by using a vernier caliper to be accurate to 0.01 mm. The antibiotics selected were as follows: cefoperazone, erythromycin, clindamycin, gentamicin, streptomycin, kanamycin, ciprofloxacin, naphthalenePyridine acid, amoxicillin, ampicillin, tetracycline and vancomycin. Escherichia coli ATCC25922 and ATCC25922 are 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 is CFP (cefoperazone) -CIP (ciprofloxacin) -TE (tetracycline) -NA (nalidixic acid) -VA (vancomycin); the drug resistance spectrum of GDMCC 60859 was CFP (cefoperazone) -DA (clindamycin) -AML (amoxicillin) -CIP (ciprofloxacin) -TE (tetracycline) -S (streptomycin) -K (kanamycin) -AMP (ampicillin) -NA (nalidixic acid) -VA (vancomycin).

Example 5 Multi-site sequence (MLST) typing analysis of Campylobacter jejuni Standard Strain

The 7 primers used were synthesized by Shanghai BioLimited, using the protocol described in the MLST official network (see Table 3 for primer sequences). The reagent for multi-site sequence typing is Takara

HS DNA Polymerase, reaction system for PCR total 50 μ L: 5 XPrimeSTAR Buffer (Mg2+ plus) 10. mu.L, dNTP mix 4. mu.L, primers 1. mu.L each, template 1. mu.L, PrimeSTAR HS DNA Polymerase (2.5U/. mu.l) 1. mu.L, with ultrapure water. And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 deg.C for 2 min; annealing at 50 deg.C for 1 min; stretching at 72 deg.C for 1 min; 35 cycles are carried out; final extension at 72 ℃ for 10 min. After the PCR was completed, gel electrophoresis was performed to confirm that the product was correct in size, and the product was subjected to bidirectional sequencing by Zemer Feishell technology (China) Co. And (3) carrying out matching analysis on the sequencing result and an MLST database, respectively obtaining allele numerical values of seven housekeeping gene loci, forming corresponding allele spectrums, and judging the sequence types of the alleles, wherein the typing results are shown in a table 4.

Table 3: campylobacter jejuni MLST typing primer sequence

Table 4: MLST typing results

The results of the type ST using the 7 housekeeping genes for MLST typing are shown in Table 4, in which ST9558 and 9560 are both new types of ST.

Example 6 signature sequence analysis of Campylobacter jejuni Standard Strain

The non-essential genes specific to the strain are mainly obtained according to the pan-genomic analysis result of the campylobacter jejuni. The genome sequences of 74 strains of campylobacter jejuni (including GDMCC 60857, GDMCC 60858 and GDMCC 60859) are selected for pan-genome analysis. The Pan-genome is analyzed by adopting a GF method in prokaryotic Pan-genome automated Analysis software (PGAP), and the Analysis result is processed by a local Perl script to obtain the core gene and non-core gene information of all strains.

Extracting a specific non-core gene protein sequence of the campylobacter jejuni, respectively comparing the protein sequence with a total protein library and an NCBI (non-redundant protein) database (NR) of the campylobacter jejuni through local Blast, removing sequences which can map to an NR library, wherein the rest sequences are specific sequences. The specific sequence of GDMCC 60857_00919 is shown as SEQ ID NO. 1, and the specific sequence of GDMCC 60858_02046 is shown as SEQ ID NO. 2. Primers were designed for these specific sequences using Primer 5, and primers were synthesized from Shanghai (see Table 5 for Primer sequences), and the reaction conditions and system thereof are shown in tables 6 and 7. The specificity of the specific gene is tested by PCR amplification, including the specificity of the molecular sequence in Campylobacter jejuni, the specificity in other strains, and the specificity in other mechanism preserving strains, and the specific PCR result is shown in the attached figures 2-4.

A special drug-resistant gene island is found in a strain GDMCC 60859, the drug-resistant island carries 9 drug-resistant related genes, the drug-resistant island is found in campylobacter jejuni for the first time, and the specific information is shown in a specific sequence SEQ ID NO. 3.

Table 5: specific target site primer

TABLE 6 PCR reaction System for specific tags

Note: a used Mix is Thermofisiher K8081

TABLE 7 PCR reaction procedure for identification of specific molecular tag molecules

The invention also provides a freeze-drying protective agent for quantitatively storing campylobacter jejuni, and the components of the freeze-drying protective agent in different embodiments and comparative examples are shown in table 8:

table 8: components of each group of freeze-drying protective agent

The parts by weight of the skim milk powder were increased in the comparative example to make up the total amount due to its lack of components.

The freeze-drying survival rate of the freeze-dried strain (GDMCC 60857) of the protective agent in the embodiment 7-9 and the comparative example 1-3 is as follows:

after the recovery of the strains, inoculating the strains into a culture medium, culturing until a proper amount of the strains are selected and added into the protective agents of the examples 7-9 and the comparative examples 1-3 at the early stage of the logarithmic phase to the stationary phase, uniformly mixing, subpackaging the mixture into penicillin bottles, taking samples, diluting and counting to obtain the bacterial content A0 before freeze-drying. Transferring the half-stoppered split penicillin bottles into a freeze dryer for pre-freezing at the temperature of minus 40 ℃ for 3 hours, starting main drying for 20-25 hours, then entering an analysis drying stage for 6-8 hours, finishing drying, pressing the stoppered split penicillin bottles in a vacuum state, moving the split penicillin bottles out of the freeze dryer, automatically capping the split penicillin bottles, ensuring the complete vacuum state of the samples, and storing the split penicillin bottles at the low temperature of minus 20 ℃. The lyophilized samples were diluted and counted, the count result is the lyophilized bacteria content a, and the lyophilized survival rate is the percentage of a to a0, and the results are shown in table 9:

table 9: comparison of freeze-drying survival rates of different compositions of lyoprotectants

As can be seen from Table 9, the lyoprotectant in example 9 is the best example of the present invention, and therefore, comparative examples 1 to 3 are prepared by taking example 9 as a comparison object, and as a result, as shown in Table 9, the protective effects of comparative examples 1 to 3 are all worse than those of the examples due to the absence of one of the components of the lyoprotectant of the present invention, respectively, thereby illustrating the synergistic effect among potassium lactate, inositol and L-cysteine hydrochloride, which are components of the lyoprotectant of the present invention, and the absence of any one of the components cannot achieve the effect of the protectant of the present invention.

The freeze-drying stability of the freeze-dried strains of the protective agents in the examples 7 to 9 and the comparative examples 1 to 3 is compared, and the specific method is as follows:

quantitative quality control bacteria were prepared using different protective agents according to the preparation method of example 7, stored at-20 ℃, and 3 samples were taken every month to check the bacterial content according to the counting method described above. In order to better compare the effect of each protective agent in the long-term storage process, the number of viable bacteria before freeze-drying is calculated according to the freeze-drying survival rate of each protective agent when quantitative quality control bacteria are prepared, so that the bacteria content of each protective agent is about 3000 cfu/bottle after freeze-drying.

As can be seen from the attached figure 5, the bacteria content protected by the cryoprotectants of examples 7 to 9 did not change significantly after 12 months of storage, while the bacteria content protected by the cryoprotectants of comparative examples 1 to 3 significantly decreased after 2 months, and was close to 0 after 12 months.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

SEQUENCE LISTING

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<213> bacterium

<400> 3

ttagaaatccctttgagaatgtttatatacattcaaggtaaccagccaactaatgacaatgattcctgaaaaaagtaataacaaattactatacagataagttgactgatcaacttccataggtaacaacctttgatcaagtaagggtatggataataaaccacctacaattgcaatacctgttccctctgataaaaagctggtaaagttaagcaaactcattccagcaccagcttcctgctgtttcaagctacttgaaacaattgttgatataactgttttggtgaacgaaagcccacctaaaacaaatacgattataattgtcatgaaccatgatgttgtttctaaaagaaaggaagcagttaaaaagctaacagaaagaaatgtaactccgatgtttaacacgtataaaggacctcttctatcaacaagtatcccaccaatgtagccgaaaataatgacactcattgttccagggaaaataattacacttccgatttcggcagtacttagctggtaaacatctttcatcatataaggaaccatagagacaaaccctgctactgttccaaatataattcccccacaaagaactccaatcataaaaggtatatttttccctaatccgggatcaacaaaaggatctgttactttcctgatatgttttacaaatatcaggaatgacagcacgctaacgataagaaaagaaatgctatatgatgttgtaaacaacataaaaaatacaatgcctacagacattagtataattcctttgatatcaaaatgaccttttatccttacttctttctttaataatttcataagaaacggaacagtgataattgttatcataggaatgagtagaagataggaccaatgaatataatgggctatcattccaccaatcgctggaccgactccttctcccatggctactatcgatccaataagaccaaatgctttacccctattttcctttggaatatagcgcgcaactacaaccattacgagtgctggaaatgcagctgcaccagccccttgaataaaacgagccataataagtaaggaaaagaaagaatggccaacaaacccaattaccgacccgaaacaatttattataattccaaataggagtaaccttttgatgcctaattgatcagatagctttccatatacagctgttccaatggaaaaggttaacataaaggctgtgttcacccagtttgtactcgcaggtggtttattaaaatcatttgcaatatcaggtaatgagacgttcaaaaccatttcatttaatacgctaaaaaaagataaaatgcaaagccaaattaaaatttggttgtgtcgtaaattcgattgtgaataggatgtattcacatttcaccctccaataatgagggcagacgtagtttatagggttaatgatacgcttccctcttttaattgaaccctgttacattaattacacttcataattaattcctcctaaacttgattaaaacattttaccacatataaactaagttttaaagtcagtatttcatcacttatacaacaatatggcccgtttgttgaactactctttaataaaataatttttccgttcccaattccacattgcaataatagaaaatccatcttcatcggctttttcgtcatcatctgtatgaatcaaatggttctgttgcaaagttttaaatctactatcaaataaggtagaataatagaaaaagatagcaggaggaatgacgatgaatcattttaaaggaaagcaatttcagcaggatgtgattattgtagccgtgggctactatcttcgttataaccttagctatcgtgaagttcaagaaatcttatatgatcgtggcattaacgtttctcatacgacgatttatcgttgggtgcaagaatatggcaaactactctatcaaatttggaaaaagaaaaataaaaaatccttttattcctggaaaatggatgaaacgtacatcaaaattaaaggaaaatggcattatttgtatcgagccatcgatgcagatggtttaaccttggatatttggttacgtaaaaaacgggacacacaagcagcctatgcttttcttaagcggttagtgaagcagtttgatgaaccgaaggttgtagtcacagataaagccccctctattacaagtgcctttaagaaactaaaagaatacggcttttatcaagggacagaacatcgtaccattaaatacctgaataatttgattgaacaagaccatcgtccagtaaagagacgcaataaattctatcgaagtttacgcactgcctctaccacgattaaaggtatggaagccatccgaggattatataagaaaacccgaaaagaaggcactctcttcgggttttcggtctgtactgaaatcaaggtattattgggaatcccagcttaaatcatagataccgtaagggattttattctttatttaaaactttgcaacagaaccgcataactcaaaaattacataacttccaattctctcatcaactgttcccattcattaaaataattaccctgacaccttttaacatcatcaattgatttttgtatttccgtcagtactatataatcagaagaattgtttggttcagaaataattttattcaatctttctaattcttcatcatattgatttattaacacttcaagtttttcaattcgtttctccttcttaaccttctccttctcaatggagttacgatcgccttttactgtgttatttttctctactttctgctcaactttaatgttattaaatggcttttcagaattcatcctttttttctcgtactcaccataagtagattgtacaaactctacaccatccttatcaaaaacaagtaatctgtcagcaatcttattagtaaaatatctatcatgtgacacaaaaataattgtaccttgataactgcataagatattctctaaattctcttttccaataatatccatgtggtttgtcggttcatctaagattaaaacattagtacgtttatataataatttacataatgtcaatctaaccttttctccacctgacaaagaggacaccggtctaaaaacatcatctccactaaactgaaatgagccaagagcagttctgacctctgtgtcatttagctcagggtattcgctttgaaaaatttcgaatagtgtatcatctccactgatttgagcaagctgttggtcaaaatagctaatttcaacattgtatccgaatttaaaatctccagacaatgctgccacaccacccataagtgttttaagcaacgtggatttaccaataccattactcccaacaattccaagcttctgtcccctttcaaggttgaaattaacctttgcaagaggagtatcatagcctatcacaagttctgaagcacttaatacttgccttgaactactgattctcggttgaaatttagacatataagtttttgtatcgtattggtctggtgcatttaatatttgcatacgctgtaataatttaattttagattgcaccattttagctttcgtaggtttataacgaaaacgttcaatcaagcgtgtaatcctttcaatttctatctgttgcaagtcgtaatctttttgctgtttgatatgattttctcgtttttgctcctcaaaggctgaataattacctttataacatttggtctctccccattcgatttcataaaccttatccacaattcgattaagaaacattctatcatgggaaataataaccaatgtagatttataacttctcaaataactctccaaccattgtattgtttctatatcaaggtggttagtaggttcatcaagtaatagaatgtctggctttgttaaaagtatttttataaaagctatcttagttcgctgaccacctgaaaattcagaaatgggttttttatcatctgcttcagtaaaacccatactacgaatcatcgtttcatattctttttggtaggttagacctccaagagccatatacctttcactgatatcagagtattcattgatgattttatcatcatattgattctccattttatctatcagctgtttcatcttgttttccatatcaataagcgtcttaaataccgtacggacttcatccaccattgatatactttcatcttcaaaaggcatctgtcttaaatagctaatataagggttaccggtctttattacttgaaactcactttcaccagttccttcttctaattcaatttcgcctataatagcttttagcaaggttgtttttccacatccatttctaccaacaatagcaatcttttcattatcattgatttcaataccgatgttttccaacaccatattaccatcatagtaaacggcaccattaattattttgtattgcattttcatatcctcctttgcgttagtactagcaattgcaaaggtggctttggacaataattattctactccatgtaaagcttgtcacagccacctatacatggagttctttaaatgttactatagttgtagatttcatttgcttctacctcctatcgattattaatataattcaattgccaccttattgtaccaagtcgctagcgcaatggctagcctaagttttagcttacaccctttctggtaaaagtaaagtaactgatctgttatctcatctggcaccagattcccgataaaccctgctgttcccgtcaacagaaaccgcagccacactggaaaaagatacaaaaggattgccgcaattgattctttgcattagccatcagctttacaaatcactctttctatattcaaaaggcgtttttcctgtttcttttttaaacacggatgtgaagtaactttgtgagttaaatataagatattcactaatttctgatattgactgattcgtatataccaatagaaatttggcatatctgggtgagaaacgatcaccaatgttttcattgtttcatccttaacatactca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gacagaagcccagaaaggatttggcgggacttctatgtggatggtatattctgcttttctaagcggtctttccatatatataacagtaggctcttttatttctgcctccacatgatatttttcctcaaggatggcacaaatgacttccatctgcacattccccaaaaaagaaagtataatctcatgcgttgtagtatccacataatattttaaaagagggtcgccatctgaaatttctgtaagtgccccaagcaatatttcccgctgttcaggtttctttactgcaatcgttgtttggagcatagggagaggattttcaataaattttctctgcggcaatagctttgtatctccaagaacactatttagcttcaaaaactcattttgcaaaataacaatttccccggaataagccttatcaattttacataattcaccgtttgtcggaacatacatatctgtgatttttattttctcttttccgatattctaaccgaatcccgcaaatgcaggattccgccataaaggcgtacatatgcaagacgttgtctttcttccgaatattcaatcttaaaaacctgcccgcatagttcagattgaccttcaggcgttgatgaataaaatttactggcaattacttctataagctgccgactccccagattgtttttagcgcttccgtgataaacgggaaataacgttccgttttggaatctcctgttttcttcctgttccagttctgacattttaaacggtttccctgacatatatttctctaatagttcatcgtttcccataattaccgcatcccactgttccatatcgtcattgtccgttacatttatatggggatgctgcccaaccttttgcttcactataatttccgaagaaagctttgctttcatttcccgatataccattggcaaatcaatcccctcttggtcaattttattgatgaaaaaaattgtcggaatcttcattatctgtagtgcatgaaacagtatacgggtctgtgcctgtatgccatcctttgcagaaactaataatactgctccgtctaatacggataaagaacggtatacttccgccaaaaaatccatatggcctggcgtatctataatgttgacttttacatcctcccactgaaaagatgtcactgctgtctggatagtgattcccctttgacgctccaaattcattgtatctgtccttgttgtgccttcatctacgctccctagttctgcaattgcaccactggtatacaataaactttccgttaatgttgtctttcctgcgtcaacgtgagccagaatgcctaagttaattattttcat

<210> 4

<211> 20

<212> DNA

<213> Synthesis

<400> 4

ccaagactta acaagtaagc 20

<210> 5

<211> 20

<212> DNA

<213> Synthesis

<400> 5

tagacgttga ctaaattgag 20

<210> 6

<211> 20

<212> DNA

<213> Synthesis

<400> 6

gcatatgaag aaagtaaagg 20

<210> 7

<211> 20

<212> DNA

<213> Synthesis

<400> 7

tctagattca tttgagcatt 20

<210> 8

<211> 20

<212> DNA

<213> Synthesis

<400> 8

GAAGGCACTC TCTTCGGGTT

<210> 9

<211> 21

<212> DNA

<213> Synthesis

<400> 9

AGGTGGCTGT GACAAGCTTT A

Claims

1. Specific molecular targets for detecting campylobacter jejuni, wherein the molecular targets are:

(b) the nucleotide sequence in (a) is subjected to substitution, deletion or addition of one or more nucleotides, and has more than 90% homology with the nucleotides in (a).

2. The primer for detecting the specific molecular target according to claim 1, wherein the PCR primer for the nucleotide sequence amplification shown as SEQ ID NO. 1 comprises: an upstream primer shown as SEQ ID NO. 4 and a downstream primer shown as SEQ ID NO. 5; the PCR primer for the nucleotide sequence amplification shown as SEQ ID NO. 2 comprises: an upstream primer shown as SEQ ID NO. 6 and a downstream primer shown as SEQ ID NO. 7; the PCR primers for the amplification of the nucleotide sequence shown as SEQ ID NO. 3 include an upstream primer shown as SEQ ID NO. 8 and a downstream primer shown as SEQ ID NO. 9.

3. A Campylobacter jejuni (Campylobacter jejuni) which is (a), (b) or (c):

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

(b) strain 3853-1A contains a nucleotide sequence shown as 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 said strain 346-1C further comprises the following virulence genes: cdtB, cdtC, ciaB, pldA, flig, dnaJ, racR, cadF, cdtA, docA, imaA, rpon, and ceuE; and resistance to the following antibiotics: cefoperazone, ciprofloxacin, tetracycline, nalidixic acid and vancomycin.

5. The campylobacter jejuni of claim 3, wherein 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 resistance to the following antibiotics: cefoperazone, ciprofloxacin, tetracycline, nalidixic acid and vancomycin.

6. The campylobacter jejuni of claim 3, wherein said 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 resistance to the following antibiotics: cefoperazone, clindamycin, amoxicillin, ciprofloxacin, tetracycline, streptomycin, kanamycin, ampicillin, nalidixic acid and vancomycin.

7. The campylobacter jejuni of claim 3, wherein strain 346-1C has the deposit number: GDMCC 60857; the preservation number of the strain 3853-1A is as follows: GDMCC 60858; the strain 542-1A has a accession number GDMCC 60859.

8. Use of campylobacter jejuni according to claim 3 for resistance to antibiotics of campylobacter jejuni. Preferably, the antibiotic resistance of strain 346-1C is resistance to cefoperazone, ciprofloxacin, tetracycline, nalidixic acid and vancomycin; the antibiotic resistance of the strain 3853-1A is the resistance to cefoperazone, ciprofloxacin, tetracycline, nalidixic acid and vancomycin; the antibiotic drug resistance of the strain 542-1A is drug resistance to cefoperazone, clindamycin, amoxicillin, ciprofloxacin, tetracycline, streptomycin, kanamycin, ampicillin, nalidixic acid and vancomycin.

9. Use of a campylobacter jejuni according to claim 3 for improving accuracy of testing a chromogenic plate for campylobacter jejuni.

10. The campylobacter jejuni freeze-drying protective agent is characterized by comprising the following components in parts by weight: 2-7 parts of fetal bovine serum, 5-20 parts of skimmed milk powder, 0.2-5 parts of potassium lactate, 1-4 parts of inositol and 0.1-2 parts of L-cysteine hydrochloride.