CN112795669B

CN112795669B - Yersinia enterocolitica standard strain containing specific molecular target and detection and application thereof

Info

Publication number: CN112795669B
Application number: CN202011615514.XA
Authority: CN
Inventors: 王涓; 吴清平; 丁郁; 陈惠元; 张菊梅; 陈谋通; 薛亮; 吴诗; 叶青华; 代京莎; 徐环; 庞锐; 曾海燕; 雷涛; 古其会; 韦献虎; 张友雄; 张淑红; 杨小鹃; 刘鸣
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: 2022-05-20
Anticipated expiration: 2040-12-30
Also published as: WO2022141943A1; CN112795669A

Abstract

The invention provides three standard strains which can be used as a reference for experiments related to the Yersinia enterocolitica (Yersinia enterocolitica), wherein the standard strains have typical colonial morphology and physiological and biochemical characteristics of the Yersinia enterocolitica, can be used for checking the accuracy of a selective culture medium of the Yersinia enterocolitica, and can be used as a reference sample in the detection of common physiological and biochemical phenotypes and molecular characteristics of the Yersinia enterocolitica; the standard strain contains a corresponding specific molecular target (such as a nucleotide sequence shown in SEQ ID NO: 1-11), and also provides a specific PCR amplification primer containing the specificity of the molecular target. The invention also provides a freeze-drying protective agent for the yersinia enterocolitica, and the freeze-drying survival rate of the protective agent can reach more than 85 percent; the strain can be preserved at the temperature of 20 ℃ below zero to ensure that the magnitude of the strain does not change for more than one year, so the strain can be used for storing the quality control strain for a long time.

Description

Yersinia enterocolitica standard strain containing specific molecular target and detection and application thereof

Technical Field

The invention belongs to the technical field of microbiological examination, and particularly relates to a standard strain of yersinia enterocolitica; physiological, biochemical and molecular characteristics of the strain; corresponding molecular target and PCR amplification primer and a method for preserving yersinia enterocolitica.

Background

Yersinia enterocolitica (y.enterocolitica), a gram-negative zoonosis pathogen, is widely distributed in nature and is a major cause of yersinia disease. Since it can grow in a low temperature environment (0-4 ℃), frozen and refrigerated foods have recently become a new important source of infection of the disease. The main symptoms of yersinia disease are gastroenteritis, pseudoappendicitis, diarrhea and the like, and a small part of patients can suffer from chronic diseases such as reactive arthritis and the like due to infection. The monitoring data of the European Union food safety agency 2013 and 2017 show that the number of the affected people of the yersinia disease is third in the livestock-related diseases within the range of the European Union, and the number of the affected people is in an increasing trend along with the improvement of a monitoring and reporting system of the European Union member states. Yersinia enterocolitica now found can be divided into six groups according to biotype: 1A, 1B and 2-5. Of these, the 1A biotype strain has been considered to be a nonpathogenic strain in the past because it does not contain the important virulence plasmid pYV, but with the progress of research, Yersinia enterocolitica of the 1A biotype has been isolated in more and more cases of Yersinia disease, and thus Yersinia enterocolitica of the 1A biotype is considered to have a potential pathogenic potential in mammals. At present, China has defects in monitoring yersinia disease, and the pollution of yersinia enterocolitica in food temporarily lacks detailed information. Therefore, the establishment of the standard bacterial strain of the yersinia enterocolitica is of great significance for perfecting the monitoring, prevention and control of yersinia disease in China.

At present, the standard strains of yersinia enterocolitica are mainly derived from American Type Culture Collection (ATCC), China center for medical Culture of Microorganisms (CMCC), and the like. Most of the standard strains are from foreign clinical separation, and the pollution and spreading condition of the strains in food cannot be well reflected. Meanwhile, the characters of the main epidemic strains in foreign countries cannot be taken as representatives of the characters of the main epidemic strains in China due to different main epidemic strain types of yersinia enterocolitica in food in different countries and regions.

Disclosure of Invention

In order to solve the technical problems, the invention provides 3 food isolated strains of yersinia enterocolitica from China, which have typical physiological and biochemical characteristics of the yersinia enterocolitica and can better reflect the genetic background of the yersinia enterocolitica in China. Yersinia enterocolitica strain C009 is a frozen duck wing sample isolated from the carinitum region of guangzhou, china with a biotype of 1A and serotypes of O:5, the preservation number is: GDMCC60852, classification name: yersinia enterocolitica; yersinia enterocolitica strain Y802 was isolated from minced meat samples of shanghai city, china, and had a biotype of 1A and a serotype of O: and 8, the preservation number is: GDMCC60853, classification name: yersinia enterocolitica; yersinia enterocolitica strain C1702 was isolated from a beef sample from the first city of the guangdong province with a biotype of 1A, unknown serotype, and a deposit number of: GDMCC60855, classification name: yersinia enterocolitica. The yersinia enterocolitica strains C009, Y802, and C1702 obtained by the above isolation are all deposited in the Guangdong province collection of microorganisms at the 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: the preservation date is 2019, 10 months and 27 days.

The technical scheme adopted by the invention is as follows:

the invention provides three standard strains of yersinia enterocolitica (y. enterocolitica), which are characterized by being (a), (b) or (c):

(a) the strain C009 contains at least one nucleotide sequence shown as SEQ ID NO. 1-5;

(b) the strain Y802 contains at least one nucleotide sequence shown as SEQ ID NO. 6-10;

(c) strain C1702, containing the nucleotide sequence shown in SEQ ID NO. 11.

Preferably, said strain C009 further comprises the following virulence genes: inv, ystB, ymoA, hrep, sat, fepA, fes, fepD and myfA; and resistance to the following antibiotics: ampicillin, cephalothin and sulfamethoxazole.

Preferably, said strain Y802 also comprises the following virulence genes: ystB, ymoA, fes, sat, inv, hrep and fepA; and resistance to the following antibiotics: ampicillin, cephalothin and cefotaxime.

Preferably, said strain C1702 further comprises the following virulence genes: ystB, ymoA, fes, sat, inv, hrep and tccC; and resistance to the following antibiotics: ampicillin, cephalothin and sulfamethoxazole.

Preferably, the strain C009 has the deposit number: GDMCC 60852; the preservation number of the strain Y802 is as follows: GDMCC 60853; the deposit number of the strain C1702 is GDMCC 60855.

The invention also provides application of the yersinia enterocolitica in researching the antibiotic resistance of the yersinia enterocolitica. Preferably, the antibiotic resistance of strain-1 is resistance against ampicillin, cephalothin or sulfamethoxazole; the antibiotic drug resistance aimed by the strain-2 is the drug resistance of ampicillin, cephalothin or cefotaxime; the antibiotic drug resistance of the strain-3 is the drug resistance of ampicillin, cephalothin or sulfamethoxazole.

The invention also provides application of the yersinia enterocolitica in improving the accuracy of the yersinia enterocolitica chromogenic plate.

The invention also provides a freeze-drying protective agent for preparing quantitative yersinia enterocolitica, which comprises the following components in parts by weight: 1-3 parts of sodium glycerophosphate, 6-12 parts of skimmed milk powder, 0.1-4 parts of polyvinylpyrrolidone, 0.1-2 parts of reducing glutathione and 0.2-2 parts of L-cysteine hydrochloride.

Preferably, the lyoprotectant comprises the following components in parts by weight: 2 parts of sodium glycerophosphate, 8 parts of skim milk powder, 0.5 part of polyvinylpyrrolidone, 0.5 part of reducing glutathione and 2 parts of L-cysteine hydrochloride.

The invention also provides a specific molecular target for detecting yersinia enterocolitica, which is characterized in that the molecular target is:

(a) 1-11 of any one of the nucleotide sequences shown in SEQ ID NO; or,

(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). The specific molecular target of the invention is obtained by comparing the newly obtained yersinia enterocolitica through pan-genome analysis, and the yersinia enterocolitica can be specifically detected, and has stronger specificity.

The invention provides a primer for detecting the specific molecular target as claimed in claim 1, which is characterized in that a PCR primer for amplifying the nucleotide sequence shown as SEQ ID NO. 1 comprises: an upstream primer shown as SEQ ID NO. 12 and a downstream primer shown as SEQ ID NO. 13; the PCR primer for the nucleotide sequence amplification shown as SEQ ID NO. 2 comprises: an upstream primer shown as SEQ ID NO. 14 and a downstream primer shown as SEQ ID NO. 15; the PCR primers for the amplification of the nucleotide sequence shown as SEQ ID NO. 3 comprise an upstream primer shown as SEQ ID NO. 16 and a downstream primer shown as SEQ ID NO. 14; the PCR primers for the amplification of the nucleotide sequence shown as SEQ ID NO. 4 comprise an upstream primer shown as SEQ ID NO. 18 and a downstream primer shown as SEQ ID NO. 19; the PCR primer for the amplification of the nucleotide sequence shown as SEQ ID NO. 5 comprises an upstream primer shown as SEQ ID NO. 20 and a downstream primer shown as SEQ ID NO. 21; the PCR primers for the amplification of the nucleotide sequence shown as SEQ ID NO. 6 comprise an upstream primer shown as SEQ ID NO. 22 and a downstream primer shown as SEQ ID NO. 23; the PCR primers for the amplification of the nucleotide sequence shown as SEQ ID NO. 7 comprise an upstream primer shown as SEQ ID NO. 24 and a downstream primer shown as SEQ ID NO. 25; the PCR primer for the amplification of the nucleotide sequence shown as SEQ ID NO. 8 comprises an upstream primer shown as SEQ ID NO. 26 and a downstream primer shown as SEQ ID NO. 27; the PCR primers for the amplification of the nucleotide sequence shown as SEQ ID NO. 9 comprise an upstream primer shown as SEQ ID NO. 28 and a downstream primer shown as SEQ ID NO. 29; the PCR primers for the amplification of the nucleotide sequence shown as SEQ ID NO. 10 comprise an upstream primer shown as SEQ ID NO. 30 and a downstream primer shown as SEQ ID NO. 31; the PCR primers for the amplification of the nucleotide sequence shown in SEQ ID NO. 11 include an upstream primer shown in SEQ ID NO. 32 and a downstream primer shown in SEQ ID NO. 33.

The invention has the beneficial effects that: the yersinia enterocolitica GDMCC60852, GDMCC60853 and GDMCC60855 have typical colony morphology and physiological and biochemical characteristics of the yersinia enterocolitica and can be used for testing the accuracy of the selective medium for the yersinia enterocolitica. The biological types and serotypes of the strains are clear, the carried virulence genes and the drug resistance spectrum are clear, and compared with the currently commonly used yersinia enterocolitica standard strains, the strains are the only strains which can accurately reflect the genetic background of the yersinia enterocolitica in food in China, so the strains can be used as the standard strains for scientific research and detection.

In addition, the yersinia enterocolitica inventors of the present invention provide a lyoprotectant having the following advantages: the molding is good, the appearance is beautiful, the water solubility is good, and the complete dissolution can be realized within 1 to 2 seconds; the freeze-drying survival rate can reach more than 85 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 colony morphology chart of Yersinia enterocolitica standard strain GDMCC60852 (gram stain, CIN plate, modified Y plate results from left to right).

FIG. 2 is a colony morphology chart of Yersinia enterocolitica standard strain GDMCC60853 (gram stain, CIN plate, modified Y plate results from left to right).

FIG. 3 is a colony morphology chart of Yersinia enterocolitica standard strain GDMCC60855 (gram stain, CIN plate, modified Y plate results from left to right).

FIG. 4 is a graph showing the molecular signature verification result of Yersinia enterocolitica standard strain GDMCC60852 (wherein the specificity of 5 specific genes of the strain is shown respectively).

FIG. 5 is a diagram showing the molecular signature verification result of Yersinia enterocolitica standard strain GDMCC60853 (wherein the specificity of 5 specific genes of the strain is shown respectively).

FIG. 6 is a diagram showing the results of molecular signature verification of Yersinia enterocolitica standard strain GDMCC60855 (wherein the specificity of 1 specific gene of the strain is shown respectively).

FIG. 7 is a graph showing the variation of bacteria content in the shelf life of different compositions of lyoprotectants.

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 and culture of Yersinia enterocolitica strains

Thoroughly shearing collected food samples under the aseptic condition, weighing 25g of the samples, diluting by 10 times, enriching by using PBS, and carrying out shake culture at 22-25 ℃ for 2-3d or standing culture for 5 d. Then, the mixture is treated by alkali (0.5 mL enrichment broth +4.5mL KOH) for 20 seconds, inoculated to CIN agar and cultured for 24-48 h at 30 ℃.5 representative colonies were picked and inoculated into nutrient agar and cultured at 30 ℃ for 24 hours. Adding the bacterial liquid into 25% glycerin tube under aseptic condition, and storing in-80 deg.C refrigerator. The purified colonies can be subjected to subsequent screening experiments as well as biochemical identification and biotyping.

Example 2 physiological and biochemical characteristics and serotype analysis of Yersinia enterocolitica Standard strains

Modified krebs test: 3 to 5 suspicious colonies obtained by separation are respectively selected and inoculated on the improved Ke-Di-saccharide iron agar, the slant surface is firstly scribed during inoculation, then the bottom layer is punctured, the culture is cultured for 24 hours at the temperature of 26 +/-1 ℃, and the culture which turns yellow and does not produce gas on the slant surface and the bottom is subjected to further biochemical identification.

Colony morphology: using modified Y plate culture, the colony morphology of Yersinia enterocolitica standard strain was a round, pink colony with bile precipitation rings at the edges (results are shown in FIGS. 1-3).

Urease test and kinetic observations: the suspected culture obtained by a full-loop modified Klebsiella biosciences test is selected by an inoculating loop and inoculated into a urea culture medium, the inoculation amount is enough, the culture is shaken for a few seconds, and the culture is carried out for 2 h-4 h at the temperature of 26 +/-1 ℃. The positive colonies of the urease test are respectively inoculated in two-tube semi-solid culture medium and cultured for 24h at 26 +/-1 ℃ and 36 +/-1 ℃. The suspicious bacteria culture with power at 26 ℃ and no power at 36 ℃ is streaked and inoculated to a nutrient agar plate for purification culture, and the purified product is used for gram staining microscopy and biochemical test.

And (3) dyeing microscopic examination: and smearing the suspicious colonies, performing gram staining, and observing the morphology by microscopic examination. All Yersinia enterocolitica are gram negative, rod-shaped or spherical, with size (0.8-3.0 μm). times.0.8 μm, no spores, no capsules, and periflagella.

API20E identifies: bovine-eye-shaped single colonies were scraped from the modified Y-plate, prepared into cell suspensions of appropriate turbidity using physiological saline, and identified using API20E biochemical identification test strips (results are shown in tables 1 to 3).

Table 1: yersinia enterocolitica standard strain GDMCC60852 API identification result

Table 2: yersinia enterocolitica standard strain GDMCC60853 API identification result

Table 3: yersinia enterocolitica standard bacterium GDMCC60855 API identification result

The strain has typical enterocolitis Yersinia biochemical characteristics: in the biochemical identification of API20E, the identification percentage of yersinia enterocolitica GDMCC60852 was 98.3% and the T value was 0.79. The identification percentage of yersinia enterocolitica GDMCC60853 was 95.4%, and the T value was 0.64; the percentage of identification of yersinia enterocolitica GDMCC60855 was 98.3% and the T value was 0.71.

Serotype analysis: five sera of O1, 2, O3, O5, O8 and O9 were used for agglutination test, and physiological saline was used for negative control. If the strain is self-coagulated in physiological saline to form a rough strain, the strain cannot be typed. 10 mu L of parting serum is taken and dropped on a sterilized empty culture dish, a proper amount of colonies are picked from an NA plate (or TSA plate) and smeared in the serum uniformly, the serum agglutination condition is observed, if the agglutination appears, the serotype is positive, if the agglutination appears, the serum test is carried out again, if the agglutination appears, O1 and 2 serum tests are carried out, and if the agglutination appears, O1 and 2 serum tests are carried out, if the agglutination appears, O3 serum tests are carried out. The identification result shows that the serotype of the standard strain GDMCC60852 is: o is 5; the standard strain GDMCC60853 serotype was: o is 8; the standard strain GDMCC60855 serotype is unknown.

Example 3 virulence factor carrying characteristics of Yersinia enterocolitica Standard strains

Identifying virulence genes carried by the strain by a PCR method. The primer used is limited by Shanghai biological organismSynthesis (primer sequences are shown in table 4). The PCR amplification system (25. mu.L) included: 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 1.5% agarose gel for electrophoretic separation (130V, 35 min) using 2000pb DNA Marker.

Table 4: primer sequences and amplified fragments for detecting enterocolitis yersinia virulence genes.

The enterocolitis yersinia standard strain carries virulence genes as follows:

virulence genes carried by the standard strain GDMCC60852 are: ystB-ymoA-fes-sat-inv-hrep-fepA-fepD-myfA.

Virulence genes carried by the standard strain GDMCC60853 are: ystB-ymoA-fes-sat-inv-hrep-fepA.

Virulence genes carried by the standard strain GDMCC60855 are: ystB-ymoA-fes-sat-inv-hrep-tccC.

Example 4 drug susceptibility characterization of Yersinia enterocolitica Standard strains

The drug sensitivity characteristic determination of the yersinia enterocolitica adopts a drug sensitivity paper sheet method, and the specific operation is as follows: activating Yersinia enterocolitica operating agar plate, adding physiological saline to dilute to final concentration of 1 × 10⁷cfu/mL is coated on an MH plate, after the bacterial liquid is dried, an antibiotic paper sheet is attached to the surface of the culture medium, and the culture is carried out for 24 hours at 37 ℃. And measuring the size of the inhibition zone by using a vernier caliper to be accurate to 0.01 mm. The antibiotics selected were as follows: ampicillin (AMP, 10. mu.g), cephalothin (CF, 30. mu.g), amoxicillin clavulanic acid (AMC, 30. mu.g), cefotaxime (CTX, 5. mu.g), ceftazidime (CAZ, 30. mu.g), chloramphenicol (CHL 30. mu.g), kanamycin (K, 30. mu.g), gentamicinElement (GEN,10 μ g), streptomycin (10 μ g), sulfanilamide (SSS, 30 μ g), sulfamethoxazole (SXT, 25 μ g) tetracycline (TCY,30 μ g) sulfamethoxazole (SMZ, 25 μ g), imipenem (IMP,10 μ g), ticarcillin (75 μ g). The experiment used Escherichia coli ATCC 25922 as the quality control strain.

The drug resistance of yersinia enterocolitica standard strains is as follows:

the antibiotics tolerated by GDMCC60852 strain were: ampicillin (AMP,10 μ g); cephalothin (CF,30 μ g); sulfamethoxazole (SXT, 25. mu.g).

The antibiotics tolerated by GDMCC60853 strain were: ampicillin (AMP,10 μ g); cephalothin (CF,30 μ g); cefotaxime (CTX, 5. mu.g).

The antibiotics tolerated by GDMCC60855 strain were: ampicillin (AMP,10 μ g); cephalothin (CF,30 μ g); sulfamethoxazole (SXT, 25. mu.g).

Example 5 Multi-site sequence (MLST) typing analysis of Yersinia enterocolitica Standard strains

The primers used in this experiment were synthesized by Ongchoma BioLimited, Guangzhou (primer sequences are shown in Table 5) using the protocol required by the MLST official network. Reagents used for fragment amplification include: PrimeSTAR Max DNA Polymerase (Takara).

Table 5: yersinia MLST PCR amplification primer table

Note: rfaE was used exclusively for amplification of O:3 serotype yersinia enterocolitica.

The PCR reaction system is 50 μ L: 2 × PrimeSTAR Max DNA Polymerase 25 μ L; 1 mu L of each upstream primer and downstream primer; 1 mu L of DNA template; the volume of the solution was made up to 50. mu.L with ultrapure water.

The PCR reaction conditions are as follows: first stage-pre-denaturation at 95 ℃ for 5 min; second stage-denaturation at 94 ℃ for 30 seconds, annealing for 30 seconds (temperature depends on primer), extension at 72 ℃ for 30 seconds; third stage-extension at 72 ℃ for 5 minutes, and finally 4 ℃. Wherein the second phase is repeated 30 times.

After the PCR amplification was completed, the product was subjected to agarose gel electrophoresis to determine the size of the product. The correct size product was sequenced (Ongzhou Onghama Biotechnologies, Inc.). And after the sequencing result is determined to be good, searching the corresponding MLST type by using the sequencing result.

The results of yersinia enterocolitica standard strain MLST are shown in table 6:

table 6: yersinia enterocolitica Standard Strain MLST typing

Example 6 signature sequence analysis of Yersinia enterocolitica Standard Strain

The screening of the characteristic sequences of the yersinia enterocolitica GDMCC60852, GDMCC60853 and GDMCC60855 is mainly based on the result of the pan-genomic analysis of the yersinia enterocolitica to screen the non-essential genes specific to the strain. The genome sequences of 253 representative yersinia enterocolitica strains (containing the yersinia enterocolitica strain of CMCC 52204) were selected for pan-genomic analysis. The Pan-genome Analysis adopts an MP 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. Then selecting the specific genes of the three strains, and removing non-specific sequences through local Blast comparison. Primers are designed aiming at the finally selected specific sequences, PCR amplification verification is carried out, and the verification result is shown in FIGS. 4-6.

Yersinia enterocolitica GDMCC60852 has 5 unique genes, respectively numbered GDMCC60852_00002, GDMCC60852_00642, GDMCC60852_00654, GDMCC60852_ 02222 and GDMCC60852_ 02225, and its nucleotides are as shown in SEQ ID NO: 1-5, can be amplified and verified by the following primers:

table 7: molecular tag primer sequence of yersinia enterocolitica standard strain GDMCC60852

Yersinia enterocolitica GDMCC60853 has 5 unique genes, which are numbered GDMCC60853_01766, GDMCC60853_01769, GDMCC60853_01770, GDMCC60853_ 01773 and GDMCC60853_01774 respectively, and the nucleotides of the genes are shown in SEQ ID NO: 6-10, the amplification verification can be carried out by the following primers:

table 8: molecular tag primer sequence of yersinia enterocolitica standard strain GDMCC60853

Yersinia enterocolitica GDMCC60855 has 1 unique gene, is respectively numbered as GDMCC 60855-04389, and has nucleotide shown as SEQ ID NO:11, the following primers can be used for amplification verification:

table 9: yersinia enterocolitica standard strain GDMCC60855 molecular tag primer sequence

The invention also provides a freeze-drying protective agent for quantitatively storing the yersinia enterocolitica standard strain, and the components of the freeze-drying protective agent in different examples and comparative examples are shown in the table 10:

table 10: 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 60852) of the protective agents in examples 7 to 9 and comparative examples 1 to 3 is tested by the following specific method:

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 freeze-dried samples were diluted and counted, the count result is the freeze-dried bacterial content A, and the freeze-drying survival rate is the percentage of A to A0, and the results are shown in Table 11:

table 11: comparison of freeze-drying survival rates of different compositions of lyoprotectants

As can be seen from Table 11, the lyoprotectant in example 8 is the best example of the present invention, and therefore, comparative examples 1 to 3 are prepared by taking example 8 as a comparison object, and as a result, as shown in Table 11, 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 in comparison with the other examples, which indicates that the components of the lyoprotectant in the present invention, namely sodium glycerophosphate, polyvinylpyrrolidone and reduced glutathione, have a synergistic effect, and the absence of any one of the components cannot achieve the effect of the protectant in comparison with the other examples.

The freeze-drying stability of the freeze-dried strains of the protective agents in the embodiments 7-9 and the comparative examples 1-3 is compared, and the specific method comprises the following steps:

quantitative quality control bacteria were prepared using different protective agents according to the preparation method of example 7, stored at-20 ℃, and 3 were extracted every month to check the bacterial content according to the aforementioned counting method. 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 7, the bacteria content of the quality control bacteria protected by the freeze-drying protective agents of the embodiments 7 to 9 has no significant change after 12 months of storage, while the bacteria content of the quality control bacteria protected by the freeze-drying protective agents of the comparative examples 1 to 3 is significantly reduced after 2 months, and the bacteria content is 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

<110> Guangdong institute for microbiological analysis and detection (Guangdong center for microbiological analysis and detection) Kyork Biotech Co., Ltd

<120> Yersinia enterocolitica standard strain containing specific molecular target, detection and application thereof

<130> 12.11

<160> 33

<170> PatentIn version 3.3

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<211> 2652

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caaatactca aaatattgcg tcgctatttc attgcagaag accgcgaaga gctgggtaaa 1320

aacctaacag cgcgcttcaa ggtggagtac atcaaaccca gtaaggggac ggccacttcc 1380

tacgcagcca aatacatcag caaaaatatt gacggttacg cactggaggg ggaaatcgac 1440

cacgaaaccg gccgcccgct ggcagaaacc gcccgccgcg ctatggcgtg ggcatccacc 1500

caccgtatcc gccaatacca gtttgtagga acaccaccgg taacaccgta tcgggagcta 1560

cgcaaactta gtaaccggat ggaagcgcaa ctgaaacacg cccgccagtg gaagcccgga 1620

caatctttgt taaccgatac agccatggac agggtgatgg ccgcagccga tgccgggtgt 1680

tttgccagct acatcaaggc acagggaggc gttctcattc cccgcaagga ttacaccgtc 1740

gcactggcct atgagcttgc cgataaaccc aatgcttacg gtgaaatcac ccccaaaatc 1800

tttggtatct ggtcacccaa taaaggggaa gacaccaaaa tctgtacgcg gcaaaagacc 1860

ttcactatcc gcaaaaaaga gaagccaaaa accgtcgacc ttatggagaa gttaggcacc 1920

ccgtgcggca ccgccgcccc ttggagtcct gtcaataact cgacagagga gaaaaaacag 1980

ccagaaaatg acgaaaacca caccacacaa gaaaccggtg aagagcagga aatcaccgac 2040

tttaacacca ttacccacaa acagcgccgc gcattgctca agcgattgcg acaaatgcca 2100

cctgtttgca ccaaagcccc aattgccacc gcaccagacg aattactggc cgcccaacgt 2160

gcacaggaaa aacgccagca ggccgcccaa cgccgtcagc accggctgca cctgctcaat 2220

acccatctgg aagatatcgg ggaaaacctg cccccgctgg cgcgcaccat tctggccgat 2280

ggcggcagct attacaccac cacacgctgc tatcgtctga ccgaacgcgg ggaactgctt 2340

tgcctgccct atcgtccacg tccggataac cagcaaaaag cggcactgtt caatgaacgc 2400

gtctcacgca tcaatcaaat actgacacag cacaacgctg caccgctgtc acaggaaagt 2460

tcactggcgg agaaaatagc ccgacataat gatttactgc aaaaacacac ccaggtaccg 2520

ctggctattc agccagtaac cacaaaacca acaccggagg aaaaacaaca agcccgaaac 2580

gtggcgcact ttgcccagaa aatgcgccac ctgtttctgc agttcaaagc aaagcaacac 2640

agtgctgact aa 2652

<210> 2

<211> 906

<212> DNA

<213> bacterium

<400> 2

atgcctaagt ataaaatgga tatattgaac aatgctattg atagtctcaa tgaatctctt 60

gataaatacg atcagggaca ggcaggtaat ataaggcaac ataaatttgc attacttcat 120

ttttgtcatt tcatggagtt agcccttaaa tactatttaa caacaaaaaa tgaaaatcta 180

atctataaaa aggtatatat ttatattaaa aataaagcta agagtgaaaa gatttctcta 240

tcagatgcat atgacaaatt agaagatgaa gattttgatt tcaatgagtt acttgttggt 300

gactctaatc cattcacaat aacagcagat caagcattag cctttattaa atcagacgat 360

gcaggtattg atgatgaact tatttcagag atatctgcta tgaaacaact gagaaataac 420

attgaacact gtcgttttga gatggatact aaagatgttc gattagctct aggccggtta 480

actcgtggat ttgatcaatt ctatgaatat gttggtttcg gtgagttaca acattcagta 540

aataaagcac agttgggtat atttcaaact ttagccaatg aatatgatca taacttatcc 600

gaagcgaaag cagatgctca ggaagctcac agggatgcat ttcgcggggt taggctgaag 660

cattatgaat tcgttaactt cacgacttat gattgtcatg aatgtaatca aactaaccta 720

atgataccta atgaagaatc tccttcagga tatcgttgta cgcattgtgg caacgaagag 780

tctgacgata ttgaggttga atgtgaaatt tgtggtggaa tttggcctaa tggggacatg 840

tgttcttggg aggatacata tgattatact tgccctgact gcaatgactt ctctagcaga 900

gattga 906

<210> 3

<211> 1800

<212> DNA

<213> bacterium

<400> 3

atgaataaga tagggaatgt gatttccagt tctcctgaac ttataagaat tcagatggaa 60

ggaaaagata ctttagaaaa acataagaca caactgcaag ttggttcctt tttaaaaata 120

gaagatggaa accataatta tgctgttgca acaataaaaa atcttactgg acaatacaat 180

gaaaaagaag atggacctaa ttggatattt ataatagagg catctcctat aggtgtccta 240

accaatgaaa atgatttgtt aattttttcc agaggaacac aagtacttcc tgtcccaaca 300

gaattagtgt atacatttga agataatgac ttgaaatcaa ttttctctga aggaggtggt 360

gattttaatt ttagaatagg tagcttatct aataatcaga aaatcccttt ttatatagat 420

ggcgacaaat tttttagcaa acatattggt gttgttggtt ctacagggtc agggaagtcc 480

tgcgcagttg caagtttgat tcaaaatatt gtaggcatat ctgaatcaaa aaatattttt 540

aaaggcagta ttaagaactc tcacgtattg atatttgata ttcattctga atactcttca 600

gcatttacta tggaaagaga agaagagttt agtttgaatg ttttaactgt tgataacttg 660

tgtttacctt actggcttat gaattcagag gaaattgagt ctttatttat tgaaagtaat 720

gagcaaaatt ctcacaatca agtatctcag ttaaaacgtg ctgttatttt aaataaagaa 780

aaattcaacc ctaaagtaga taatataacg tatgacagtc cagtctattt tagcattaat 840

gaagtttata attatatctg caataaaaat gatttaaccg tttatgaaaa agattcaaag 900

ctatatctag ccacattcga ttttgatgaa gaatataaag aggacttgct tttttcaaaa 960

ataaactttg aaaaagccac aggaaatgca aaacataaaa tatttgatga aaaagtatct 1020

aaatatggtg gtttcaccgg cgatttcgaa agattcatat cgagatttga aacaaaacta 1080

catgatagac gacttaagtt tcttttatct ccagaattac cccctttaaa agagataaga 1140

tctaaagatt ttgaaacgat attaaaacaa ttcttaggat ataataacaa ggctaacatt 1200

tcgatagtag atttgagtgg aattccgttt gaagttttga gcttaacagt tagtttaatt 1260

tcaagattaa tgtttgattt tgcatttcac tattcaaaaa tgatgcattc aaaaaacaaa 1320

acaaatgata taccgttctt aatagtatgc gaggaagccc ataattacat acctaaagat 1380

aactcttttg aatttaaagc atcacgaaaa tctatcgaac gtattgctaa agaaggaagg 1440

aaatatggtt taagcctaat ggtggtaagc cagcgacctt ccgaagtttc cgatacaata 1500

ttttcccaat gtaataactt tttatcattg agacttacaa ataagaacga tcaaggatat 1560

ataaaagcat tattgccaga tggtgccaat gggttagttg atttattgcc tagtttagga 1620

caaggcgagg cctttattgt tggagatgcg attttaatgc ctagtttagt aaaattacct 1680

cgcccttccc cagaaccaag ctcagcaagt ataaaagttt ataaggaatg gaactcagtt 1740

tggaaagaag ctgaatttaa taagcttgtc gaacgatggg gtaaaaatag tgataattaa 1800

<210> 4

<211> 1107

<212> DNA

<213> bacterium

<400> 4

atgtcaattt cacttgaact gcaaaaaatt gctgataatt taaccgcatt tgagaataat 60

gaattagatg agattgatca tttaatcgat ataacagaag aaatatctaa gtcattttca 120

ggttcatggt ttggctatca ttctcgagtc tattacagag atttcaacac tcctccatca 180

ggtgccatat ttagtgctga atgggggtta atggattcat tctcaatggg gtcggttggt 240

gattgggtgg aataccagta tgatgccgtt attaattata ttcataatga atggaatgat 300

aatgagttag atgaatatat aaaaatttct aataaggcaa actcaatatt cgaagtgtgt 360

aaaagtgaca ctttatctct tatttattca aataaagaga ctcttgatga agataagttc 420

ttgatggatt taatcgagaa aatagaaaaa acaaaagtga ttgttgaaaa ccaatttata 480

agttattacc gaccaaatcg tcaaataatg actcgtgata tgaatgcgat aaacaatggt 540

ttacaaacgc cacctcatat cgcagtgtat tgcaaggcaa tgagtataaa atcaccatat 600

atgtcttgtg gagaattaaa ggtaaatctg ataaagcttt ctaatcatat taaaaataaa 660

gaaaagaaat cagttattga agagagaatc ggtgtgaata ttttcatcgg tcatggacgc 720

tctcatatgt ggagagagct taaagatttt gtccaggata agctaagact accttatgat 780

gaatttaatc gtgttccagt ggccggagtt acaaatataa ctcggttagc tcaaatgctg 840

gatcaagcat gcattgcctt tttaatcatg acagctgaag acgagttact ggatggcaat 900

aagcaagcta gaatgaacgt tattcatgaa gtaggattat ttcaaggacg tctgggtttt 960

gagcgagcta tagtcttatt ggaagagggt tgtgaggagt ttacgaacat taatggtctt 1020

ggccaaataa gatttcctaa aggtaacatt tccgcagtat ttcaagatat tcgagaagtc 1080

ttggaacgag aggatataat aaagtag 1107

<210> 5

<211> 1389

<212> DNA

<213> bacterium

<400> 5

atgggaaatg ataagtccat ctgccatcaa tgtgtgggtg aaaagtttat cagtgccatg 60

attaaaaaaa agggtagtgc taatgatcgt tgtagctact gtaggagcag aagaaaaaca 120

atctctctgc ccgagttagc tgaatccatg cacacagtgt tttctaccta ttacagatgt 180

agagctgatg agggactcta tcccggctac agtttcggta atccagcgga ggatattatt 240

gctgaatgcc tcgaagtaga tgaagatatt tccgcagaga ttcatgaagc tctgaaggaa 300

aactataatg acgggtttga cggtgatgtt tacagcgagg attacgttta caaagaaaaa 360

acccactctt ccgaagctct ggataataaa tgggaagaga taaaaaagtc cctccagaac 420

gaagcacgat tttttaataa tcacgtaaaa gatttttttg ataaaatatt tagcggtatt 480

gaaacacacc ggacgcaaga cggcaggagc gcggttaatt atattaataa tgatatgcca 540

gtatttaggg cgagggtctt tgacagctat gacaaggtag aagaagcact tcaacatcct 600

gagagacact tcggcccgcc cccgcatttg ctagctactt cgggccgcat gaatgcgcaa 660

ggcattcccg tcttctacgg agcctcgtcc cctaatatcg ccatcgcaga agtaagacca 720

gcggtaggta gccttgtcgt cgtagcgcag ttcagccctt tgaagccctt acggattttg 780

gaaatttccg ctctggatga actgagtact atcaaaaata gtttattcga tcctcagacg 840

gctgaaaagt ttgccacggc ctgctttctg cgaaagcttt cccgcaaact gacactacca 900

gtttcaggga aaaaaactga cactgaatat ctcattactc aggctatttc cgaataccta 960

tccttatcag acatatataa acttgacgga atcagtttta aatcaacaca gcaaacctat 1020

gagaaaaatg aattctccac tccctacaac attgttctct tcaatagatc atcagcagtt 1080

ctgaatgcgg atgttcaaaa cacgagatat tcagtaaaca tttttgaata cgacgatgac 1140

gacgagacga gttattcctg gttggaacca gaaatacgga aaattgaaac cgtttctgat 1200

aaattatcca tatcccgtca acatatatcc ctgaaagact attcccttca gttaaatgcc 1260

agtgagatga ctttccatat aattaaaggg gttgtatacc agaagactga gtacccaatc 1320

aggctgggta aacctaagct tatgaaagca gatagctctc atgagttttt tggtgacgaa 1380

ccattttga 1389

<210> 6

<211> 756

<212> DNA

<213> bacterium

<400> 6

atgtcattcg ctaaatgccc tatctctggg accactaagc cagcccctca cagtgtccct 60

aagttcctga ttgacccatt gaccaatgtg ttcactacac gcagtgctct ctctaaagaa 120

tccagagcct atgctcgtat ctatggagca actgctatca ctggtactct ctataaagag 180

tcattacctc ctaaggcccc tacagttaag actaacagta acgctctgac ccatgaggac 240

attaagaccc gcttcgttat cagtgagtca tcaccctcag gtctcgcaga tattcgcagt 300

ggtaagcctg taggctctaa ggggtcttac tggcgtgtag ctgtaagtgc tggtgttacc 360

cagatggtcc ctgactatcg cactggtggc ctccgtaagg cctataagaa cctcacagcc 420

gcagctcacc gagtggtctt tatgttgaac cataagagag acattaaggt aggctgtatc 480

gttactcacg ttaatggtga ctcatatgac aatagaccct gtaacctcct tgaggtgtcc 540

cagagcgcca cagcgttgca ccgtaaggct tcccagagag ttagcgataa ggcctatgtt 600

ggagttggct acgtgaagcg cagagagtgc tatgtgggcc gctgtaaggt ccaaggtatc 660

atctattaca ctaagggcta taaggatatc aatatgtgtc ataacgcctt agtgaagctc 720

agagagactg taggcgcacc tgctgtagcc ctttaa 756

<210> 7

<211> 1158

<212> DNA

<213> bacterium

<400> 7

atgagtagca ataaattcac ttctgctggc aacaccaacg ctaacactcc acgagtaggc 60

aataaggcct ctaatctgtc cgagctgggc cgtcacctgc aagactctga tggtatggct 120

gtaccgaact ccgtatgggg tactgacatt gaccgcactg agtacgtgga gatggtcggt 180

gagaacacat ctatccttaa cttaggtcat acagaccgct caggtcgccc tatggcattc 240

ggtaatgact ctcatgacta tgtgcaagcc ttagaggcct ctctggatgc aacctttaac 300

gatggtgaat tcgctcaggt catgcaggag catattttcc cttgtgagtt ccgtccgttt 360

ggtacacagc ctaccccatt gaacatccag ttacttgaag accgctcagt gacctttaac 420

ccagagggta agctggaccg ctctaagttg cctaaaggta aagccattgc tggtagttca 480

atcatggtcg ctcctgtact tcatggtgac gataagaccg agacaggtat tctgtgctca 540

gtggctactc acgtatcaga ctttgatatg atgggcggtt ggactgatgg cagcttacgc 600

caaactgtag gtaacatgca gctgcaattc tgccgtgtcc tgtcaggcca agtagccaag 660

gtcctatcta agaccccaga cctacagacc cttgagtgtg aagctctaag cgttaaacct 720

gctgaggccg ctgaggacct gctggatgct ctggctacct ctatgcctac tcacttaggt 780

gctaccttgg acctatacgc cttgatggtc ccagagaagc tggaaggtgt cttagagcgt 840

gctgctcaac gtgcaggtca tgaagatgcc tctgagctgt tcggttgtac catcatgggt 900

tatgtaggtg aagatactgg tgtctacttg ctgcctaaag cattcgctat gatttctttc 960

cgttcaacca aagagggtga cacagttaag attgttatca ctcgtgacac taaccgctca 1020

ggctatgctg tagagatggt ctcagtgttg gacgttatgg caactggcac cgttaaggtt 1080

aaacatggtg agttcgataa ggaatccgta gcggagttcc cagtggtcca tcgcctgacc 1140

tttaaggcaa gctcatag 1158

<210> 8

<211> 411

<212> DNA

<213> bacterium

<400> 8

atgagtatct cattccgtaa attagaccta actgtatccg cagatggtca aaccgtcttg 60

gtgttcggcc aagagttcgc cgctaagtat ttctctgagg ttgtggtaac taccatgctg 120

aatagcgtag gtgacccatc attcaagtct aacaatatcc tcaataactt acatgctgct 180

ggccttaatg ctggtgacta tggggtttac tctaagttct ggggtatgag caatcaagaa 240

gctcgtgagc aagcagagca tgaacgccgt gaagcccaga aacacttgga gcgtgtgcgt 300

gacatgatgg caacacctgc tgacatcgct aaggaagcct ctaagaaacg tgagactgct 360

gaccgtatgg aacgtatgta cggacgtaaa ggtgcagctt tcggcctgta a 411

<210> 9

<211> 246

<212> DNA

<213> bacterium

<400> 9

atgattatcg acattattgc tatcagcctc atcttagtgg tcatgtatat ccgtagaagc 60

caatccgctg acctgcattg tagatactta cagctatcca gcttgggcta tgagtccata 120

gagctgatgt gtacagatga tagattcgct aaggcctaca gacacactaa gttactgaca 180

acggtcctaa ggattctcgt agttggctat gctgcctcat gggtggtaac tctgctcagt 240

cgatag 246

<210> 10

<211> 231

<212> DNA

<213> bacterium

<400> 10

atgtttacta accctatcac cttaatcatg accaccttca atggtcttat agtggtcctt 60

ggcttgtcta atggttccac tccaagtgta gccttaggcg ctggtctgtt cgccttactg 120

tttatcaatt gggtgactga cggtaactct aagttctggg agtccttggc tatgtggtcc 180

tacccgctca ccgtagcgtg tgcctttctg agcctgagct tggctttctg a 231

<210> 11

<211> 999

<212> DNA

<213> bacterium

<400> 11

atgatttttg agcttgcttt tctgtttttc tcttcattca ctaccttgtt tttaatgaga 60

aaagtcgcaa aacgcattgg tttggttgac aaacccaatg caagaaaact acacgaggga 120

gcgattcctc ttgtcggcgg aatttctatc acattggtgt tagctcaatt tcttatttat 180

aaacctgatc taatcaagca cagcgggtta tttattattt caattatggt gctgacttta 240

attggcgctc ttgatgataa atttgaccta agcgttcgcg tgcgtattgg ggtgcaaacg 300

gttctctctt tagtcatgat gcatattggc gggctagagc ttcaccacat tggtaatatt 360

ttaggctttg gtgatattta tctaggctgg ataggaagcg tcattaccat tatggcagtg 420

attggagcaa ttaatgcctt caatatggtg gatgggattg acggcctgtt aggcgggctt 480

tcgattgtga cttttggtgc attagccttc ttattgaaag tagacagcca acatgggttg 540

gcttatctat gcgtggtgat tattgtggcg atgctacctt atatttgtat gaacctcggc 600

atattaggac gtaaacgtaa agtctttatg ggtgatgcgg gaagcatgat gataggcttt 660

actgtgattt ggatgctatt aggcgtcagc caagttgatg caaaaccact catgcgacca 720

gttacggctt tatggcttat cgcggtaccg ctcatggaca tgactgcaat tatgatccgt 780

cgcatacgac gcggagactc accatttaaa ccagacagag aacatttgca tcatatttgt 840

cagcgtttag gccttagctc tcgacaaaca ctagtagcca tttgctctgt tgccattctg 900

ttcgccagtt ttggtattta cggtgaagcg gttaatttaa ctgaagctgt catgttttat 960

agctttatcg gcagcttttt atttactcac tactattaa 999

<210> 12

<211> 19

<212> DNA

<213> Synthesis

<400> 12

atggccaaca gacatcaca 19

<210> 13

<211> 21

<212> DNA

<213> Synthesis

<400> 13

ttagtcagca ctgtgttgct t 21

<210> 14

<211> 19

<212> DNA

<213> Synthesis

<400> 14

tgcctaagta taaaatgga 19

<210> 15

<211> 21

<212> DNA

<213> Synthesis

<400> 15

agagaagtca ttgcagtcag g 21

<210> 16

<211> 21

<212> DNA

<213> Synthesis

<400> 16

agggaatgtg atttccagtt c 21

<210> 17

<211> 20

<212> DNA

<213> Synthesis

<400> 17

ttttacccca tcgttcgaca 20

<210> 18

<211> 21

<212> DNA

<213> Synthesis

<400> 18

tgtcaatttc acttgaactg c 21

<210> 19

<211> 21

<212> DNA

<213> Synthesis

<400> 19

tcctctcgtt ccaagacttc t 21

<210> 20

<211> 20

<212> DNA

<213> Synthesis

<400> 20

tccatctgcc atcaatgtgt 20

<210> 21

<211> 21

<212> DNA

<213> Synthesis

<400> 21

ggttcgtcac caaaaaactc a 21

<210> 22

<211> 21

<212> DNA

<213> Synthesis

<400> 22

tcattcgcta aatgccctat c 21

<210> 23

<211> 20

<212> DNA

<213> Synthesis

<400> 23

ttaaagggct acagcaggtg 20

<210> 24

<211> 20

<212> DNA

<213> Synthesis

<400> 24

attcacttct gctggcaaca 20

<210> 25

<211> 20

<212> DNA

<213> Synthesis

<400> 25

tgagcttgcc ttaaaggtca 20

<210> 26

<211> 21

<212> DNA

<213> Synthesis

<400> 26

tctcattccg taaattagac c 21

<210> 27

<211> 20

<212> DNA

<213> Synthesis

<400> 27

aaagctgcac ctttacgtcc 20

<210> 28

<211> 19

<212> DNA

<213> Synthesis

<400> 28

ttgctatcag cctcatctt 19

<210> 29

<211> 20

<212> DNA

<213> Synthesis

<400> 29

agcagagtta ccacccatga 20

<210> 30

<211> 21

<212> DNA

<213> Synthesis

<400> 30

ctaaccctat caccttaatc a 21

<210> 31

<211> 19

<212> DNA

<213> Synthesis

<400> 31

aaagccaagc tcaggctca 19

<210> 32

<211> 20

<212> DNA

<213> Synthesis

<400> 32

tgcatattgg cgggctagag 20

<210> 33

<211> 20

<212> DNA

<213> Synthesis

<400> 33

cagtcatgtc catgagcggt 20

Claims

1. Application of primer for detecting specific molecular target in preparation of yersinia enterocolitica: (Y. enterocolitica) The application of the strain detection reagent is characterized in that the specific molecular targets are as follows:

1-10 of any one or more nucleotide sequences shown in SEQ ID NO;

wherein, the primer for detecting the specific molecular target shown in SEQ ID NO. 1 is used for preparing the yersinia enterocoliticaY. enterocolitica) The detection reagent of the strain C009 with the collection number GDMCC 60852;

wherein, the primer for detecting the specific molecular target shown in SEQ ID NO. 2 is used for preparing the yersinia enterocoliticaY. enterocolitica) The detection reagent of the strain C009 with the collection number GDMCC 60852;

wherein, the primer for detecting the specific molecular target shown in SEQ ID NO. 3 is used for preparing the yersinia enterocoliticaY. enterocolitica) The detection reagent of the strain C009 with the preservation number of GDMCC 60852;

wherein, the primer for detecting the specific molecular target shown in SEQ ID NO. 4 is used for preparing the yersinia enterocolitica: (yersinia enterocolitica)Y. enterocolitica) The detection reagent of the strain C009 with the collection number GDMCC 60852;

wherein, the primer for detecting the specific molecular target shown in SEQ ID NO. 5 is used for preparing the yersinia enterocoliticaY. enterocolitica) The detection reagent of the strain C009 with the collection number GDMCC 60852;

wherein, the primer for detecting the specific molecular target shown in SEQ ID NO. 6 is used for preparing the yersinia enterocoliticaY. enterocolitica) The detection reagent of the strain Y802 with the collection number of GDMCC 60853;

wherein, the primer for detecting the specific molecular target shown in SEQ ID NO. 7 is used for preparing the yersinia enterocoliticaY. enterocolitica) The detection reagent of the strain Y802 with the collection number of GDMCC 60853;

wherein SEQ ID NO 8 is detectedPrimers specific for the molecular targets shown for the preparation of Yersinia enterocolitica: (Y. enterocolitica) The detection reagent of the strain Y802 with the collection number of GDMCC 60853;

wherein, the primer for detecting the specific molecular target shown in SEQ ID NO. 9 is used for preparing the yersinia enterocoliticaY. enterocolitica) The detection reagent of the strain Y802 with the collection number of GDMCC 60853;

wherein, the primer for detecting the specific molecular target shown in SEQ ID NO. 10 is used for preparing the yersinia enterocoliticaY. enterocolitica) The detection reagent of the strain Y802 with the collection number of GDMCC 60853.

2. The use according to claim 1,

the primer aiming at detecting the specific molecular target shown as SEQ ID NO. 1 comprises: an upstream primer shown as SEQ ID NO. 12 and a downstream primer shown as SEQ ID NO. 13;

the primer aiming at detecting the specific molecular target shown as SEQ ID NO. 2 comprises: an upstream primer shown as SEQ ID NO. 14 and a downstream primer shown as SEQ ID NO. 15;

the primers for detecting the specific molecular target shown as SEQ ID NO. 3 comprise: an upstream primer shown as SEQ ID NO. 16 and a downstream primer shown as SEQ ID NO. 17;

the primers for detecting the specific molecular target shown as SEQ ID NO. 4 comprise: the upstream primer shown as SEQ ID NO. 18 and the downstream primer shown as SEQ ID NO. 19;

the primers for detecting the specific molecular target shown as SEQ ID NO. 5 comprise: an upstream primer shown as SEQ ID NO. 20 and a downstream primer shown as SEQ ID NO. 21;

the primers for detecting the specific molecular target shown as SEQ ID NO. 6 comprise: an upstream primer shown as SEQ ID NO. 22 and a downstream primer shown as SEQ ID NO. 23;

the primers for detecting the specific molecular target shown as SEQ ID NO. 7 comprise: an upstream primer shown as SEQ ID NO. 24 and a downstream primer shown as SEQ ID NO. 25;

the primers for detecting the specific molecular target shown as SEQ ID NO. 8 comprise: the upstream primer shown as SEQ ID NO. 26 and the downstream primer shown as SEQ ID NO. 27;

the primers for detecting the specific molecular target shown as SEQ ID NO. 9 comprise: an upstream primer shown as SEQ ID NO. 28 and a downstream primer shown as SEQ ID NO. 29;

the primer aiming at detecting the specific molecular target shown as SEQ ID NO. 10 comprises: the upstream primer shown as SEQ ID NO. 30 and the downstream primer shown as SEQ ID NO. 31.