WO2022141944A1 - Standard strains of campylobacter jejuni containing specific molecular targets, examination therefor, and application thereof - Google Patents
Standard strains of campylobacter jejuni containing specific molecular targets, examination therefor, and application thereof Download PDFInfo
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- WO2022141944A1 WO2022141944A1 PCT/CN2021/087079 CN2021087079W WO2022141944A1 WO 2022141944 A1 WO2022141944 A1 WO 2022141944A1 CN 2021087079 W CN2021087079 W CN 2021087079W WO 2022141944 A1 WO2022141944 A1 WO 2022141944A1
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- campylobacter jejuni
- strain
- seq
- gdmcc
- strains
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- 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.
- 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.
- Campylobacter jejuni 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.
- ATCC American Type Culture Collection
- CMCC Medical Microbiology Center
- 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.
- 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.
- 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.
- 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 present invention provides specific molecular targets for detecting Campylobacter jejuni, the molecular targets are:
- 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).
- the present invention provides primers for detecting the specific molecular target
- 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):
- strain 346-1C contains the nucleotide sequence shown in SEQ ID NO: 1;
- strain 3853-1A contains the nucleotide sequence shown in SEQ ID NO:2;
- Strain 542-1A contains the nucleotide sequence shown in SEQ ID NO:3.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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 °C 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.
- 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.
- 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.
- Listeria monocytogenes [LM19115, LM54002, LM54004
- Figure 5 is a schematic diagram of changes in the storage period of Campylobacter jejuni quantitative quality control bacteria.
- 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°C ⁇ 1°C for 24h ⁇ 48h under microaerophilic conditions.
- 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.
- 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.
- 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.
- 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 ⁇ L) contained: 2 ⁇ Ferment PCRmix, 12.5 ⁇ L; 0.4 ⁇ M upstream and downstream primers; ddH 2 O, 8.5 ⁇ L and genomic DNA, 2 ⁇ L. 8-10 ⁇ L of PCR products were loaded on 2.0% agarose gel for electrophoresis separation (120V, 40min), using 2000pb DNA Marker.
- the virulence factors carried by the strain GDMCC 60857 are:
- 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:
- 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.
- 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).
- the reagent for multilocus sequence typing is Takara's HS DNA Polymerase, total 50 ⁇ L of PCR reaction system: 10 ⁇ L of 5 ⁇ PrimeSTAR Buffer (Mg2+plus), 4 ⁇ L of dNTP Mixture, 1 ⁇ L of each primer, 1 ⁇ L of template, 1 ⁇ L of PrimeSTAR HS DNA Polymerase (2.5U/ ⁇ l), 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.
- 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.
- 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.
- 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 .
- 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:
- the weight part of skim milk powder was increased to make up the total amount due to its lack of components.
- 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:
- strains 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.
- the pre-freezing temperature is -40°C
- the time is 3 hours
- the main drying is turned on
- the time is 20-25 hours
- the analysis and drying stage is entered
- 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 °C low temperature.
- 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.
- 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:
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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
本发明属于微生物检验技术领域，具体涉及检测空肠弯曲菌特异性分子靶标以及含有该靶标的标准菌。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.
腹泻性疾病是世界范围内的一个重要公共卫生问题，每年因感染性腹泻死亡的人数超过200万，而在发展中国家，每年因腹泻性疾病死亡的人数高达1000万，给全球公共卫生行业造成了巨大经济损失，也给人民的生命健康构成了重要威胁。空肠弯曲菌是一种人畜共患病病原菌，可以引起人和动物发生多种疾病，并且是一种食物源性病原菌，被认为是引起全世界人类细菌性腹泻的主要原因。空肠弯曲菌广泛存在肉与肉制品中，尤其是禽肉中，是我国常见重要的食源性致病菌。空肠弯曲菌的致病机理以及其致病因素包括粘附、侵袭、产生毒素和分子模拟机制等四个方面，通过分子模拟机制可以引起最严重的并发症一格林一巴利综合征。空肠弯曲菌可以通过产生细胞紧张性肠毒素、细胞毒素和细胞致死性膨胀毒素而致病。了解该菌的流行特征及遗传进化是有效防控该菌所致的食源性疾病的基础，而是否使用了合适的标准菌株决定了研究结果的可靠性，目前空肠弯曲菌的标准菌株主要来源美国的美国微生物菌株保藏中心(American Type Culture Collection，ATCC)，我国医学微生物菌种中心(CMCC)并没有保存空肠弯曲菌。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.
2015年5月1日后，我国食品中空肠弯曲菌检验主要执行GB/T4789.9—2014《食品安全国家标准食品微生物学检验空肠弯曲菌检验》国家标准。空肠弯曲菌的鉴定主要采用传统的生化鉴定方法。主要通过氧化酶、过氧化氢酶、马尿酸盐水解和吲哚乙酸酯水解试验，进而确认是否为空肠弯曲菌。目前，我国仍缺乏代表性的食品分离株作为标准对照菌株，现用标准菌株大部分源于ATCC，ATCC的标准菌株一般是从临床上分离得到的，与食源性空肠弯曲菌菌株在致病能力和基因型都不同，且与中国分离得到的菌株在性状上有所差别，不能代表所有空肠弯曲菌的特性，更不能代表中国空肠弯曲菌的特征，尤其是食源性空肠弯曲菌。在中国CMCC网站上不能检索到弯曲菌属的任何菌株，具有中国本土来源的标准菌株资源十分缺乏，具有食品源的菌株更是缺乏，因此，构建国内具有代表性的空肠弯曲菌标准菌株是十分有必要的。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.
发明内容SUMMARY OF THE INVENTION
为解决上述技术问题，本发明提供了3株空肠弯曲菌中国地区的食品分离菌株，该菌株具有典型的空肠弯曲菌生理生化特性，且能较好地反映该菌在中国地区的遗传背景，能弥补中国地区无食源性空肠弯曲菌标准菌株可用的空缺。空肠弯曲菌菌株346-1C是分离于中国杭州市万寿亭农贸市场的鸽子肉样品，其保藏编号为： GDMCC 60857，分类名为：Campylobacterjejuni；空肠弯曲菌菌株3853-1A是分离于中国银川市北京华联南门广场店鸡肉样品，保藏编号为：GDMCC 60858，分类名为：Campylobacterjejuni；空肠弯曲菌菌株542-1A是分离于中国拉萨药王山农贸市场鸽子肉样品，其保藏编号为：GDMCC 60859，分类名为：Campylobacter jejuni。上述分离获得的空肠弯曲菌菌株346-1C、3853-1A、542-1A均保藏于广东省微生物菌种保藏中心，地址：中国广州市先烈中路100号省微生物所实验楼五楼，保藏日期：2019年10月27日。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.
本发明采用的技术方案为：The technical scheme adopted in the present invention is:
本发明提供了用于检测空肠弯曲菌的特异性分子靶标，所述分子靶标为：The present invention provides specific molecular targets for detecting Campylobacter jejuni, the molecular targets are:
(a)如SEQ ID NO:1～3所示的任意一种或几种核苷酸序列；或者，(a) any one or several nucleotide sequences shown in SEQ ID NOs: 1 to 3; or,
(b)在(a)中的核苷酸序列经过取代、缺失或添加一个或几个核苷酸且与(a)中核苷酸具有90％以上同源性的核苷酸序列。在发现3株新的空肠弯曲菌的同时还发现这三株菌分别含有一段特异的分子靶标，能使其区别与其他菌株，具有较强的特异性。(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.
本发明提供了检测所述的特异性分子靶标的引物，针对如SEQ ID NO:1所示的核苷酸序列扩增的PCR引物包括：如SEQ ID NO:4所示的上游引物和如SEQ ID NO:5所示的下游引物；针对如SEQ ID NO:2所示的核苷酸序列扩增的PCR引物包括：如SEQ ID NO:6所示的上游引物和如SEQ ID NO:7所示的下游引物；针对如SEQ ID NO:3所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:8所示的上游引物和如SEQ ID NO:9所示的下游引物。发明人针对上述特异性分子靶标设计了引物，能特异扩增出上述片段，因此，可作为检测上述分子靶标的特异性引物。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.
本发明还提供了一种空肠弯曲菌(Campylobacterjejuni)，是(a)、(b)或(c)：The present invention also provides a Campylobacter jejuni, which is (a), (b) or (c):
(a)菌株346-1C含有如SEQ ID NO:1所示的核苷酸序列；(a) strain 346-1C contains the nucleotide sequence shown in SEQ ID NO: 1;
(b)菌株3853-1A含有如SEQ ID NO:2所示的核苷酸序列；(b) strain 3853-1A contains the nucleotide sequence shown in SEQ ID NO:2;
(c)菌株542-1A含有如SEQ ID NO:3所示的核苷酸序列。(c) Strain 542-1A contains the nucleotide sequence shown in SEQ ID NO:3.
优选地，所述菌株346-1C还包含了以下毒力基因：cdtB、cdtC、ciaB、pldA、flig、dnaJ、racR、cadF、cdtA、docA、imaA、rpon和ceuE；并且对以下抗生素具有耐药性：头孢哌酮、环丙沙星、四环素、萘啶酸和万古霉素。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.
优选地，所述菌株3853-1A还包含了以下毒力基因：cdtB、cdtC、ciaB、pldA、flig、dnaJ、racR、cadF、cdtA、docA、imaA、rpon、flaA、wlaN和ceuE；并且对以下抗生素具有耐药性：头孢哌酮、环丙沙星、四环素、萘啶酸和万古霉素。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.
优选地，所述菌株542-1A还包含了以下毒力基因：cdtB、cdtC、ciaB、pldA、flig、dnaJ、racR、cadF、cdtA、docA、imaA、rpon、flaA和ceuE；并且对以下抗生素具有耐药性：头孢哌酮、克林霉素、阿莫西林、环丙沙星、四环素、链霉素、卡那霉素、氨苄西林、奈啶酸和万古霉素。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.
优选地，所述菌株346-1C的保藏编号为：GDMCC 60857；所述菌株3853-1A的保藏编号为：GDMCC 60858；所述菌株542-1A的保藏编号为GDMCC 60859。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.
本发明还提供了所述的空肠弯曲菌在研究空肠弯曲菌抗生素耐药性中的应用。优选地，所述菌株-1的抗生素耐药性为对头孢哌酮、环丙沙星、四环素、萘啶酸和万古霉素的耐药性；所述菌株-2的抗生素耐药性为对头孢哌酮、环丙沙星、四环素、萘啶酸和万古霉素的耐药性；所述菌株-3的抗生素耐药性为对头孢哌酮、克林霉素、阿莫西林、环丙沙星、四环素、链霉素、卡那霉素、氨苄西林、奈啶酸和万古霉素的耐药性。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.
优选地，所述抗生素包括以下抗生素中的任意一种或几种：头孢哌酮、环丙沙星、四环素、奈啶酸、万古霉素、链霉素、卡那霉素、氨苄西林、克林霉素或和阿莫西林。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.
本发明还提供了所述的空肠弯曲菌在提高检验空肠弯曲菌显色平板的准确性中的应用。The invention also provides the application of the said Campylobacter jejuni in improving the accuracy of testing the color plate of Campylobacter jejuni.
本发明还提供了一种用于制备定量空肠弯曲菌质控菌的冻干保护剂，包括以下重量份的组分：胎牛血清2～7份、脱脂奶粉5～20份、乳酸钾0.2-5份、肌醇1-4份、L-半胱氨酸盐酸盐0.1-2份。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.
优选地，包括以下重量份的组分：胎牛血清5份、脱脂奶粉20份、乳酸钾5份、肌醇2份、L-半胱氨酸盐酸盐1份。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.
本发明之所以选择上述组分是由于肌醇具有很强的亲水性，在冷冻或干燥过程中，可与菌体细胞膜磷脂中的磷酸基团或与菌体蛋白质极性基团形成氢键，保护细胞膜和蛋白质结构与功能的完整性，脱脂奶粉能够包裹在菌体细胞外层保护菌体。胎牛血清对菌悬液起到稳定作用，乳酸钾和L-半胱氨酸盐酸盐在冷冻干燥过程和长期储存中降低细胞氧化酶的活性，防止冻干品的氧化变质。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.
本发明的有益效果为：本发明空肠弯曲菌GDMCC 60857、GDMCC 60858、GDMCC 60859，具有标准的空肠弯曲菌的菌体显微形态和生理生化特征，可用于检验空肠弯曲菌显色平板的准确性，以及相关试剂验证的标准菌株。这些菌株为中国国内本土的食品来源菌株，且来源清晰可靠，遗传背景清晰。从API Campy反应的结果可见，所述菌株为空肠弯曲菌，但从典型值T可知，其性状和API所选择的标准菌株差别较大，是具有中国地区遗传背景和典型性状的菌株，因此这些菌株可以作为中国空肠弯曲菌检测的特异性的参考菌株。GDMCC 60857和GDMCC 60858具有特异性的分子靶标位点，可以使用PCR直接鉴定，具有快速、便宜、易操作等特点。GDMCC 60859具有一个较大的耐药岛，也可以通过PCR或者测序鉴定。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.
另外，针对本发明的空肠弯曲菌，发明人提供了一种空肠弯曲菌冻干保护剂，所述保护剂具有以下优点：成型好，外观漂亮且水溶性好，1-2秒内能够完全溶解；冻干存活率能达到40％以上；在-20℃条件下可以保存至少一年以上，能够保证其数量级不发生变化，可用于长期储存质控菌株。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.
图1为空肠弯曲菌在skirrow血平板和改良CCD平板上菌落形态图，及其革兰氏染色后镜检观察形态图；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;
图2为空肠弯曲菌中靶标位点具有特异性示意图(a：图中编号346-5和346-7能扩增出目的条带，其他编号的空肠弯曲菌菌株无目的条带，编号346-5和346-7为保藏号GDMCC 60857的菌株(菌株-1)；b：图中编号24(+)能扩增出目的条带，其他编号的空肠弯曲菌菌株无目的条带，编号24为保藏号GDMCC 60858的菌株(菌株-2)；c：图中“+”能扩增出目的条带，其他编号的空肠弯曲菌菌株无目的条带，编号“+”为保藏号GDMCC 60859的菌株(菌株-3)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)
图3为本发明保藏号GDMCC 60857的空肠弯曲菌菌株与其他种属相比具有特异性的示意图，图中显示其他菌属(包括苏云金杆菌、蜡样芽胞杆菌、大肠杆菌、小肠结肠炎耶尔森氏菌、金黄色葡萄球菌、单增李斯特氏菌、克罗诺杆菌、副溶血性弧菌和沙门氏菌)没有扩增出目的大小条带(279bp)，而本发明的空肠弯曲菌具有目的条带。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.
图4为本发明保藏号GDMCC 60857的空肠弯曲菌菌株与其他机构保存的标准菌株相比具有特异性的示意图，图中显示其他菌属(单核增生李斯特菌[LM19115、LM54002、LM54004]、金黄色葡萄球菌[Sa25923]、铜绿假单胞菌[Pa9027、Pa27853、Pa10104]、沙门氏菌[sal150335、Sal1402]、大肠杆菌[Ec25922]、小肠结肠炎耶尔森氏菌[Ye5402、JC2、Y802、C009]、蜡样芽胞杆菌[Bc2068]和空肠弯曲菌[Cj33291])没有扩增出目的条带，而本发明的空肠弯曲菌具有目的条带。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.
图5为空肠弯曲杆菌定量质控菌储存期内的变化示意图。Figure 5 is a schematic diagram of changes in the storage period of Campylobacter jejuni quantitative quality control bacteria.
为了更加简洁明了的展示本发明的技术方案、目的和优点，下面结合具体实施例和附图详细说明本发明的技术方案。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.
实施例1 空肠弯曲菌标准菌株的分离、鉴定和培养Example 1 Isolation, identification and cultivation of standard strains of Campylobacter jejuni
取25g(mL)样品(水果、蔬菜或水产品为50g)加入盛有225mLBolton肉汤的有滤网的均质袋中(若为无滤网均质袋可使用无菌纱布过滤)，用拍击式均质器均质1min～2min，经滤网或无菌纱布过滤，将滤过 液进行培养。在微需氧条件下，36℃±1℃培养4h，如条件允许配以100r/min的速度进行振荡。必要时测定增菌液的pH值并调整至7.4±0.2，42℃±1℃继续培养24h～48h。将24h增菌液、48h增菌液及对应的1:50稀释液分别划线接种于Skirrow血琼脂与mCCDA琼脂平板上，微需氧条件下42℃±1℃培养24h～48h。另外可选择使用空肠弯曲菌显色平板作为补充。观察24h培养与48h培养的琼脂平板上的菌落形态，空肠弯曲菌显色培养基上的可疑菌落按照说明进行判定。挑取5个(如少于5个则全部挑取)或更多的可疑菌落接种到哥伦比亚血琼脂平板上，微需氧条件下42℃±1℃培养24h～48h。按照GB4789.9-2014进行鉴定。将目标菌落从血平板上，用布氏肉汤进行重悬，加入新鲜的甘油肉汤(终浓度为25％)，并加入0.025％FBP，保存于-80℃冰箱。纯化后的菌株可进行形态特征、生理生化和分子生物学等鉴定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.
实施例2 空肠弯曲菌标准菌株的生理生化特征鉴定Example 2 Identification of physiological and biochemical characteristics of standard strains of Campylobacter jejuni
(1)选择性培养基生长状况(1) Growth status of selective medium
mCCDA琼脂平板上的可疑菌落通常为淡灰色，有金属光泽、潮湿、扁平，呈扩散生长的倾向。Skirrow血琼脂平板上的第一型可疑菌落为灰色、扁平、湿润有光泽，呈沿接种线向外扩散的倾向；第二型可疑菌落常呈分散凸起的单个菌落，边缘整齐、发亮，结果如图1所示。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)镜检(2) Microscopic examination
染色镜检：将可疑菌落涂片，进行革兰氏染色，镜检观察形态。所有空肠弯曲菌为革兰氏阴性，染色为红色，菌体轻度弯曲呈逗点状，无芽孢和荚膜，有鞭毛，结果如图1所示。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鉴定(3) API Campy identification
从空肠弯曲菌从skirrow平板上上刮取菌落，用生理盐水制备成浊度适当的细胞悬浮液，使用API Campy生化鉴定试剂条鉴定，鉴定结果如表1所示：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:
表1：API Campy鉴定结果Table 1: API Campy identification results
从表1 API Campy反应的结果可知，上述筛选出的菌株为空肠弯曲菌，但从典型值T可知，其性状和API所选择的标准菌株差别较大，是具有中国地区遗传背景和典型性状的菌株，因此这些菌株可以作为中国空肠弯 曲菌检测的特异性的参考菌株。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.
实施例3 空肠弯曲菌标准菌株的毒力因子携带特征的鉴定Example 3 Identification of virulence factor carrying characteristics of standard strains of Campylobacter jejuni
采用PCR的方法鉴定菌株携带的毒力基因。所使用的引物由上海生工生物有限公司合成(引物序列见表2)。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).
PCR扩增体系(25μL)包含：2×Ferment PCRmix，12.5μL；0.4μM上下游引物；ddH
2O，8.5μL和基因组DNA，2μL。8-10μL的PCR产物上样于2.0％琼脂糖凝胶进行电泳分离(120V，40min)，使用2000pb DNA Marker。
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.
经过PCR及凝胶电泳确认，所述菌株GDMCC 60857携带的毒力因子有：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；cdtB-cdtC-ciaB-pldA-flig-dnaJ-racR-cadF-cdtA-docA-imaA-rpon-ceuE;
GDMCC 60859携带的毒力因子为：cdtB-cdtC-ciaB-pldA-flig-dnaJ-racR-cadF-cdtA-docA-imaA-rpon-flaA-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携带的毒力因子为：The virulence factors carried by GDMCC 60858 are:
cdtB-cdtC-ciaB-pldA-flig-dnaJ-racR-cadF-cdtA-docA-imaA-rpon-flaA-wlaN-ceuE。cdtB-cdtC-ciaB-pldA-flig-dnaJ-racR-cadF-cdtA-docA-imaA-rpon-flaA-wlaN-ceuE.
表2.毒力相关基因引物Table 2. Virulence-related gene primers
实施例4 空肠弯曲菌标准菌株的药敏特征鉴定Example 4 Identification of drug susceptibility of standard strains of Campylobacter jejuni
空肠弯曲菌在布氏肉汤中培养约40小时后，调整OD
600为0.1～0.3，吸取100μL的菌液，涂布于含5％羊血的MH平板，待菌液干了之后将抗生素纸片贴在培养基表面，42℃培养24h。采用游标卡尺测定抑菌圈大小，精确至0.01mm。选用的抗生素如下：头孢哌酮、红霉素、克林霉素、庆大霉素、链霉素、卡那霉素、环丙沙星、萘啶酸、阿莫西林、氨苄西林、四环素、万古霉素。Escherichia coli ATCC25922和ATCC25922作为质控菌株。空肠弯曲菌GDMCC 60857的耐药谱为CFP(头孢哌酮)-CIP(环丙沙星)-TE(四环素)-NA(萘啶酸)-VA(万古霉素)；GDMCC 60858的耐药谱为CFP(头孢哌酮)-CIP(环丙沙星)-TE(四环素)-NA(萘啶酸)-VA(万古霉素)；GDMCC 60859的耐药谱为CFP(头孢哌酮)-DA(克林霉素)-AML(阿莫西林)-CIP(环丙沙星)-TE(四环素)-S(链霉素)-K(卡那霉素)-AMP(氨苄西林)-NA(奈啶酸)-VA(万古霉素)。
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).
实施例5 空肠弯曲菌标准菌株的多位点序列(MLST)分型分析Example 5 Multi-locus sequence (MLST) typing analysis of standard strains of Campylobacter jejuni
采用MLST官网所述的实验方案，所使用的7对引物由上海生工生物有限公司合成(引物序列见表3)。多位点序列分型的试剂为Takara的
HS DNA Polymerase，PCR的反应体系总50μL：5×PrimeSTAR Buffer(Mg2+plus)10μL，dNTP Mixture 4μL，引物各1μL，模板1μL，PrimeSTAR HS DNA Polymerase(2.5U/μl)1μL，加超纯水补齐。PCR反应条件：95℃预变性，5min；94℃变性，2min；50℃退火，1min；72℃延伸，1min；共进行35循环；最后72℃终延伸，10min。PCR结束之后进行凝胶电泳，确认产物大小正确，将产物交赛默飞世尔科技(中国)有限公司进行双向测序。将测序结果与MLST数据库中进行匹配分析，分别获取七个管家基因位点的等位基因数值，并形成相应的等位基因谱，判断其序列型，分型结果如表4所示。
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.
表3：空肠弯曲菌MLST分型引物序列Table 3: Sequences of primers for MLST typing of Campylobacter jejuni
表4：MLST分型结果Table 4: MLST typing results
使用上述7个管家基因进行MLST分型，其ST型结果如表4所示，其中ST9558以及9560均为ST新的型别。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.
实施例6 空肠弯曲菌标准菌株的特征序列分析Example 6 Characteristic sequence analysis of standard strains of Campylobacter jejuni
主要根据空肠弯曲菌的泛基因组分析结果获得菌株特有的非必需基因。共选取了74株空肠弯曲菌(含GDMCC 60857、GDMCC 60858、GDMCC 60859菌株)的基因组序列来进行泛基因组分析。泛基因组采用原核生物泛基因组自动化分析软件(Pan-GenomicsAnalysis Pipeline，PGAP)中的GF方法来分析，通过本地Perl脚本对分析结果进行处理，得到所有菌株的核心基因及非核心基因信息。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.
提取空肠弯曲菌特有的非核心基因蛋白序列，通过本地Blast分别将其比对回空肠弯曲菌的蛋白总库及NCBI非冗余蛋白数据库(NR)，去除能mapping到nr库的序列，剩余序列即为特异性序列。GDMCC 60857_00919的特异序列如SEQ ID NO:1所示，GDMCC 60858_02046的特异序列如SEQ ID NO:2所示。使用Primer 5针对这些特异性序列设计引物，由上海生工合成引物(引物序列见表5)，其反应条件和体系见表6和表7。特有基因通过PCR扩增检验其特异性，包括该分子序列在空肠弯曲菌中的特异性，其他菌株中的特异性，其他机构保存菌株中的特异性，具体PCR结果见附图2-4。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.
菌株GDMCC 60859中发现了较为特殊的耐药基因岛，该耐药岛携带有9种耐药相关基因，该耐药岛在空肠弯曲菌中首次发现，其具体信息见特异序列SEQ ID NO:3。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 .
表5：特异性靶标位点引物Table 5: Primers specific to target sites
表6.特异性标签的PCR反应体系Table 6. PCR reaction system for specific tags
注：a所使用Mix为Thermofisher K8081Note: a Mix used is Thermofisher K8081
表7 特异性分子标签分子鉴定PCR反应程序Table 7 PCR reaction program for molecular identification of specific molecular tags
本发明还提供一种空肠弯曲菌定量保存的冻干保护剂，不同实施例以及对比例中冻干保护剂的组分如表8所示：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:
表8：各组冻干保护剂的组分Table 8: Components of each group of lyoprotectants
在对比例中增加脱脂奶粉的重量份以补足由于其缺少组分的总分量。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.
将以实施例7～9与对比例1～3保护剂的冻干菌种(GDMCC 60857)其冻干存活率，具体方法如下：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:
将菌种复苏后接入培养基中培养至对数后期至稳定期前期选取合适的菌量加入到实施例7～9与对比例1～3保护剂中，混合均匀后分装至西林瓶中，并取样进行稀释计数，为冻干前含菌量A0。将分装好的西林瓶半加塞转入冻干机中进行预冻，预冻温度为-40℃，时间为3小时，开启主干燥，时间20-25h，之后进入解析干燥阶段，时间为6-8小时，结束干燥，在真空状态下压塞并移出冻干机，进行自动轧盖，保证样品的完全真空状态，并置于-20℃低温保存。取冻干后的样品进行稀释计数，计数结果为冻干后含菌量A，冻干存活率为A与A0的百分比，结果如表9所示：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:
表9：不同组成的冻干保护剂冻干存活率的比较Table 9: Comparison of lyophilized survival rates of lyoprotectants with different compositions
由表9可以看出，实施例9的冻干保护剂是本发明的最佳实施例，因此，以实施例9为比较对象，制备了对比例1～3冻干保护剂，结果如表9所示，由于对比例1～3分别缺少本发明冻干保护剂的其中一种组分，其保护效果均比实施例要差，由此说明本发明的冻干保护剂的中的组分乳酸钾、肌醇和L-半胱氨酸盐酸盐之间存在协同增效效应，缺少其中任何一种均不能达到本发明保护剂的效果。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.
将以实施例7～9与对比例1～3保护剂的冻干菌种比较其冻干稳定性，具体方法如下：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:
按照实施例7的制备方法使用不同的保护剂进行制备定量质控菌，并置于-20℃条件下储存，每个月抽取3支按照前述计数方法进行检验含菌量。为了更好的比较各保护剂在长期储存过程中的效果，在制备定量质控菌时按照各保护剂的冻干存活率进行计算冻干前的活菌数，使各保护剂冻干后，菌含量均约为3000cfu/瓶。各保护剂制备的定量质控菌经12个月储存后含菌量变化如下附图5所示。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.
由附图5可知，经12个月储存后由实施例7～9的冻干保护剂保护的含菌量无显著变化，而由对比例1～3冻干保护剂保护的含菌量在经2个月后菌含量显著下降，到12个月后菌含量接近0。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.
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。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)
- 用于检测空肠弯曲菌的特异性分子靶标，其特征在于，所述分子靶标为：A specific molecular target for detecting Campylobacter jejuni, characterized in that the molecular target is:(a)如SEQ ID NO:1～3所示的任意一种或几种核苷酸序列；或者，(a) any one or several nucleotide sequences shown in SEQ ID NOs: 1 to 3; or,(b)在(a)中的核苷酸序列经过取代、缺失或添加一个或几个核苷酸且与(a)中核苷酸具有90％以上同源性的核苷酸序列。(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).
- 检测如权利要求1所述的特异性分子靶标的引物，其特征在于，针对如SEQ ID NO:1所示的核苷酸序列扩增的PCR引物包括：如SEQ ID NO:4所示的上游引物和如SEQ ID NO:5所示的下游引物；针对如SEQ ID NO:2所示的核苷酸序列扩增的PCR引物包括：如SEQ ID NO:6所示的上游引物和如SEQ ID NO:7所示的下游引物；针对如SEQ ID NO:3所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:8所示的上游引物和如SEQ ID NO:9所示的下游引物。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.
- 一种空肠弯曲菌(Campylobacter jejuni)，其特征在于，是(a)、(b)或(c)：A kind of Campylobacter jejuni (Campylobacter jejuni) is characterized in that, is (a), (b) or (c):(a)菌株346-1C含有如SEQ ID NO:1所示的核苷酸序列；(a) strain 346-1C contains the nucleotide sequence shown in SEQ ID NO: 1;(b)菌株3853-1A含有如SEQ ID NO:2所示的核苷酸序列；(b) strain 3853-1A contains the nucleotide sequence shown in SEQ ID NO:2;(c)菌株542-1A含有如SEQ ID NO:3所示的核苷酸序列。(c) Strain 542-1A contains the nucleotide sequence shown in SEQ ID NO:3.
- 如权利要求3所述的空肠弯曲菌，其特征在于，所述菌株346-1C还包含了以下毒力基因：cdtB、cdtC、ciaB、pldA、flig、dnaJ、racR、cadF、cdtA、docA、imaA、rpon和ceuE；并且对以下抗生素具有耐药性：头孢哌酮、环丙沙星、四环素、萘啶酸和万古霉素。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.
- 如权利要求3所述的空肠弯曲菌，其特征在于，所述菌株3853-1A还包含了以下毒力基因：cdtB、cdtC、ciaB、pldA、flig、dnaJ、racR、cadF、cdtA、docA、imaA、rpon、flaA、wlaN和ceuE；并且对以下抗生素具有耐药性：头孢哌酮、环丙沙星、四环素、萘啶酸和万古霉素。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.
- 如权利要求3所述的空肠弯曲菌，其特征在于，所述菌株542-1A还包含了以下毒力基因：cdtB、cdtC、ciaB、pldA、flig、dnaJ、racR、cadF、cdtA、docA、imaA、rpon、flaA和ceuE；并且对以下抗生素具有耐药性：头孢哌酮、克林霉素、阿莫西林、环丙沙星、四环素、链霉素、卡那霉素、氨苄西林、奈啶酸和万古霉素。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.
- 如权利要求3所述的空肠弯曲菌，其特征在于，所述菌株346-1C的保藏编号为：GDMCC 60857；所述菌株3853-1A的保藏编号为：GDMCC 60858；所述菌株542-1A的保藏编号为GDMCC 60859。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.
- 如权利要求3所述的空肠弯曲菌在空肠弯曲菌抗生素耐药性中的应用。优选地，所述菌株346-1C的抗生素耐药性为对头孢哌酮、环丙沙星、四环素、萘啶酸和万古霉素的耐药性；所述菌株3853-1A的抗生素耐药性为对头孢哌酮、环丙沙星、四环素、萘啶酸和万古霉素的耐药性；所述菌株542-1A的抗生素耐药性为对头 孢哌酮、克林霉素、阿莫西林、环丙沙星、四环素、链霉素、卡那霉素、氨苄西林、奈啶酸和万古霉素的耐药性。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.
- 如权利要求3所述的空肠弯曲菌在提高检验空肠弯曲菌显色平板的准确性中的应用。The application of the Campylobacter jejuni according to claim 3 in improving the accuracy of testing the color plate of Campylobacter jejuni.
- 一种空肠弯曲菌冻干保护剂，其特征在于，包括以下重量份的组分：胎牛血清2～7份、脱脂奶粉5～20份、乳酸钾0.2-5份、肌醇1-4份、L-半胱氨酸盐酸盐0.1-2份。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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