WO2022141937A1 - 含有特异性分子靶标的沙门氏菌标准菌株及其检测和应用 - Google Patents

含有特异性分子靶标的沙门氏菌标准菌株及其检测和应用 Download PDF

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WO2022141937A1
WO2022141937A1 PCT/CN2021/087010 CN2021087010W WO2022141937A1 WO 2022141937 A1 WO2022141937 A1 WO 2022141937A1 CN 2021087010 W CN2021087010 W CN 2021087010W WO 2022141937 A1 WO2022141937 A1 WO 2022141937A1
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salmonella
fscc
seq
enterica
enteritidis
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PCT/CN2021/087010
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French (fr)
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叶青华
吴清平
杨小鹃
张菊梅
丁郁
陈谋通
薛亮
陈惠元
王涓
代京莎
吴诗
曾海燕
徐环
古其会
雷涛
庞锐
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广东省科学院微生物研究所(广东省微生物分析检测中心)
广东环凯生物科技有限公司
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Definitions

  • the invention relates to the field of bioengineering, in particular to a Salmonella standard strain containing a specific molecular target and its detection and application.
  • Salmonella (Salmonella spp.) is one of the most common food-borne pathogens, and the food poisoning cases caused by it are often ranked first in the world. Because of its low nutritional requirements, it can survive in feces, soil, food (meat, eggs, milk, bread, etc.), and water for as long as 5 months to 2 years, and there is no obvious sensory change after contaminating the food, so it is very easy to Infectious diseases caused by animal and human consumption, such as animal diseases such as typhoid fever, pullorum, swine cholera, and infectious food poisoning such as human gastroenteritis, typhoid fever, septicemia, and colds.
  • animal diseases such as typhoid fever, pullorum, swine cholera
  • infectious food poisoning such as human gastroenteritis, typhoid fever, septicemia, and colds.
  • the initial symptoms of human infection with Salmonella are nausea, vomiting, diarrhea, fever, etc. If not treated in time, the mortality rate is as high as 10%. Under normal circumstances, the consumption of Salmonella more than 10 5 CFU can cause human infection, and for highly susceptible people 15-20 CFU can cause Salmonella infection. Therefore, Salmonella is the key monitoring target of food-borne pathogens. Understanding the transmission law and pathogenic 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 strain is used is used to determine the reliability of the research results. At present, the standard strains of Salmonella are mainly from international or domestic culture collection centers, and the genetic characteristics have been confirmed, guaranteed and traceable, such as American Type Culture Collection (ATCC), my country Medical Microorganisms Center (CMCC) et al.
  • ATCC American Type Culture Collection
  • CMCC my country Medical Microorganisms Center
  • the research team systematically completed the risk survey of pathogenic microorganisms in 4300 foods in 43 major cities across the country, and has isolated and preserved more than 1500 strains of Salmonella with more than 60 serotypes, which is currently the strain resource bank with the most serotypes of Salmonella in food sources in my country.
  • the detection rates of Salmonella enteritidis and Salmonella typhimurium were 17% and 12% respectively, which were the serotypes with the largest number of strains.
  • most of the standard strains used to study the evolutionary law and pathogenicity of the bacteria are from the American Collection of Microbial Strains, which cannot well reflect the spread of the bacteria in food. The emergence rate has been at a high level, but there is a lack of representative isolates to study the genetic structure and transmission law of the bacteria in China.
  • the protective agent used in the current report is basically used for qualitative storage of Salmonella, and it is enough to ensure that there are still viable bacteria within the shelf life.
  • Ke Lu et al. used 20% skimmed milk, 10% sucrose , 8% polyvinylpyrrolidone and 3% sodium L-glutamate
  • the freeze-drying survival rate is only 38%, and it can only be stored for a short time at -20 °C, and its bacterial content drops from 1800cfu/part to 1000cfu/serving.
  • Another example is Chinese patent CN102140423B using 0.1-10 parts by mass of water-soluble sugar, 0.1-5 parts by mass of skim milk powder, 0.1-20 parts by mass of gelatin, and 0-10 parts by mass of activated carbon as protective agents for quantitative preservation.
  • the agent does not mention the level of freeze-drying survival rate, and because the protective agent contains a certain concentration of gelatin, its solubility is affected under normal temperature conditions or when used in winter, and the dissolution rate is slow; in addition, the protective agent contains activated carbon, Activated carbon is insoluble in aqueous solution, sinks to the bottom of the bottle after lyophilization, and is scattered on the surface of the plate during recovery, which affects the appearance and the automatic counting of colonies. Therefore, there is an urgent need for a long-term quantitative preservation method of strains that can accurately control the number of bacteria, has good stability and is convenient to use.
  • the purpose of the present invention is to overcome the deficiencies of the prior art, and to provide a Salmonella standard strain and a specific molecular target and application for detecting the standard strain.
  • the present invention provides 5 strains of Salmonella (2 strains of Salmonella enteritidis, 2 strains of Salmonella typhimurium and 1 strain of Salmonella paratyphi B) as food isolates in China.
  • the strains have typical physiological and biochemical characteristics of Salmonella and can better reflect Genetic background of the bacteria in China.
  • the technical scheme adopted in the present invention is: a specific molecular target for detecting 5 strains of Salmonella is provided, and the molecular target is:
  • 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 also provides primers for detecting the specific molecular target
  • PCR primers for amplification of the nucleotide sequence shown in SEQ ID NO: 1 include: an upstream primer shown in SEQ ID NO: 6 and a downstream primer shown in SEQ ID NO: 7;
  • PCR primers for amplification of the nucleotide sequence shown in SEQ ID NO:2 include: an upstream primer shown in SEQ ID NO:8 and a downstream primer shown in SEQ ID NO:9;
  • 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:10 and the downstream primer set forth in SEQ ID NO:11.
  • PCR primers for amplification of the nucleotide sequence shown in SEQ ID NO:4 include: an upstream primer shown in SEQ ID NO:12 and a downstream primer shown in SEQ ID NO:13;
  • PCR primers for amplification of the nucleotide sequence shown in SEQ ID NO:5 include: an upstream primer shown in SEQ ID NO:14 and a downstream primer shown in SEQ ID NO:15;
  • the present invention also provides a Salmonella, which is (a), (b), (c), (d) or (e):
  • Salmonella enteritidis (Salmonella enterica subsp.enterica serovar Enteritidis) FSCC (I) 215456, containing the nucleotide sequence shown in SEQ ID NO: 1;
  • Salmonella enteritidis (Salmonella enterica subsp.enterica serovar Enteritidis) FSCC (I) 215467, containing the nucleotide sequence shown in SEQ ID NO: 2;
  • Salmonella typhimurium (Salmonella enterica subsp.enterica serovar Typhimurium) FSCC (I) 215032, containing the nucleotide sequence shown in SEQ ID NO: 3;
  • Salmonella typhimurium (Salmonella enterica subsp.enterica serovar Typhimurium) FSCC (I) 21501206, containing the nucleotide sequence shown in SEQ ID NO: 4;
  • the Salmonella enterica subsp.enterica serovar Enteritidis FSCC (I) 215456 further comprises at least one of the following virulence genes: avrA, ssaQ, mgtC, siiD, sopB, bcfC, steB, sodC1, sopE1, gtgB, spvC, pefA, rck.
  • the Salmonella enterica subsp.enterica serovar Enteritidis FSCC (I) 215467 further comprises at least one of the following virulence genes: avrA, ssaQ, mgtC, siiD, sopB, bcfC, steB, sodC1, sopE1, gtgB, spvC, pefA, rck.
  • the Salmonella enterica subsp. enterica serovar Typhimurium FSCC (I) 215032 further comprises at least one of the following virulence genes: gipA and sodC1.
  • the Salmonella enterica subsp. enterica serovar Typhimurium FSCC (I) 21501206 further comprises at least one of the following virulence genes: gipA, sodC1, spvC and pefA-rck.
  • the Salmonella enterica subsp.enterica serovar Paratyphi B FSCC (I) 215947 further comprises at least one of the following virulence genes: avrA, ssaQ, mgtC, siiD, sopB, bcfC and steB.
  • the Salmonella enterica (Salmonella enterica subsp.enterica serovar Enteritidis) FSCC (I) 215456, which is classified as Salmonella enterica subsp.Enterica (Salmonella enterica subsp.Enterica), has been deposited in Guangdong City Microbial Bacteria on October 27, 2019 Species Collection Center, Address: Building 59, Yard, No. 100, Middle Xianlie Road, Guangzhou City, Guangdong province, China, Postal Code: 510075, and the deposit number is GDMCC 60847.
  • the Salmonella enterica subsp. enterica serovar Enteritidis (Salmonella enterica subsp. enterica serovar Enteritidis) FSCC (I) 215467, which is classified as Salmonella enterica subsp. enterica serovar Enteritidis (Salmonella enterica subsp. enterica serovar Enteritidis), has been deposited in Guangdong City on October 27, 2019 Microbial Culture Collection Center, Address: Building 59, Yard, No. 100, Middle Xianlie Road, Guangzhou City, Guangdong Province, China, Postal Code: 510075, preservation number: GDMCC 60848.
  • the Salmonella enterica subsp.entenca serovar Typhimurium (Salmonella enterica subsp.entenca serovar Typhimurium) FSCC (I) 215032, which is classified as Salmonella enterica subsp.enterica serovar Typhimurium (Salmonella enterica subsp.enterica serovar Typhimurium), has been deposited on Guangdong Provincial Microbial Culture Collection Center, Address: Building 59, Yard, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong province, China, Postal Code: 510075, preservation number: GDMCC 60849.
  • the Salmonella enterica subsp.enterica serovar Tvphimurium FSCC (I) 21501206 whose classification name is Salmonella enterica subsp.enteriva serovar Typhimurium, has been deposited on October 27, 2019 in Guangdong Provincial Microbial Culture Collection Center, the preservation number is GDMCC 60850.
  • Salmonella enterica subsp.enterica serovar Paratyphi B (Salmonella enterica subsp.enterica serovar Paratyphi B) FSCC (I) 215947, its classification is named as Salmonella enterica subsp.enterica serovar Paratyphi B, in 2019 On October 27th, it was deposited in the Guangdong Provincial Microbial Culture Collection Center, Address: Building 59, Yard, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong province, China, zip code: 510075, and the deposit number is GDMCC 60851.
  • the present invention also provides the application of the Salmonella in antibiotic resistance of Salmonella.
  • the invention also provides the application of the Salmonella in improving the accuracy of detecting the Salmonella color plate.
  • the present invention also provides a freeze-drying protective agent for Salmonella, comprising the following components in parts by weight: 1-5 parts of fetal bovine serum, 7-15 parts of skim milk powder, 1-7 parts of stachyose, 0.1-2 parts of ascorbic acid, 3 parts of -(N-morpholine)propanesulfonic acid 0.01-0.5 part.
  • stachyose has strong hydrophilicity, and can form a hydrogen bond with the phosphate group in the phospholipid of the cell membrane of the cell or with the polar group of the cell protein during the freezing or drying process.
  • skim milk powder can be wrapped in the outer layer of bacterial cells to protect bacteria, while fetal bovine serum can stabilize cell activity, 3-(N-morpholine) propanesulfonic acid buffer can stabilize bacteria pH of the suspension.
  • ascorbic acid reduces the activity of cellular oxidase during freeze-drying and long-term storage, preventing oxidative deterioration of freeze-dried products.
  • Salmonella enteritidis strain FSCC (I) 215456 of the present invention Salmonella enteritidis strain FSCC (I) 215467, Salmonella typhimurium strain FSCC (I) 215032, Salmonella typhimurium strain FSCC (I) 21501206, Salmonella paratyphi B strain FSCC (I) 215947 has standard Salmonella colony morphology and physiological and biochemical characteristics, which can be used to test the accuracy of Salmonella selective medium. Compared with other standard strains of this species, it is the only strain that can reflect the genetic background in China and can be used as a reference strain for scientific research. Research.
  • the quantitative Salmonella prepared by the freeze-drying protective agent of the present invention has the following advantages: 1) good shape, beautiful appearance and good water solubility, can be completely dissolved within 1-2 seconds; 2) freeze-drying survival rate can reach more than 90 percent. 3) It can be stored for at least one year under the condition of 2-8 °C, and the value does not change, which can be used for long-term storage of quantitative quality control strains.
  • a strain of Salmonella enterica (Salmonella enterica subsp.enterica serovar Enteritidis) FSCC(I) 215467, its taxonomic name is Salmonella enterica (Salmonella enterica subsp.enterica serovar Enteritidis), which has been deposited in Guangdong City Microorganisms on October 27, 2019 Collection Center, Address: Building 59, Yard, No. 100, Middle Xianlie Road, Guangzhou City, Guangdong province, China, Postal Code: 510075, and the collection number is GDMCC 60848.
  • Fig. 1 is the microscopic observation morphological diagram of Salmonella strains; wherein, A: Salmonella enteritidis strain FSCC(I)215456; B: Salmonella enteritidis strain FSCC(I)215467; C: Salmonella typhimurium strain FSCC(I)215032; D: Salmonella typhimurium strain FSCC(I) 21501206; E: Salmonella paratyphi beta strain FSCC(I) 215947.
  • Figure 2 is the colony morphology diagram of Salmonella strains on the chromogenic medium; wherein A: Salmonella enteritidis strain FSCC(I)215456; B: Salmonella enteritidis strain FSCC(I)215467; C: Salmonella typhimurium strain FSCC(I)215032 ; D: Salmonella typhimurium strain FSCC(I) 21501206; E: Salmonella paratyphi B strain FSCC(I) 215947.
  • Fig. 3 is the colony morphology diagram of Salmonella strain on XLD medium; wherein A: Salmonella enteritidis strain FSCC(I)215456; B: Salmonella enteritidis strain FSCC(I)215467; C: Salmonella typhimurium strain FSCC(I)215032; D: Salmonella typhimurium strain FSCC(I) 21501206; E: Salmonella paratyphi B strain FSCC(I) 215947.
  • Figure 4 is the colony morphology diagram of Salmonella strains on HE medium; wherein A: Salmonella enteritidis strain FSCC(I)215456; B: Salmonella enteritidis strain FSCC(I)215467; C: Salmonella typhimurium strain FSCC(I)215032; D: Salmonella typhimurium strain FSCC(I) 21501206; E: Salmonella paratyphi B strain FSCC(I) 215947.
  • Fig. 5 is the colony morphology diagram of Salmonella strains on BS medium; wherein A: Salmonella enteritidis strain FSCC(I)215456; B: Salmonella enteritidis strain FSCC(I)215467; C: Salmonella typhimurium strain FSCC(I)215032; D: Salmonella typhimurium strain FSCC(I) 21501206; E: Salmonella paratyphi B strain FSCC(I) 215947.
  • Fig. 6 is the PCR amplification diagram of the unique gene fragment of Salmonella strain.
  • Figure 7 shows the changes in bacterial content of Salmonella strains during quantitative storage.
  • the round purple colonies on the Salmonella cyclamen color plate are Salmonella enteritidis strain FSCC(I)215456, Salmonella enteritidis strain FSCC(I)215467, Salmonella typhimurium strain FSCC(I)215032, Salmonella typhimurium strain FSCC(I) ) 21501206, Salmonella paratyphi B strain FSCC (I) 215947.
  • the target colonies were transferred from TSA to tryptone soy broth and recovered at 37°C overnight. Under sterile conditions, the bacterial solution was added to a tube with a final concentration of 30% glycerol, stored in a -80°C refrigerator, and stored in a freeze-dried tube.
  • the purified colonies can be identified in terms of morphological characteristics, physiology and biochemistry, serotype and molecular biology.
  • the preservation date is 2019/10/27
  • the preservation numbers are GDMCC 60847, GDMCC 60848 , GDMCC 60849, GDMCC 60850, GDMCC 60851.
  • Salmonella Enteritidis strain FSCC(I)215456 was isolated from roast duck samples in Beijing, China
  • Salmonella Enteritidis strain FSCC(I)215467 was isolated from cold noodle samples in Jinan City, Shandong province, China
  • Salmonella typhimurium strain FSCC(I)215032 was isolated from From pork samples from Shaoguan City, Guangdong province, China
  • Salmonella typhimurium strain FSCC(I)21501206 was isolated from pork samples from Sanya City, Hainan province, China
  • Salmonella paratyphi B strain FSCC(I)215947 was isolated from Jinan City, Shandong province, China. in fresh fish.
  • Salmonella is a gram-negative bacillus, straight and slender, mostly with periflagellates, no spores, and no capsule ( Figure 1).
  • Circular purple colonies on Salmonella cyclamens chromogenic medium red colonies with black center in the middle on XLD selective medium; blue-green colonies on HE selective medium with black center in the middle; selective culture in BS Black colonies appear on the top with metallic luster (Fig. 2-Fig. 5).
  • the strain of the present invention has standard Salmonella biochemical characteristics.
  • the identification percentages of Salmonella Enteritidis strain FSCC(I)215456 and Salmonella Enteritidis strain FSCC(I)215467 are both 89.4%, and the T value is 1, indicating that it is typical of Salmonella
  • the strains are exactly the same; the identification percentage of Salmonella typhimurium strain FSCC(I)215032 and Salmonella typhimurium strain FSCC(I)21501206 is 99.9%, and the T value is 0.97; the identification percentage of Salmonella typhimurium strain FSCC(I)215947 is 99.9% , the T value is 0.65.
  • A-F polyvalent O serum was used for slide agglutination test, while normal saline was used as control. Those that self-aggregate in normal saline are rough strains and cannot be typed.
  • the agglutination test was performed with O4; O3, O10; O7; O8; O9; O2 and O11 factor serum in turn. Based on the test results, the O group was determined.
  • strains agglutinated by O3 and O10 serum use O10, O15, O34, O19 single-factor serum for agglutination test to determine E1 and E4 subgroups.
  • the final determination of each O antigen component should be based on the inspection of O single-factor serum. As a result, those without O single factor serum were checked with two O complex factor serums.
  • H polyvalent 1 a, b, c, d, i
  • H polyvalent 2 eh, enx, enz15, fg, gms, gpu, gp, gq, mt, gz51
  • H Multivalent 3 k, r, y, z, z10, lv, lw, lzl3, lz28, lz40
  • H polyvalent 4 1, 2; 1, 5; 1, 6; 1, 7; z6
  • H Multivalent 5 z4z23, z4z24, z4z32, z29, z35, z36, z38
  • H Multi-price 8 z56, z57, z60, z61, z62
  • each H antigen component should be based on the test results of H single factor serum. If there is no H single factor serum, two H complex factor serums should be checked. If the first phase H antigen is detected but the second phase H antigen is not detected, or the second phase H antigen is detected but the first phase H antigen is not detected, it can be transplanted on the agar slant for 1 to 2 generations. examine. If only one phase of H antigen is still detected, use the method of phase variation to check the other phase. Monophasic bacteria do not need to be checked for phase variation.
  • Embodiment 3 Virulence factor carrying characteristics of Salmonella strains
  • the virulence factor carrying of Salmonella strains was confirmed by PCR.
  • primers and amplification methods refer to previous literature reports.
  • the 15 pairs of primers used were synthesized by Shanghai Sangon Biological Co., Ltd. (see Table 6 for primer sequences).
  • the total reaction system is 25 ⁇ L: 2 ⁇ HS Mix 12.5 ⁇ L (Dongsheng Innovation), primer 0.5 ⁇ L, template 1-2 ⁇ L, supplemented with ultrapure water.
  • Reaction conditions pre-denaturation at 95°C for 5 min; denaturation at 95°C for 1 min; annealing at 56°C for 1 min; extension at 72°C for 1 min; a total of 35 cycles; final extension at 72°C for 10 min.
  • S. Enteritidis strain FSCC(I)215456 and S. Enteritidis strain FSCC(I)215467 carried avrA-ssaQ-mgtC-siiD-sopB-bcfC-steB-sodC1-sopE1-gtgB-spvC-pefA-rck virulence factor; Salmonella typhimurium strain FSCC(I)215032 carries gipA-sodC1 virulence factor, Salmonella typhimurium strain FSCC(I)21501206 carries gipA-sodC1-spvC-pefA-rck virulence factor; Salmonella paratyphi beta strain FSCC(I) 215947 carries the avrA-ssaQ-mgtC-siiD-sopB-bcfC-steB virulence factor.
  • the Salmonella strains were activated on TSA plates and diluted with physiological saline to a final concentration of 1 ⁇ 10 7 cfu/mL and spread on MH plates. After the bacteria liquid was dry, the antibiotic paper was attached to the surface of the medium and incubated at 37°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: ampicillin (AMP, 10 ⁇ g), amoxicillin-clavulanate (2:1; Amc, 30 ⁇ g), cefazolin (Cf, 30 ⁇ g), cefazolin (KZ, 30 ⁇ g), cefoxitin ( FOX, 30 ⁇ g), ceftriaxone (CRO, 30 ⁇ g), cefotaxime (CTX, 30 ⁇ g), ceftazidime (CAZ, 30 ⁇ g), cefoperazone (CFP, 75 ⁇ g), cefepime (FEP, 30 ⁇ g), chloramphenicol Ciprofloxacin (C, 30 ⁇ g), tetracycline (TE, 30 ⁇ g), nalidixic acid (NA, 30 ⁇ g), ciprofloxacin (CIP, 5 ⁇ g), amikacin (AK, 30 ⁇ g), gentamicin (GEN , 10 ⁇ g), streptomycin (S, 10 ⁇ g), kanamycin (K, 30 ⁇ g
  • the primers and amplification methods of the 7 housekeeping genes of Salmonella MLST typing refer to previous literature reports.
  • the 7 pairs of primers used were synthesized by Shanghai Sangon Biological Co., Ltd. (see Table 7 for primer sequences).
  • the total reaction system of PCR for multi-locus sequence typing is 25 ⁇ L: 2 ⁇ HS Mix 12.5 ⁇ L (Dongsheng Innovation), 0.5 ⁇ L of primers, 1-2 ⁇ L of template, and supplemented with ultrapure water.
  • PCR reaction conditions pre-denaturation at 95°C for 5 min; denaturation at 95°C for 1 min; annealing at 56°C for 1 min; extension at 72°C for 1 min; a total of 35 cycles; final extension at 72°C for 10 min.
  • Salmonella paratyphi strain FSCC (I) 215947 was used for pan-genome analysis.
  • pan-genome was analyzed by the MP method in the prokaryotic pan-genome automated analysis software (Pan-Genomics Analysis Pipeline, PGAP), and the analysis was performed by a local Perl script. The results were processed to obtain the core gene and non-core gene information of all strains.
  • the specific non-core gene protein sequences of the 5 strains were extracted and aligned back to the Salmonella total protein library and the NCBI non-redundant protein database (NR) by local Blast. Sequences that aligned with known Salmonella proteins were removed, and the rest were genes specific to the five strains. The specificity of the unique gene was tested by PCR amplification in the corresponding 5 strains and other Salmonella.
  • NR NCBI non-redundant protein database
  • Salmonella enteritidis strain FSCC(I)215456 Salmonella enteritidis strain FSCC(I)215467, Salmonella typhimurium strain FSCC(I)215032, Salmonella typhimurium strain FSCC(I)21501206, Salmonella paratyphi B Strain FSCC (I) 215947 carries a unique gene whose sequence is shown in SEQ ID Nos: 1 to 5. The gene can be amplified and tested by the following primers:
  • the present invention also provides a freeze-drying protective agent for Salmonella, and provides several specific embodiments (Examples 7-9) and comparative examples (Comparative Examples 1-3) of the freeze-drying protective agent.
  • a freeze-drying protective agent for preparing Salmonella quantitative quality control bacteria comprising 3 parts by mass of fetal bovine serum, 12 parts by mass of skimmed milk powder, 4 parts by mass of stachyose, 2 parts by mass of ascorbic acid, 0.03 parts by mass of 3 -(N-morpholine)propanesulfonic acid.
  • a freeze-drying protective agent for preparing Salmonella quantitative quality control bacteria comprising 5 parts by mass of fetal bovine serum, 10 parts by mass of skimmed milk powder, 5 parts by mass of stachyose, 1 part by mass of ascorbic acid, 0.1 part by mass of 3 -(N-morpholine)propanesulfonic acid.
  • a freeze-dried protective agent for preparing Salmonella quantitative quality control bacteria comprising 1 mass part of fetal bovine serum, 15 mass parts of skimmed milk powder, 7 mass parts of stachyose, 2 mass parts of ascorbic acid, 0.2 mass parts of 3 -(N-morpholine)propanesulfonic acid.
  • the freeze-drying protection agent does not contain fetal bovine serum, and only uses 12 parts by mass of skim milk powder, 4 parts by mass of stachyose, 2 parts by mass of ascorbic acid, and 0.03 parts by mass of 3-(N-morpholine ) propanesulfonic acid.
  • the freeze-drying protective agent does not contain stachyose, but only contains 3 parts by mass of fetal bovine serum, 12 parts by mass of skim milk powder, 2 parts by mass of ascorbic acid, and 0.03 parts by mass of 3-(N-morpholine ) propanesulfonic acid.
  • the freeze-drying protective agent contained only 12 parts by mass of skimmed milk powder.
  • the preparation method of bacterial powder is as follows: after the bacterial strain is recovered, it is inserted into a shake flask and cultivated until the late logarithmic stage to the early stage of the stable stage. Select an appropriate amount of bacteria and add it to the protective agent. Dilution count was performed, which was the bacterial content A0 before lyophilization.
  • 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 the freeze dryer, perform automatic capping, ensure the complete vacuum state of the sample, and store it at 2-8 °C.
  • Example 11 Changes in bacterial content during shelf life of freeze-dried protective agents with different compositions

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Abstract

发明提供了用于检测5株沙门氏菌的特异性分子靶标，所述分子靶标为：(a)如SEQ ID NO：1～5所示的任意一种或几种核苷酸序列；或者，(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。本发明还提供了5株含有所述特异性分子靶标的沙门氏菌标准菌株，其保藏号分别为：GDMCC 60847、GDMCC 60848、GDMCC 60849和GDMCC 60850。

Description

含有特异性分子靶标的沙门氏菌标准菌株及其检测和应用 技术领域
本发明涉及生物工程领域，尤其涉及含有特异性分子靶标的沙门氏菌标准菌株及其检测和应用。
背景技术
沙门氏菌(Salmonella spp.)是最常见的食源性致病菌之一，由其引起的食物中毒病例在世界范围内常常排在首位。因其营养要求低，在粪便、土壤、食品(肉、蛋、奶、面包等)、水中存活时间长达5个月至2年之久，且污染食品后没有明显的感官变化，因而极易被动物及人类食用造成感染性疾病，如禽伤寒、鸡白痢、猪霍乱等动物性疾病，以及人类胃肠炎、类伤寒、败血症、感冒等感染型食物中毒。人类感染沙门氏菌后初期症状为恶心、呕吐、腹泻、发烧等，若不及时治疗死亡率高达10％。通常情况下沙门氏菌食用量超过10 5CFU即可造成人类感染，而对于高度易感人群15-20CFU即可引起沙门氏菌感染。因此，沙门氏菌是食源性致病菌重点监控对象，了解该菌的传播规律以及致病性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。目前对于沙门氏菌标准菌株主要来源于国际或国内菌种保藏中心保藏的，遗传学特性得到确认和保证并可追溯，如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)等。
本研究团队首次系统完成了全国43个主要城市4300份食品中致病微生物风险调查，已分离保存了60多种血清型1500多株沙门氏菌，为目前我国食品源沙门氏菌血清型最多的菌种资源库，其中肠炎沙门氏菌、鼠伤寒沙门氏菌的检出率分别为17％、12％，为菌株数最多的血清型。目前研究该菌的进化规律和致病性使用的标准菌株大部分为美国微生物菌株保藏中心来源的菌株，并不能很好地反映该菌在食品中的传播特征，且在我国食品中沙门氏菌的检出率一直处于较高的水平，却缺乏代表性的分离株来研究该菌在中国的遗传结构和传播规律。
在菌株的长期保藏方法研究方面，目前报道所采用的保护剂基本用于定性储存沙门氏菌，在保质期内能够保证还有活菌存在即可，如柯璐等人使用20％脱脂牛奶、10％蔗糖、8％聚乙烯吡咯烷酮和3％L-谷氨酸钠，其冻干存活率仅为38％，且在-20℃只能短期保存，在一个月内其含菌量由1800cfu/份下降至1000cfu/份。又如中国专利CN102140423B采用0.1-10质量份的水溶性糖、0.1-5质量份的脱脂奶粉、0.1-20质量份的明胶、0-10质量份的活性炭作为定量化保存的保护剂，该保护剂并没有提到冻干存活率的高低，且由于保护剂中含有一定浓度的明胶，在常温条件下或在冬天使用时其溶解度受到影响，溶解速度较慢；此外该保护剂中含有活性炭，活性炭不溶于水溶液，冻干后沉在瓶底，且复苏时，散落在平板表面，影响美观以及菌落的自动计数。因此，亟需一种能准确控制菌数，稳定性好，使用便捷的菌株长期定量保藏方法。
发明内容
本发明的目的在于克服现有技术的不足，提供沙门氏菌标准菌株及检测该标准菌株的特异性分子靶标和应用。本发明提供5株沙门氏菌(2株肠炎沙门氏菌、2株鼠伤寒沙门氏菌及1株乙型副伤寒沙门氏菌)为中国地区的食品分离菌株，该菌株具有典型的沙门氏菌生理生化特性，且能较好地反映该菌在中国地区的遗传背景。
为实现上述目的，本发明采取的技术方案为：提供一种用于检测5株沙门氏菌的特异性分子靶标，所述分子靶标为：
(a)如SEQ ID NO：1～5所示的任意一种或几种核苷酸序列；或者，
(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。
本发明还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：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：2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；
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：10所示的上游引物和如SEQ ID NO：11所示的下游引物。
针对如SEQ ID NO：4所示的核苷酸序列扩增的PCR引物包括：如SEQ ID NO：12所示的上游引物和如SEQ ID NO：13所示的下游引物；
针对如SEQ ID NO：5所示的核苷酸序列扩增的PCR引物包括：如SEQ ID NO：14所示的上游引物和如SEQ ID NO：15所示的下游引物；
本发明还提供了一种沙门氏菌，是(a)、(b)、(c)、(d)或(e)：
(a)肠炎沙门氏菌(Salmonella enterica subsp.enterica serovar Enteritidis)FSCC(I)215456，含有如SEQ ID NO：1所示的核苷酸序列；
(b)肠炎沙门氏菌(Salmonella enterica subsp.enterica serovar Enteritidis)FSCC(I)215467，含有如SEQ ID  NO：2所示的核苷酸序列；
(c)鼠伤寒沙门氏菌(Salmonella enterica subsp.enterica serovar Typhimurium)FSCC(I)215032，含有如SEQ ID NO：3所示的核苷酸序列；
(d)鼠伤寒沙门氏菌(Salmonella enterica subsp.enterica serovar Typhimurium)FSCC(I)21501206，含有如SEQ ID NO：4所示的核苷酸序列；
(e)乙型副伤寒沙门氏菌(Salmonella enterica subsp.enterica serovar Paratyphi B)FSCC(I)215947，含有如SEQ ID NO：5所示的核苷酸序列。
优选的，所述肠炎沙门氏菌(Salmonella enterica subsp.enterica serovar Enteritidis)FSCC(I)215456还包含了以下毒力基因中的至少一种：avrA、ssaQ、mgtC、siiD、sopB、bcfC、steB、sodC1、sopE1、gtgB、spvC、pefA、rck。
优选的，所述肠炎沙门氏菌(Salmonella enterica subsp.enterica serovar Enteritidis)FSCC(I)215467还包含了以下毒力基因中的至少一种：avrA、ssaQ、mgtC、siiD、sopB、bcfC、steB、sodC1、sopE1、gtgB、spvC、pefA、rck。
优选的，所述鼠伤寒沙门氏菌(Salmonella enterica subsp.enterica serovar Typhimurium)FSCC(I)215032还包含了以下毒力基因中的至少一种：gipA和sodC1。
优选的，所述鼠伤寒沙门氏菌(Salmonella enterica subsp.enterica serovar Typhimurium)FSCC(I)21501206还包含了以下毒力基因中的至少一种：gipA、sodC1、spvC和pefA-rck。
优选的，所述乙型副伤寒沙门氏菌(Salmonella enterica subsp.enterica serovar Paratyphi B)FSCC(I)215947还包含了以下毒力基因中的至少一种：avrA、ssaQ、mgtC、siiD、sopB、bcfC和steB。
[根据细则91更正 21.06.2021]　
优选的，所述肠炎沙门氏菌(Salmonella enterica subsp.enterica serovar Enteritidis)FSCC(I)215456，其分类命名为肠炎沙门氏菌(Salmonella enterica subsp.Enterica)，已于2019年10月27号保藏于广东省微生物菌种保藏中心，地址：中国广东省广州市先烈中路100号大院59号楼，邮政编码：510075，保藏编号为GDMCC 60847。
[根据细则91更正 21.06.2021]　
优选的，所述肠炎沙门氏菌(Salmonella enterica subsp.enterica serovar Enteritidis)FSCC(I)215467，其分类命名为肠炎沙门氏菌(Salmonella enterica subsp.enterica serovar Enteritidis)，已于2019年10月27号保藏于广东省微生物菌种保藏中心，地址：中国广东省广州市先烈中路100号大院59号楼，邮政编码：510075，保藏编号为GDMCC 60848。
[根据细则91更正 21.06.2021]　
优选的，所述鼠伤寒沙门氏菌(Salmonella enterica subsp.entenca serovar Typhimurium)FSCC(I)215032，其分类命名为鼠伤寒沙门氏菌(Salmonella enterica subsp.enterica serovar Typhimurium)，已于2019年10月27号保藏于广东省微生物菌种保藏中心，地址：中国广东省广州市先烈中路100号大院59号楼，邮政编码：510075，保藏编号为GDMCC 60849。
优选的，所述鼠伤寒沙门氏菌(Salmonella enterica subsp.enterica serovar Tvphimurium)FSCC(I)21501206，其分类命名为鼠伤寒沙门氏菌(Salmonella enterica subsp.enteriva serovar Typhimurium)，已于2019年10月27号保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC 60850。
[根据细则91更正 21.06.2021]　
优选的，所述乙型副伤寒沙门氏菌(Salmonella enterica subsp.enterica serovar Paratyphi B)FSCC(I)215947，其分类命名为乙型副伤寒沙门氏菌(Salmonella enterica subsp.enterica serovar Paratyphi B)，已于2019年10月27号保藏于广东省微生物菌种保藏中心，地址：中国广东省广州市先烈中路100号大院59号楼，邮政编码：510075，保藏编号为GDMCC 60851。
本发明还提供所述的沙门氏菌在沙门氏菌抗生素耐药性中的应用。
本发明还提供所述的沙门氏菌在提高检验沙门氏菌显色平板的准确性中的应用。
本发明还提供一种沙门氏菌冻干保护剂，包括以下重量份的组分：胎牛血清1-5份、脱脂奶粉7-15份、水苏糖1-7份、抗坏血酸0.1-2份、3-(N-吗啉)丙磺酸0.01-0.5份。
本发明的冻干保护剂中，水苏糖具有很强的亲水性，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细胞外层保护菌体，而胎牛血清能稳定细胞的活性，3-(N-吗啉)丙磺酸缓冲液能稳定菌悬液的pH值。抗坏血酸作为抗氧化剂，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。
本发明的有益效果：
(1)本发明肠炎沙门氏菌菌株FSCC(I)215456、肠炎沙门氏菌菌株FSCC(I)215467、鼠伤寒沙门氏菌菌株FSCC(I)215032、鼠伤寒沙门氏菌菌株FSCC(I)21501206、乙型副伤寒沙门氏菌菌株FSCC(I)215947具有标准的沙门氏菌菌落形态和生理生化特征，可用于检验沙门氏菌选择性培养基的准确性。该菌血清型明确、携带毒力基因情况具体，耐药性明了，遗传背景清晰，相比其它该物种的标准菌株，是目前唯一能反映中国地区遗传背景的菌株，可作为参考菌株用于科学研究。
(2)本发明的冻干保护剂制备的定量化的沙门氏菌，具有以下优点：1)成型好，外观漂亮且水溶性好，1-2秒内能够完全溶解；2)冻干存活率能达到90％以上。3)在2-8℃条件下可以保存至少一年以上，量值不发生变化，能够用于定量化质控菌株的长期保存。
生物材料保藏
[根据细则91更正 21.06.2021]　
一株肠炎沙门氏菌(Salmonella enterica subsp.enterica serovar Enteritidis)FSCC(I)215456，其分类命名为 肠炎沙门氏菌(Salmonella enterica subsp.enterica serovar Enteritidis)，已于2019年10月27号保藏于广东省微生物菌种保藏中心，地址：中国广东省广州市先烈中路100号大院59号楼，邮政编码：510075，保藏编号为GDMCC 60847。
[根据细则91更正 21.06.2021]　
一株肠炎沙门氏菌(Salmonella enterica subsp.enterica serovar Enteritidis)FSCC(I)215467，其分类命名为肠炎沙门氏菌(Salmonella enterica subsp.enterica serovar Enteritidis)，已于2019年10月27号保藏于广东省微生物菌种保藏中心，地址：中国广东省广州市先烈中路100号大院59号楼，邮政编码：510075，保藏编号为GDMCC 60848。
[根据细则91更正 21.06.2021]　
一株鼠伤寒沙门氏菌(Salmonella typhimurium)FSCC(I)215032，其分类命名为鼠伤寒沙门氏菌(Salmonella typhimurium)，已于2019年10月27号保藏于广东省微生物菌种保藏中心，地址：中国广东省广州市先烈中路100号大院59号楼，邮政编码：510075，保藏编号为GDMCC 60849。
[根据细则91更正 21.06.2021]　
一株鼠伤寒沙门氏菌(Salmonella typhimurium)FSCC(I)21501206，其分类命名为鼠伤寒沙门氏菌(Salmonellatyphimurium)，已于2019年10月27号保藏于广东省微生物菌种保藏中心，地址：中国广东省广州市先烈中路100号大院59号楼，邮政编码：510075，保藏编号为GDMCC 60850。
[根据细则91更正 21.06.2021]　
一株乙型副伤寒沙门氏菌(Salmonella enterica subsp.enterica serovar Paratyphi B)FSCC(I)215947，其分类命名为乙型副伤寒沙门氏菌(Salmonella enterica subsp.enterica serovar Paratyphi B)，已于2019年10月27号保藏于广东省微生物菌种保藏中心，地址：中国广东省广州市先烈中路100号大院59号楼，邮政编码：510075，保藏编号为GDMCC 60851。
附图说明
图1为沙门氏菌菌株的镜检观察形态图；其中，A：肠炎沙门氏菌菌株FSCC(I)215456；B：肠炎沙门氏菌菌株FSCC(I)215467；C：鼠伤寒沙门氏菌菌株FSCC(I)215032；D：鼠伤寒沙门氏菌菌株FSCC(I)21501206；E：乙型副伤寒沙门氏菌菌株FSCC(I)215947。
图2为沙门氏菌菌株在显色培养基上的菌落形态图；其中A：肠炎沙门氏菌菌株FSCC(I)215456；B：肠炎沙门氏菌菌株FSCC(I)215467；C：鼠伤寒沙门氏菌菌株FSCC(I)215032；D：鼠伤寒沙门氏菌菌株FSCC(I)21501206；E：乙型副伤寒沙门氏菌菌株FSCC(I)215947。
图3为沙门氏菌菌株在XLD培养基上的菌落形态图；其中A：肠炎沙门氏菌菌株FSCC(I)215456；B：肠炎沙门氏菌菌株FSCC(I)215467；C：鼠伤寒沙门氏菌菌株FSCC(I)215032；D：鼠伤寒沙门氏菌菌株FSCC(I)21501206；E：乙型副伤寒沙门氏菌菌株FSCC(I)215947。
图4为沙门氏菌菌株在HE培养基上的菌落形态图；其中A：肠炎沙门氏菌菌株FSCC(I)215456；B：肠炎沙门氏菌菌株FSCC(I)215467；C：鼠伤寒沙门氏菌菌株FSCC(I)215032；D：鼠伤寒沙门氏菌菌株FSCC(I)21501206；E：乙型副伤寒沙门氏菌菌株FSCC(I)215947。
图5为沙门氏菌菌株在BS培养基上的菌落形态图；其中A：肠炎沙门氏菌菌株FSCC(I)215456；B：肠炎沙门氏菌菌株FSCC(I)215467；C：鼠伤寒沙门氏菌菌株FSCC(I)215032；D：鼠伤寒沙门氏菌菌株FSCC(I)21501206；E：乙型副伤寒沙门氏菌菌株FSCC(I)215947。
图6为沙门氏菌菌株的特有基因片段PCR扩增图。
图7为沙门氏菌菌株定量保存期内含菌量变化。
具体实施方式
为更清楚地表述本发明的技术方案，下面结合具体实施例进一步说明，但不能用于限制本发明，此仅是本发明的部分实施例。
实施例1沙门氏菌菌株的分离、鉴定和培养
采集的食品样品，以无菌操作取样品25g(mL)分别加入到含有225mLTTB增菌液和SC增菌液的均质袋中，在拍击式均质器上连续均质1-2min。于42℃±1℃培养24h。取原增菌液划线于XLT4选择性平板上，于37℃±1℃培养18-24h，典型菌落在XLT4选择性平板上呈粉红色，带黑色中心。挑取XLT4平板上的典型菌落二次分离划线于环凯显色培养基，于37℃培养18h-24h。同时划线TSA斜面，临时保存可疑菌落。
在环凯沙门氏菌显色平板上呈圆形紫色菌落即为肠炎沙门氏菌菌株FSCC(I)215456、肠炎沙门氏菌菌株FSCC(I)215467、鼠伤寒沙门氏菌菌株FSCC(I)215032、鼠伤寒沙门氏菌菌株FSCC(I)21501206、乙型副伤寒沙门氏菌菌株FSCC(I)215947。将目标菌落从TSA上转接到胰蛋白胨大豆肉汤中，37℃过夜复苏。在无菌条件下将菌液加入终浓度为30％甘油管中，保存于-80℃冰箱，并进行冻干管保存。纯化后的菌落可进行形态特征、生理生化、血清型以及分子生物学等方面的鉴定。
[根据细则91更正 21.06.2021]　
分离得到肠炎沙门氏菌菌株FSCC(I)215456、肠炎沙门氏菌菌株FSCC(I)215467、鼠伤寒沙门氏菌菌株FSCC(I)215032、鼠伤寒沙门氏菌菌株FSCC(I)21501206、乙型副伤寒沙门氏菌菌株FSCC(I)215947，保藏于广东省微生物菌种保藏中心，地址为中国广州市先烈中路100号大院59号楼，邮政编码：510075，保藏日为2019/10/27，保藏编号分别为GDMCC 60847、GDMCC 60848、GDMCC 60849、GDMCC 60850、GDMCC 60851。
其中肠炎沙门氏菌菌株FSCC(I)215456分离自中国北京的烧鸭样本中，肠炎沙门氏菌菌株FSCC(I)215467分离自中国山东省济南市的凉面样本中，鼠伤寒沙门氏菌菌株FSCC(I)215032分离自中国广东省韶关市的猪肉样本中，鼠伤寒沙门氏菌菌株FSCC(I)21501206分离自中国海南省三亚市猪肉样本中，乙型副伤寒沙门氏菌菌株FSCC(I)215947分离自中国山东省济南市的新鲜鱼肉中。
实施例2沙门氏菌菌株的生理生化特征和血清型分析
(1)镜检
将可疑菌落涂片，进行革兰氏染色，镜检观察形态。沙门氏菌为革兰阴性杆菌，直而细长，多有周鞭毛、无芽孢、无荚膜(图1)。
(2)沙门氏菌选择性培养基生长情况
在环凯沙门氏菌属显色培养基上产生紫色圆形的菌落；在XLD选择性培养上呈红色菌落，中间黑心；在HE选择性培养基上呈蓝绿色菌落，中间黑心；在BS选择性培养上呈黑色菌落，有金属光泽(图2-图5)。
(3)API 20E鉴定
从沙门氏菌显色平板上刮取紫色的单个菌落接种到TSA上37℃培养18h-24h后，挑取适量菌落用生理盐水制备成浊度适当的菌悬浮液，使用API 20E生化鉴定试剂条鉴定(表1-5)。
表1肠炎沙门氏菌菌株FSCC(I)215456的API 20E生化鉴定结果
Figure PCTCN2021087010-appb-000001
表2肠炎沙门氏菌菌株FSCC(I)215467的API 20E生化鉴定结果
Figure PCTCN2021087010-appb-000002
表3鼠伤寒沙门氏菌菌株FSCC(I)215032的API 20E生化鉴定结果
Figure PCTCN2021087010-appb-000003
表4鼠伤寒沙门氏菌菌株FSCC(I)21501206的API 20E生化鉴定结果
Figure PCTCN2021087010-appb-000004
表5乙型副伤寒沙门氏菌菌株FSCC(I)215947的API 20E生化鉴定结果
Figure PCTCN2021087010-appb-000005
本发明的菌株具有标准的沙门氏菌生化特征，在API20E生化鉴定中，肠炎沙门氏菌菌株FSCC(I)215456与肠炎沙门氏菌菌株FSCC(I)215467鉴定百分率均为89.4％，T值为1，说明与沙门典型菌株完全一样；鼠伤寒沙门氏菌菌株FSCC(I)215032与鼠伤寒沙门氏菌菌株FSCC(I)21501206鉴定百分率为99.9％，T值为0.97；乙型副伤寒沙门氏菌菌株FSCC(I)215947鉴定百分率为99.9％，T值为0.65。
(4)血清型分析
1)O抗原的鉴定
用A-F多价O血清做玻片凝集试验，同时用生理盐水做对照。在生理盐水中自凝者为粗糙型菌株，不能分型。被A-F多价O血清凝集者，依次用O4；O3、O10；O7；O8；O9；O2和O11因子血清做凝集试验。根据试验结果，判定O群。被O3、O10血清凝集的菌株，再用O10、O15、O34、O19单因子血清做凝集试验，判定E1、E4各亚群，每一个O抗原成分的最后确定均应根据O单因子血清的检查结果，没有O单因子血清的要用两个O复合因子血清进行核对。
结果表明，鼠伤寒沙门氏菌菌株FSCC(I)215032、鼠伤寒沙门氏菌菌株FSCC(I)21501206及乙型副伤寒沙门氏菌菌株FSCC(I)215947的O抗原鉴定为O4；肠炎沙门氏菌菌株FSCC(I)215456与肠炎沙门氏菌菌株FSCC(I)215467的O抗原鉴定为O9。
2)H抗原的鉴定
先用8种多价H血清检查，如有其中一种或两种血清凝集，则再用这一种或两种血清所包括的各种H因子血清逐一检查。8种多价H血清所包括的H因子如下：
H多价1：a，b，c，d，i
H多价2：eh，enx，enz15，fg，gms，gpu，gp，gq，mt，gz51
H多价3：k，r，y，z，z10，lv，lw，lzl3，lz28，lz40
H多价4：1，2；1，5；1，6；1，7；z6
H多价5：z4z23，z4z24，z4z32，z29，z35，z36，z38
H多价6：z39，z41，z42，z44
H多价7：z52，z53，z54，z55
H多价8：z56，z57，z60，z61，z62
每一个H抗原成分的最后确定均应根据H单因子血清的检查结果，没有H单因子血清的要用两个H复合因子血清进行核对。检出第1相H抗原而未检出第2相H抗原的或检出第2相H抗原而未检出第1相H抗原的，可在琼脂斜面上移种1代-2代后再检查。如仍只检出一个相的H抗原，要用位相变异的方法检查其另一个相。单相菌不必做位相变异检查。
结果表明，鼠伤寒沙门氏菌菌株FSCC(I)215032与鼠伤寒沙门氏菌菌株FSCC(I)21501206的H抗原第1相鉴定为i，第二相鉴定为1，2；乙型副伤寒沙门氏菌菌株FSCC(I)215947的H抗原第1相鉴定为b，第二相鉴定为1，2；肠炎沙门氏菌菌株FSCC(I)215456与肠炎沙门氏菌菌株FSCC(I)215467的第1相鉴定为g，m。
实施例3沙门氏菌菌株的毒力因子携带特征
采用PCR的方法确认沙门氏菌菌株的毒力因子携带情况。引物与扩增方法参考先前文献报道。所使用的15对引物由上海生工生物有限公司合成(引物序列见表6)。反应体系总25μL：2×HS Mix 12.5μL(东盛创新)，引物0.5μL，模板1-2μL，加超纯水补齐。
反应条件：95℃预变性，5min；95℃变性，1min；56℃退火，1min；72℃延伸，1min；共进行35循环；最后72℃终延伸，10min。
表6沙门氏菌毒力因子引物序列
Figure PCTCN2021087010-appb-000006
Figure PCTCN2021087010-appb-000007
结果表明，肠炎沙门氏菌菌株FSCC(I)215456与肠炎沙门氏菌菌株FSCC(I)215467携带avrA-ssaQ-mgtC-siiD-sopB-bcfC-steB-sodC1-sopE1-gtgB-spvC-pefA-rck毒力因子；鼠伤寒沙门氏菌菌株FSCC(I)215032携带gipA-sodC1毒力因子、鼠伤寒沙门氏菌菌株FSCC(I)21501206携带gipA-sodC1-spvC-pefA-rck毒力因子；乙型副伤寒沙门氏菌菌株FSCC(I)215947携带avrA-ssaQ-mgtC-siiD-sopB-bcfC-steB毒力因子。
实施例4沙门氏菌菌株的药敏特征
沙门氏菌菌株经TSA平板活化后加入生理盐水稀释至终浓度为1×10 7cfu/mL涂布于MH平板上，待菌液干了之后将抗生素纸片贴在培养基表面，37℃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：氨苄西林(AMP，10μg)、阿莫西林克拉维酸(2∶1；Amc，30μg)、头孢噻吩(Cf，30μg)、头孢唑啉(KZ，30μg)、头孢西丁(FOX，30μg)、头孢曲松(CRO，30μg)、头孢噻肟(CTX，30μg)、头孢他啶(CAZ，30μg)、头孢哌酮(CFP，75μg)、头孢吡肟(FEP，30μg)、氯霉素(C，30μg)、四环素(TE，30μg)、萘啶酮酸(NA，30μg)、环丙沙星(CIP，5μg)、阿米卡星(AK，30μg)、庆大霉素(GEN，10μg)、链霉素(S，10μg)、卡那霉素(K，30μg)、复方新诺明(SXT，1.25/23.75μg)。金黄色葡萄球菌ATCC 25923和大肠埃希氏菌ATCC 25922作为质量控制菌株。
结果表明，肠炎沙门氏菌菌株FSCC(I)215456的耐药谱为NA-C、肠炎沙门氏菌菌株FSCC(I)215467的耐药谱为NA-AMP-cfp-S；鼠伤寒沙门氏菌菌株FSCC(I)215032的耐药谱AMP-C-S-Su-TE-NA-CN-SXT-K、鼠伤寒沙门氏菌菌株FSCC(I)21501206的耐药谱AMP-S-Su-TE-NA-amc-kf-CN-SXT-K。乙型副伤寒沙门氏菌菌株FSCC(I)215947对所测药敏纸片均敏感。
实施例5沙门氏菌菌株的多位点序列(MLST)分型分析
沙门氏菌MLST分型的7个管家基因的引物与扩增方法参考先前文献报道。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7)。多位点序列分型的PCR的反应体系总25μL：2×HS Mix 12.5μL(东盛创新)，引物0.5μL，模板1-2μL，加超纯水补齐。PCR反应条件：95℃预变性，5min；95℃变性，1min；56℃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，并将胶回收产物交上海生物工程技术有限公司进行双向测序。将测序结果用DNA Star软件根据Pub MLST的相关要求进行修正，使7个管家基因均符合国际上关于沙门氏菌多位点序列分型的序列大小要求，将修正好的7个管家基因序列与MLST数据库中序列进行比对分析，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。
表7沙门氏菌MLST分型引物序列
Figure PCTCN2021087010-appb-000008
Figure PCTCN2021087010-appb-000009
结果表明，肠炎沙门氏菌菌株FSCC(I)215456与肠炎沙门氏菌菌株FSCC(I)215467的MLST分型中均为ST11型；鼠伤寒沙门氏菌菌株FSCC(I)215032与鼠伤寒沙门氏菌菌株FSCC(I)21501206的MLST分型中均为ST19型；乙型副伤寒沙门氏菌菌株FSCC(I)215947的MLST分型中为ST42型。
实施例6沙门氏菌菌株的特征序列分析
主要根据沙门氏菌的泛基因组分析结果获得5菌株特有的非必需基因。共选取了1196株代表性沙门氏菌(含肠炎沙门氏菌菌株FSCC(I)215456、肠炎沙门氏菌菌株FSCC(I)215467、鼠伤寒沙门氏菌菌株FSCC(I)215032、鼠伤寒沙门氏菌菌株FSCC(I)21501206、乙型副伤寒沙门氏菌菌株FSCC(I)215947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-Genomics Analysis Pipeline，PGAP)中的MP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。
提取5株菌株特有的非核心基因蛋白序列，通过本地Blast分别将其比对回沙门氏菌的蛋白总库及NCBI非冗余蛋白数据库(NR)。去除能比对到已知的沙门氏菌蛋白的序列，剩下的则为5株菌株特有的基因。特有基因在相应的5株菌株及其它沙门氏菌中通过PCR扩增检验其特异性。
结果如图6所示，肠炎沙门氏菌菌株FSCC(I)215456、肠炎沙门氏菌菌株FSCC(I)215467、鼠伤寒沙门氏菌菌株FSCC(I)215032、鼠伤寒沙门氏菌菌株FSCC(I)21501206、乙型副伤寒沙门氏菌菌株FSCC(I)215947均携带一个特有的基因，序列如SEQ ID No：1～5所示，该基因可通过以下引物进行扩增检验：
表10本发明所述菌株的基因岛扩增引物序列
Figure PCTCN2021087010-appb-000010
本发明还提供一种沙门氏菌的冻干保护剂，并提供了冻干保护剂的几个具体实施例(实施例7～9)和对比例(对比例1～3)。
实施例7
一种用于制备沙门氏菌定量质控菌的冻干保护剂，含有3质量份的胎牛血清，12质量份的脱脂奶粉，4质量份的水苏糖，2质量份的抗坏血酸，0.03质量份3-(N-吗啉)丙磺酸。
实施例8
一种用于制备沙门氏菌定量质控菌的冻干保护剂，含有5质量份的胎牛血清，10质量份的脱脂奶粉，5质量份的水苏糖，1质量份的抗坏血酸，0.1质量份3-(N-吗啉)丙磺酸。
实施例9
一种用于制备沙门氏菌定量质控菌的冻干保护剂，含有1质量份的胎牛血清，15质量份的脱脂奶粉，7质量份的水苏糖，2质量份的抗坏血酸，0.2质量份3-(N-吗啉)丙磺酸。
对比例1
冻干保护剂与实施例1相比，不含有胎牛血清，仅使用12质量份的脱脂奶粉，4质量份的水苏糖，2质量份的抗坏血酸，0.03质量份3-(N-吗啉)丙磺酸。
对比例2
冻干保护剂与实施例1相比，不含有水苏糖，仅含有3质量份的胎牛血清，12质量份的脱脂奶粉，2质量份的抗坏血酸，0.03质量份3-(N-吗啉)丙磺酸。
对比例3
冻干保护剂与实施例1相比，仅含有12质量份的脱脂奶粉。
实施例10沙门氏菌标准菌株定量冻干存活率及稳定性比较
菌粉的制备方式为：将菌种复苏后接入摇瓶中进行培养至对数后期至稳定期前期选取合适的菌量加入到保护剂中，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2-8℃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。
结果表明，本发明(实施例7～9)提供的制备沙门氏菌标准菌株的定量冻干保护剂的定量冻干存活率均维持在90％以上，而对比例1、2、3的存活率下降至25.9％-52.8％(表11)。
表11沙门氏菌标准菌株不同组成的冻干保护剂定量冻干存活率的比较
Figure PCTCN2021087010-appb-000011
实施例11不同组成的冻干保护剂保质期内菌含量的变化
按照实施例7～9与对比例1、2、3的方法使用不同的保护剂进行制备定量沙门氏菌标准菌，并置于2-8℃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，在制备定量标准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1000cfu/瓶。
经12个月储存后，本发明(实施例7～9)提供的制备沙门氏菌标准菌株的定量冻干保护剂含菌量维持在800cfu/瓶以上，而对比例1、2、3的含菌量下降至200cfu/瓶以下(图7)。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。

Claims (12)

  1. 用于检测5株沙门氏菌的特异性分子靶标，其特征在于，所述分子靶标为：
    (a)如SEQ ID NO：1～5所示的任意一种或几种核苷酸序列；或者，
    (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。
  2. 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：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：2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；
    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：10所示的上游引物和如SEQ ID NO：11所示的下游引物。
    针对如SEQ ID NO：4所示的核苷酸序列扩增的PCR引物包括：如SEQ ID NO：12所示的上游引物和如SEQ ID NO：13所示的下游引物；
    针对如SEQ ID NO：5所示的核苷酸序列扩增的PCR引物包括：如SEQ ID NO：14所示的上游引物和如SEQ ID NO：15所示的下游引物。
  3. 一种沙门氏菌，其特征在于，是(a)、(b)、(c)、(d)或(e)：
    (a)肠炎沙门氏菌(Salmonella enterica subsp.enterica serovar Enteritidis)FSCC(I)215456，含有如SEQ ID NO：1所示的核苷酸序列；
    (b)肠炎沙门氏菌(Salmonella enterica subsp.enterica serovar Enteritidis)FSCC(I)215467，含有如SEQ ID NO：2所示的核苷酸序列；
    (c)鼠伤寒沙门氏菌(Salmonella enterica subsp.enterica serovar Typhimurium)FSCC(I)215032，含有如SEQ ID NO：3所示的核苷酸序列；
    (d)鼠伤寒沙门氏菌(Salmonella enterica subsp.enterica serovar Typhimurium)FSCC(I)21501206，含有如SEQ ID NO：4所示的核苷酸序列；
    (e)乙型副伤寒沙门氏菌(Salmonella enterica subsp.enterica serovar Paratyphi B)FSCC(I)215947，含有如SEQ ID NO：5所示的核苷酸序列。
  4. 如权利要求3所述的沙门氏菌，其特征在于，所述肠炎沙门氏菌(Salmonella enterica subsp.enterica serovar Enteritidis)FSCC(I)215456还包含了以下毒力基因中的至少一种：avrA、ssaQ、mgtC、siiD、sopB、bcfC、steB、sodC1、sopE1、gtgB、spvC、pefA、rck。
  5. 如权利要求3所述的沙门氏菌，其特征在于，所述肠炎沙门氏菌(Salmonella enterica subsp.enterica serovar Enteritidis)FSCC(I)215467还包含了以下毒力基因中的至少一种：avrA、ssaQ、mgtC、siiD、sopB、bcfC、steB、sodC1、sopE1、gtgB、spvC、pefA、rck。
  6. 如权利要求3所述的沙门氏菌，其特征在于，所述鼠伤寒沙门氏菌(Salmonella enterica subsp.enterica serovar Typhimurium)FSCC(I)215032还包含了以下毒力基因中的至少一种：gipA和sodC1。
  7. 如权利要求3所述的沙门氏菌，其特征在于，所述鼠伤寒沙门氏菌(Salmonella enterica subsp.enterica serovar Typhimurium)FSCC(I)21501206还包含了以下毒力基因中的至少一种：gipA、sodC1、spvC和pefA-rck。
  8. 如权利要求3所述的沙门氏菌，其特征在于，所述乙型副伤寒沙门氏菌(Salmonella enterica subsp.enterica serovar Paratyphi B)FSCC(I)215947还包含了以下毒力基因中的至少一种：avrA、ssaQ、mgtC、siiD、sopB、bcfC和steB。
  9. 如权利要求3所述的沙门氏菌，其特征在于：
    所述肠炎沙门氏菌(Salmonella enterica subsp.enterica serovar Enteritidis)FSCC(I)215456，其分类命名为肠炎沙门氏菌(Salmonella enterica subsp.enterica serovar Enteritidis)，已于2019年10月27号保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC 60847。
    所述肠炎沙门氏菌(Salmonella enterica subsp.enterica serovar Enteritidis)FSCC(I)215467，其分类命名为肠炎沙门氏菌(Salmonella enterica subsp.enterica serovar Enteritidis)，已于2019年10月27号保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC 60848。
    所述鼠伤寒沙门氏菌(Salmonella enterica subsp.enterica serovar Typhimurium)FSCC(I)215032，其分类命名为鼠伤寒沙门氏菌(Salmonella enterica subsp.enterica serovar Typhimurium)，已于2019年10月27号保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC 60849。
    所述鼠伤寒沙门氏菌(Salmonella enterica subsp.enterica serovar Typhimurium)FSCC(I)21501206，其分类命名为鼠伤寒沙门氏菌(Salmonella enterica subsp.enterica serovar Typhimurium)，已于2019年10月27号保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC 60850。
    所述乙型副伤寒沙门氏菌(Salmonella enterica subsp.enterica serovar Paratyphi B)FSCC(I)215947，其分类命名为乙型副伤寒沙门氏菌(Salmonella enterica subsp.enterica serovar Paratyphi B)，已于2019年10月27号保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC 60851。
  10. 如权利要求3所述的沙门氏菌在沙门氏菌抗生素耐药性中的应用。
  11. 如权利要求3所述的沙门氏菌在提高检验沙门氏菌显色平板的准确性中的应用。
  12. 一种沙门氏菌冻干保护剂，其特征在于，包括以下重量份的组分：胎牛血清1-5份、脱脂奶粉7-15份、水苏糖1-7份、抗坏血酸0.1-2份、3-(N-吗啉)丙磺酸0.01-0.5份。
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