WO2022141934A1 - Staphylococcus aureus standard strains containing specific molecular target, and detection and use thereof - Google Patents

Staphylococcus aureus standard strains containing specific molecular target, and detection and use thereof Download PDF

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WO2022141934A1
WO2022141934A1 PCT/CN2021/086878 CN2021086878W WO2022141934A1 WO 2022141934 A1 WO2022141934 A1 WO 2022141934A1 CN 2021086878 W CN2021086878 W CN 2021086878W WO 2022141934 A1 WO2022141934 A1 WO 2022141934A1
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staphylococcus aureus
seq
nucleotide sequence
gdmcc
parts
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Chinese (zh)
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吴诗
吴清平
代京莎
丁郁
陈谋通
张菊梅
薛亮
王涓
叶青华
曾海燕
古其会
杨小鹃
张淑红
韦献虎
张友雄
庞锐
卢勉飞
张峰
黄嘉慧
陈鲁
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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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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N1/04—Preserving or maintaining viable microorganisms
    • 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

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  • the invention relates to the technical field of bioengineering, in particular to a standard strain of Staphylococcus aureus containing a specific molecular target and its detection and application.
  • Staphylococcus aureus is one of the important food-borne pathogens that cause bacterial food poisoning. In my country, 20 to 25% of bacterial food poisoning cases are caused by Staphylococcus aureus . Staphylococcus aureus can produce a variety of toxins, some of which can withstand high temperatures, and a few micrograms of human body can cause food poisoning; some toxins can cause damage to human leukocytes and macrophages, and some can cause fiber in the blood. Protein deposition can also lead to a variety of infections such as toxic shock syndrome, osteomyelitis, suppurative endocarditis, bacteremia and fatal pneumonia in severe cases, threatening human health. Understanding the transmission rules and pathogenic evolution of this bacterium is the basis for effective prevention and control of food-borne diseases caused by this bacterium, and whether the appropriate standard strain is used determines the reliability of the research results.
  • the currently reported protective agent is basically used for qualitative storage of Staphylococcus aureus, and it is enough to ensure that there are still viable bacteria within the shelf life.
  • Xue Lei et al. used soluble starch, skimmed milk powder, trehalose, maltodextrin, sodium glutamate, sodium ascorbate, and phosphate buffer as the freeze-drying protective agent for Staphylococcus aureus to prepare proficiency test samples. Only 51.74%, and can not be quantitatively stored.
  • Another example is Chinese patent CN106497791A, which uses 2%-6% trehalose, 12%-18% sodium glutamate, 0.5%-1.5% L-cysteine hydrochloride, and 0.25%-0.75% bovine serum albumin.
  • the lyophilized survival rate of Staphylococcus aureus was 99.82%, but the protective agent was not investigated for long-term storage.
  • Another example is Chinese patent CN102140423B, which uses 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, it is of great significance to provide a lyophilized protective agent that can store quantitative Staphylococcus aureus for a long time.
  • the present invention provides 4 standard strains of Staphylococcus aureus and applications thereof.
  • the four food-borne Staphylococcus aureus isolates provided by the present invention have typical physiological and biochemical characteristics of Staphylococcus aureus, and the information of sample source, genetic background, drug resistance, virulence gene and molecular detection target is clear , which can better reflect the genetic background of the bacteria in China.
  • the technical scheme adopted in the present invention is:
  • Staphylococcus aureus which is Staphylococcus aureus Sta3702B3, isolated from chicken wing samples, and its classification name is Staphylococcus aureus, which has been preserved in Guangdong Province Microorganisms on January 8, 2020 Culture Collection Center, the deposit number is GDMCC 60841.
  • Staphylococcus aureus which is Staphylococcus aureus Sta403, isolated from beef samples, and its classification name is Staphylococcus aureus, which has been preserved in Guangdong Province Microorganisms on October 27, 2019 Culture Collection Center, the deposit number is GDMCC 60843.
  • Staphylococcus aureus Sta4127A1 isolated from prawn samples, and its classification name is Staphylococcus aureus, which has been preserved in Guangdong Province Microorganisms on October 27, 2019 Culture Collection Center, the deposit number is GDMCC 60844.
  • Staphylococcus aureus Sta1942-0 isolated from frozen chicken wing samples, and its classification name is Staphylococcus aureus, which has been preserved on October 27, 2019 in Guangdong Provincial Microbial Culture Collection Center, the preservation number is GDMCC 60845.
  • the staphylococcus aureus whose deposit number is GDMCC60841 is methicillin-resistant staphylococcus aureus (MRSA), and its molecular type is ST9-t899-SCCmec_type_IVb, carrying the following virulence genes: sec, seg , seh, sei, sem, sen, seo, sep, and ser; antibiotics tolerated include: amoxicillin/clavulanate, ampicillin, cefepime, cefoxitin, penicillin G, ceftazidime, gentamicin , kanamycin, streptomycin, chloramphenicol, clindamycin, erythromycin, telithromycin, ciprofloxacin, norfloxacin, tetracycline and co-trimoxazole.
  • MRSA methicillin-resistant staphylococcus aureus
  • the staphylococcus aureus whose deposit number is GDMCC60843 is methicillin-resistant staphylococcus aureus (MRSA), and its molecular type is ST9-t899-SCCmec_type_IVb, carrying the following virulence genes: sea, seg , sei, sek, sem, seo, sep, and seq; antibiotics tolerated include: amoxicillin/clavulanic acid, ampicillin, cefepime, cefoxitin, penicillin G, ceftazidime, gentamicin, carbamazepine Anamycin, streptomycin, chloramphenicol, clindamycin, erythromycin, telithromycin, ciprofloxacin, norfloxacin, tetracycline, and quinupristin/dalfopristin.
  • MRSA methicillin-resistant staphylococcus aureus
  • the staphylococcus aureus whose deposit number is GDMCC60844 is methicillin-resistant staphylococcus aureus (MRSA), and its molecular type is ST59-t437-SCCmec_type_IVa, carrying the following virulence genes: seb, sec , seg, sei, sej, sek, sel, sem, sep, seq, and ser; antibiotics tolerated include: amoxicillin/clavulanate, ampicillin, cefoxitin, penicillin G, ceftazidime, kanamycin , streptomycin, clindamycin, erythromycin, telithromycin, and fenomycin.
  • MRSA methicillin-resistant staphylococcus aureus
  • the molecular type of Staphylococcus aureus with the deposit number GDMCC60845 is ST4456-t13849, carrying the following virulence genes: seb, sec, seg, sei, sej, sek, sel, sem, sep, seq and ser; antibiotics to which it is tolerated include: ampicillin, penicillin G, streptomycin, quinupristin/dalfopristin, and brownomycin.
  • the newly obtained Staphylococcus aureus of the present invention is analyzed and compared by pan-genome to obtain a specific molecular target of the present invention, which can specifically detect the Staphylococcus aureus of the present invention, and has strong specificity, and the molecular target includes: The nucleotide sequences shown in SEQ ID NO.1 ⁇ 4.
  • the staphylococcus aureus with the deposit number GDMCC60841 contains the nucleotide sequence as shown in SEQ ID NO.1; the staphylococcus aureus with the deposit number GDMCC 60843 contains as shown in SEQ ID NO.2
  • the nucleotide sequence of the staphylococcus aureus with the deposit number GDMCC 60844 contains the nucleotide sequence shown in SEQ ID NO.3;
  • the staphylococcus aureus with the deposit number GDMCC 60845 contains the nucleotide sequence as shown in SEQ ID NO. .4 Nucleotide sequence shown.
  • the present invention also provides a set of primers for detecting the specific molecular target, and the PCR primer sequences for the amplification of the nucleotide sequence shown in SEQ ID NO.1 are shown in SEQ ID NO.5-6;
  • the PCR primer sequences amplified for the nucleotide sequence shown in SEQ ID NO.2 are shown in SEQ ID NO.7-8;
  • the PCR primers amplified for the nucleotide sequence shown in SEQ ID NO.3 The sequences are shown in SEQ ID NO.9-10;
  • the PCR primer sequences amplified for the nucleotide sequence shown in SEQ ID NO.4 are shown in SEQ ID NO.11-12.
  • the present invention also provides a freeze-drying protective agent for preparing and quantifying the standard strain of Staphylococcus aureus of the present invention, which is characterized by comprising 0.1-10 parts by mass of sodium alginate and 3-15 parts by mass of skimmed milk powder , 0.1-3 parts by mass of phytic acid and 0.1-3 parts by mass of reduced glutathione, and 0.2-4 parts by mass of glycine.
  • the sodium alginate has a polyhydroxy structure, which can interact with the phosphoric acid group in the cell membrane phospholipid of the bacterial cell or with the bacterial cell during the freezing or drying process.
  • Protein polar groups form hydrogen bonds that protect the integrity of cell membranes and protein structure and function.
  • Skim milk powder can be wrapped in the outer layer of bacterial cells to protect the bacteria.
  • Glycine ions are acid-base amphoteric and therefore suppress pH changes of the solution during freeze-drying. Reduced glutathione and phytate act as antioxidants, reducing the activity of cellular oxidase during freeze-drying process and long-term storage, preventing oxidative deterioration of freeze-dried products.
  • the Staphylococcus aureus strain of the invention has the standard microscopic morphology and physiological and biochemical characteristics of the Staphylococcus aureus, and can be used to test the accuracy of the Staphylococcus aureus color plate. Its genetic background is clear, including the source of strain isolation, sampling date and location, biochemical identification characteristics, virulence gene carrying status, antibiotic resistance characteristics, MLST typing characteristics, etc. It is convenient for scientific research, production and other fields to be used as a reference for different research directions and controls; in addition, these strains carry specific gene targets and are identifiable.
  • strains Compared with other types of standard strains of this species, these strains have the most typical biochemical reactions of the food source Staphylococcus aureus isolated in China, which can reflect the strains of the food source epidemic background in China, and are representative to a certain extent. Strains are used in scientific research.
  • the inventor provides a long-term storage lyoprotectant.
  • the protective agent has the following advantages: good molding, beautiful appearance and good water solubility, can be completely dissolved within 1-2 seconds; freeze-drying survival rate can reach more than 90%; The quantitative value does not change, and it can be used for long-term preservation of quantitative quality control strains.
  • Fig. 1 is the colony morphology diagram of the standard strain of Staphylococcus aureus.
  • Figure 2 is a morphological view of the standard strain of Staphylococcus aureus strains observed under microscope.
  • Figure 3 is a schematic diagram of the biochemical identification of API Staph of Staphylococcus aureus strains Sta3702B3, Sta403, Sta4127A1 and Sta1942-0.
  • Figure 4 shows the unique gene fragments of Staphylococcus aureus strains Sta3702B3, Sta403, Sta4127A1 and Sta1942-0 and PCR amplification of other Staphylococcus aureus; M: DL2000Marker; Lane 1-9: MRSA food isolate; Lane 10- 19: MSSA food isolate; Lane 20: Staphylococcus aureus ATCC6538; Lane 20: Staphylococcus aureus ATCC25923; Lane 21: Staphylococcus aureus ATCC29213; +: Staphylococcus aureus standard strain; C: blank control.
  • Figure 5 shows the unique gene fragments of Staphylococcus aureus strains Sta3702B3, Sta403, Sta4127A1 and Sta1942-0 and the PCR amplification map of other Staphylococcus; M: DL2000Marker; Lane 1: Staphylococcus saprophyticus; Lane 2: Secondary Staphylococcus ; Lane 3: Staphylococcus squirrels; Lane 4: Staphylococcus ischia; Lane 5: Staphylococcus stutzeri; Lane 6: Staphylococcus simianus; Lane 7: Staphylococcus pasteureus; Staphylococcus hemolyticus; Lane 11: Staphylococcus wornerii; Lane 12: Staphylococcus hominis; Lane 13: Staphylococcus rostri; Lane 14: Staphylococcus ludens; Lane 15-16: Staphylococcus suis; Lane 17: Staphylococcus epidermidis Coccus; Lane 18: St
  • Figure 6 is a PCR amplification diagram of the unique gene fragments and other strains of Staphylococcus aureus strains Sta3702B3, Sta403, Sta4127A1 and Sta1942-0.
  • M DL 2000Marker; Lane 1-2: Vibrio parahaemolyticus; Lane 3-6: Listeria monocytogenes; Lane 7-9: Pseudomonas aeruginosa; Lane 10-13: Escherichia coli; Lane 14- 16: Yersinia enterica; Lane 17-19: Salmonella; Lane 20-22: Cronobacter sakazakii; Lane 23-24: Campylobacter jejuni; Lane 25-27: Bacillus cereus; Lane 28: Bacillus thuringiensis; Lane29: Bacillus subtilis; +: Standard strain of Staphylococcus aureus; C: blank control.
  • Figure 7 is the change curve of the quantitative quality control bacteria of Staphylococcus aureus during the storage period.
  • the target colonies were transferred from the NA plates to brain heart infusion nutrient broth (BHI) and recovered at 37°C overnight. Under sterile conditions, the bacterial solution was added to a tube with a final concentration of 40% glycerol, stored in a -40°C refrigerator, and stored in a freeze-dried tube.
  • BHI brain heart infusion nutrient broth
  • the purified colonies can be identified in terms of morphological characteristics, physiology and biochemistry, serotype and molecular biology.
  • Staphylococcus aureus A total of 6 strains of Staphylococcus aureus were isolated, namely Sta3702B3, Sta403, Sta4127A1 and Sta1942-0, which were stored in the Guangdong Provincial Microbial Culture Collection Center, and the address is the Provincial Institute of Microbiology, No. 100, Xianlie Middle Road, Guangzhou, China.
  • the preservation dates are October 27, 2019 and January 8, 2020, and the preservation numbers are GDMCC 60841, GDMCC 60843, GDMCC60844, and GDMCC 60845.
  • Staining microscopy smear suspicious colonies, carry out Gram staining, and observe the morphology by microscopy. Staphylococcus aureus was Gram-positive and formed a prototype grape cluster under the microscope ( Figure 2).
  • Plasma coagulase test inoculate a single colony into 5 mL of BHI medium and culture at 37°C for 18-24h. Aspirate 1 mL of the culture medium, add it to plasma coagulase, and incubate at 37°C. After 2.5 hours, observe once every hour whether the coagulation is coagulated. If it does not coagulate after 6 hours, incubate overnight to observe and verify.
  • API Staph identification scrape a single pink colony from the chromogenic plate, prepare a cell suspension with appropriate turbidity with normal saline, and use the API Staph biochemical identification reagent strip for identification. The results are shown in Figure 3 and Table 2.
  • the KB method was used to confirm the drug susceptibility of Staphylococcus aureus strains. After activation by NA plate, add physiological saline to dilute to a final concentration of 1 ⁇ 10 7 cfu/mL and spread on MH plate. After the bacterial solution is dry, paste the antibiotic paper on the surface of the medium and cultivate 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: amoxicillin-clavulanic acid (AMC, 30 ⁇ g), ampicillin (AMP, 10 ⁇ g), cefepime (FEP, 10 ⁇ g), cefoxitin (FOX, 30 ⁇ g), penicillin (P, 10U), Ceftazidime (CAZ, 30 ⁇ g), Amikacin (AK, 30 ⁇ g), Gentamicin (CN, 10 ⁇ g), Kanamycin (K, 30 ⁇ g), Streptomycin (S, 25 ⁇ g), Chloramphenicol ( C, 30 ⁇ g), clindamycin (DA, 2 ⁇ g), erythromycin (E, 15 ⁇ g), telithromycin (TEL, 15 ⁇ g), ciprofloxacin (CIP, 5 ⁇ g), norfloxacin (NOR, 10 ⁇ g), tetracycline (TE, 30 ⁇ g), linezolid (LZD, 30 ⁇ g), rifampicin (RD, 5 ⁇ g), co-trim
  • Multi-locus sequence typing (MLST), Staphylococcus protein A (SPA) typing, SCCmec typing and related gene detection were performed on Staphylococcus aureus strains respectively.
  • MLST typing is mainly based on the sequence analysis of seven S. aureus housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi and yqil).
  • the 7 pairs of primers used were synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd. (see Table 3 for primer sequences).
  • PCR amplification conditions were as follows: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 2 min, and 35 cycles; the final extension was 72°C for 10 min.
  • the reaction system (25 ⁇ L) contains: 12.5 ⁇ l 2 ⁇ DreamTaq mastermix, 8.5 ⁇ l ultrapure water, 80 ng template DNA, 0.5 ⁇ M upstream and downstream primers.
  • the DNA product was purified with a PCR purification kit (Qiagen, Genman), and the purified PCR product was entrusted to Beijing Liuhe Huada Gene Technology Co., Ltd. for sequence determination.
  • the number and ST type of each housekeeping gene were aligned with the Staphylococcus aureus MLST database ( https://pubmlst.org/saureus/ ).
  • Staphylococcus protein A is an integral part of the cell wall of Staphylococcus aureus, including the Fc binding region, the X domain and the C-terminal 3 regions.
  • the X region is composed of a variable number of 24bp repeat sequences, showing gene polymorphisms .
  • primers and amplification methods refer to previous literature reports. The primers used were synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd. (see Table 3 for primer sequences).
  • PCR amplification conditions were as follows: pre-denaturation at 80 °C for 5 min, denaturation at 94 °C for 45 s, annealing at 60 °C for 45 s, extension at 72 °C for 2 min, a total of 35 cycles, and finally extended at 72 °C for 10 min.
  • the reaction system (25 ⁇ L) contains: 12.5 ⁇ l 2 ⁇ DreamTaq mastermix, 9.5 ⁇ l ultrapure water, 40 ng template DNA, 0.5 ⁇ M upstream and downstream primers.
  • the amplified product was sequenced, and the repeat sequence of the spa gene (24p was a unit) was compared with the existing type in the database ( http://spaserver2.ridom.de ) to obtain the spa type.
  • PCR was used to confirm whether these strains contained mecA/mecC genes.
  • the mecA/mecC gene encodes penicillin-binding protein 2a (PBP2a), which makes the related strains have a very low affinity for ⁇ -lactam antibiotics.
  • PBP2a penicillin-binding protein 2a
  • PBP2a is not inhibited and replaces other PBPs, which play a role in catalyzing cell wall synthesis, allowing bacteria to survive.
  • the primers and amplification methods of mecA and mecC genes were reported in the previous literature.
  • PCR amplification conditions are as follows: 95°C pre-denaturation for 5 min, 95°C denaturation for 45s, 56°C annealing for 45s, 72°C extension for 50s, for 35 cycles; and a final extension at 72°C for 8 min.
  • the reaction system (25 ⁇ L) contains: 12.5 ⁇ l 2 ⁇ DreamTaq mastermix, 9.5 ⁇ l ultrapure water, 40 ng template DNA, 0.5 ⁇ M upstream and downstream primers. 5 ⁇ L of PCR product was loaded on 2.0% agarose gel for electrophoresis separation (120V, 25min), using 2000bp DNA Marker.
  • the 16 pairs of primers used were synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd. (see Table 5 for primer sequences). These 16 pairs of primers can be used for TypeI, TypeII, TypeIII, TypeIVa, TypeIVb, TypeIVc, TypeIVd and TypeV identification.
  • the reaction system (50 ⁇ L) contains: 23 ⁇ l 2 ⁇ DreamTaq mastermix, 9 ⁇ l ultrapure water, 80ng template DNA, 0.5 ⁇ M upstream and downstream primers.
  • PCR amplification conditions are as follows: 94°C pre-denaturation for 5 min, 95°C denaturation for 45s, 65°C annealing for 45s, 72°C extension for 90s, for 10 cycles; 95°C denaturation for 45s, 55°C annealing for 45s, 72°C extension for 90s, 25 cycles Cycle; final extension at 72°C for 10 min. 10 ⁇ L of PCR product was loaded on 2.0% agarose gel for electrophoresis separation (120V, 40min), using 100pb DNA ladder Marker.
  • enterotoxins and enterotoxoids common to Staphylococcus aureus (sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq, ser, seu), toxic shock syndrome toxin 1 (tsst-1) and genes encoding leukocidin (pvl) were detected by PCR respectively.
  • enterotoxins and enterotoxoids common to Staphylococcus aureus (sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq, ser, seu)
  • tsst-1 toxic shock syndrome toxin 1
  • pvl leukocidin
  • the reaction system (50 ⁇ L) contains: 23 ⁇ l 2 ⁇ DreamTaq mastermix, 9 ⁇ l ultrapure water, 80 ng template DNA, 0.5 ⁇ M upstream and downstream primers.
  • PCR amplification conditions are as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 45s, annealing at 65°C [ ⁇ T-1°C] for 45s, extension at 72°C for 90s, for 15 cycles; denaturation at 94°C for 45s, annealing at 50°C for 45s, 72 90 s extension at °C for 20 cycles; a final extension at 72 °C for 10 min.
  • 10 ⁇ L of the PCR product was loaded on a 2.0% agarose gel for electrophoresis separation (120V, 40min) using a 2000bp DNA Marker.
  • strains can be grown in TSA, BHI and NA media.
  • Sta3702B3, Sta403 and Sta4127A1 were methicillin-resistant Staphylococcus aureus, the related strains contained mecA gene, the sequence was shown in SEQ ID No: 13, and the SCCmec types were TypeIII, TypeIVb and TypeIVa respectively.
  • the non-essential genes peculiar to these four strains were obtained mainly based on the results of pan-genome analysis of Staphylococcus aureus.
  • a total of 564 representative Staphylococcus aureus (including analysis strains) genome sequences were selected for pan-genome analysis.
  • the pan-genome was analyzed by the MP method in the prokaryotic pan-genome automated analysis software (Pan-Genomics Analysis Pipeline, PGAP), and the analysis results were processed by the local Perl script to obtain the core genes and non-core gene information of all strains.
  • the unique gene islands (characteristic sequences) carried by 2 strains of Staphylococcus aureus of the present invention are as follows:
  • serial number strain number virulence gene carrier 1 Sta3702B3 sec-seg-seh-sei-sem-sen-seo-sep-ser 2 Sta403 sea-seg-sei-sek-sem-seo-sep-seq 3 Sta4127A1 seb-sec-seg-sei-sej-sek-sel-sem-sep-seq-ser 4 Sta1942-0 seb-sec-seg-sei-sej-sek-sel-sem-sep-seq-ser
  • the present invention also provides a lyophilized protective agent for Staphylococcus aureus, and provides several specific examples (Exaphylococcus aureus, and provides several specific examples (Exaphylococcus aureus, and provides several specific examples (Exaphylococcus aureus, and provides several specific examples (Exaphylococcus aureus, and provides several specific examples (Exaphylococcus aureus, and provides several specific examples (Exaphylococcus aureus, and provides several specific examples (Exaphylococcus aureus, and provides several specific examples (Exaphylococcus aureus, and provides several specific examples (Exaphylococcus aureus, and provides several specific examples (Exaphylococcus aureus, and provides several specific examples (Exaphylococcus aureus, and provides several specific examples (Exaphylococcus aureus, and provides several specific examples (Exaphylococcus aureus, and provides several specific examples (Exaphylococcus aureus, and provides several specific examples (
  • a freeze-drying protective agent for Staphylococcus aureus comprising 3 parts by mass of sodium alginate, 10 parts by mass of skimmed milk powder, 0.2 parts by mass of phytic acid and 0.1 part by mass of reduced glutathione, 0.2 part by mass of glycine.
  • a lyophilized protective agent for Staphylococcus aureus comprising 5 parts by mass of sodium alginate, 12 parts by mass of skimmed milk powder, 0.5 parts by mass of phytic acid and 0.2 parts by mass of reduced glutathione, 2 parts by mass of glycine.
  • a lyophilized protective agent for Staphylococcus aureus comprising 5 parts by mass of sodium alginate, 9 parts by mass of skimmed milk powder, 1 part by mass of phytic acid and 0.3 part by mass of reduced glutathione, 1 part by mass of glycine.
  • the lyophilized protective agent does not contain reduced glutathione, but only contains 3 parts by mass of sodium alginate, 10 parts by mass of skim milk powder, 0.2 parts by mass of phytic acid, 0.2 parts by mass of Glycine.
  • the freeze-drying protection agent does not contain sodium alginate, but only contains 10 parts by mass of skim milk powder, 0.2 parts by mass of phytic acid, 0.1 parts by mass of reduced glutathione, and 0.2 parts by mass of glycine .
  • the freeze-drying protective agent contained only 10 parts by mass of skimmed milk powder.
  • the preparation method of the standard strain bacteria powder is as follows: after the bacteria are recovered, it is inserted into the shake flask and cultivated to 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. bottle, 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 the freeze dryer, perform automatic capping, ensure the complete vacuum state of the sample, and store it at 2-8 °C.

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Abstract

Disclosed in the present invention are Staphylococcus aureus standard strains, which respectively have deposit numbers of GDMCC 60841, GDMCC 60843, GDMCC 60844 and GDMCC 60845, and can be used in different fields such as food, medicine and clinical examination. The present invention also relates to a specific target gene for detecting and identifying the aforementioned four Staphylococcus aureus standard strains and a corresponding PCR primer thereof. Further provided in the present invention is a lyoprotectant for Staphylococcus aureus, which can be used for long-term storage of the standard strains of the present invention.

Description

含有特异性分子靶标的金黄色葡萄球菌标准菌株及其检测和应用Standard Strain of Staphylococcus aureus Containing Specific Molecular Target and Its Detection and Application 技术领域technical field
本发明涉及生物工程技术领域，具体涉及含有特异性分子靶标的金黄色葡萄球菌标准菌株及其检测和应用。The invention relates to the technical field of bioengineering, in particular to a standard strain of Staphylococcus aureus containing a specific molecular target and its detection and application.
背景技术Background technique
金黄色葡萄球菌(Staphylococcus aureus,S.aureus)是引起细菌性食物中毒的重要食源性致病菌之一，在我国，20～25％的细菌性食物中毒病例是由金黄色葡萄球菌引起的。金黄色葡萄球菌可产生多种毒素，有些毒素可耐高温，人体摄入几微克便可导致食物中毒；有的毒素可以导致人的白细胞和巨噬细胞的损坏，有的可以导致血液中的纤维蛋白沉积，严重时还可导致中毒性休克综合征、骨髓炎、化脓性心内膜炎、菌血症及致死性肺炎等多种感染，威胁人类健康。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。Staphylococcus aureus (S. aureus) is one of the important food-borne pathogens that cause bacterial food poisoning. In my country, 20 to 25% of bacterial food poisoning cases are caused by Staphylococcus aureus . Staphylococcus aureus can produce a variety of toxins, some of which can withstand high temperatures, and a few micrograms of human body can cause food poisoning; some toxins can cause damage to human leukocytes and macrophages, and some can cause fiber in the blood. Protein deposition can also lead to a variety of infections such as toxic shock syndrome, osteomyelitis, suppurative endocarditis, bacteremia and fatal pneumonia in severe cases, threatening human health. Understanding the transmission rules and pathogenic evolution of this bacterium is the basis for effective prevention and control of food-borne diseases caused by this bacterium, and whether the appropriate standard strain is used determines the reliability of the research results.
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国外临床来源的菌株，并不能很好地反映该菌在食品中的传播特征，中国作为世界重要的食品消费国，食品中金黄色葡萄球菌的检出率一直处于不低的水平，却缺乏代表性的分离株作为具有自主知识产权的标准菌株来研究该菌在中国食品中的遗传结构和传播规律。At present, most of the standard strains used to study the evolutionary law and pathogenicity of the bacteria are clinically sourced from abroad, which cannot well reflect the spread of the bacteria in food. China is an important food consumer in the world. The detection rate of Staphylococcus aureus has always been at a high level, but there is a lack of representative isolates as standard strains with independent intellectual property rights to study the genetic structure and transmission rules of this bacteria in Chinese food.
目前报道的所采用的保护剂基本用于定性储存金黄色葡萄球菌，在保质期内能够保证还有活菌存在即可。如薛蕾等人采用可溶性淀粉、脱脂奶粉、海藻糖、麦芽糊精、谷氨酸钠、抗坏血酸钠、磷酸盐缓冲液作为金黄色葡萄球菌冷冻干燥保护剂制备能力验证样品，其冻干存活率仅为51.74％，且不能定量储存。又如中国专利CN106497791A采用2％-6％海藻糖,谷氨酸钠12％-18％，L-半胱氨酸盐酸盐0.5％-1.5％，牛血清白蛋白0.25％-0.75％可使金黄色葡萄球菌冻干存活率达99.82％，但该保护剂并未进行长期储存的考察。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，提供一种可长期保存定量化金黄色葡萄球菌的冻干保护剂具有重要意义。The currently reported protective agent is basically used for qualitative storage of Staphylococcus aureus, and it is enough to ensure that there are still viable bacteria within the shelf life. For example, Xue Lei et al. used soluble starch, skimmed milk powder, trehalose, maltodextrin, sodium glutamate, sodium ascorbate, and phosphate buffer as the freeze-drying protective agent for Staphylococcus aureus to prepare proficiency test samples. Only 51.74%, and can not be quantitatively stored. Another example is Chinese patent CN106497791A, which uses 2%-6% trehalose, 12%-18% sodium glutamate, 0.5%-1.5% L-cysteine hydrochloride, and 0.25%-0.75% bovine serum albumin. The lyophilized survival rate of Staphylococcus aureus was 99.82%, but the protective agent was not investigated for long-term storage. Another example is Chinese patent CN102140423B, which uses 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, it is of great significance to provide a lyophilized protective agent that can store quantitative Staphylococcus aureus for a long time.
发明内容SUMMARY OF THE INVENTION
为克服现有技术的不足，本发明提供了4株金黄色葡萄球菌标准菌株及其应用。本发明提供的4株中国地区食源性金黄色葡萄球菌分离菌株，具有典型的金黄色葡萄球菌生理生化特性，且样品来源、遗传背景、耐药性、毒力基因和分子检测靶标等信息清晰，能较好地反映该菌在中国地区的遗传背景。In order to overcome the deficiencies of the prior art, the present invention provides 4 standard strains of Staphylococcus aureus and applications thereof. The four food-borne Staphylococcus aureus isolates provided by the present invention have typical physiological and biochemical characteristics of Staphylococcus aureus, and the information of sample source, genetic background, drug resistance, virulence gene and molecular detection target is clear , which can better reflect the genetic background of the bacteria in China.
为实现上述目的，本发明采取的技术方案为：To achieve the above object, the technical scheme adopted in the present invention is:
提供一株金黄色葡萄球菌，是金黄色葡萄球菌(Staphylococcus aureus)Sta3702B3，分离自鸡翅样品，其分类命名为金黄色葡萄球菌(Staphylococcus aureus)，已于2020年1月8号保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC 60841。Provide a Staphylococcus aureus, which is Staphylococcus aureus Sta3702B3, isolated from chicken wing samples, and its classification name is Staphylococcus aureus, which has been preserved in Guangdong Province Microorganisms on January 8, 2020 Culture Collection Center, the deposit number is GDMCC 60841.
提供一株金黄色葡萄球菌，是金黄色葡萄球菌(Staphylococcus aureus)Sta403，分离自牛肉样品，其分类命名为金黄色葡萄球菌(Staphylococcus aureus)，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 60843。Provide a Staphylococcus aureus, which is Staphylococcus aureus Sta403, isolated from beef samples, and its classification name is Staphylococcus aureus, which has been preserved in Guangdong Province Microorganisms on October 27, 2019 Culture Collection Center, the deposit number is GDMCC 60843.
提供一株金黄色葡萄球菌，是金黄色葡萄球菌(Staphylococcus aureus)Sta4127A1，分离自对虾样品，其分类命名为金黄色葡萄球菌(Staphylococcus aureus)，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 60844。Provide a strain of Staphylococcus aureus, which is Staphylococcus aureus Sta4127A1, isolated from prawn samples, and its classification name is Staphylococcus aureus, which has been preserved in Guangdong Province Microorganisms on October 27, 2019 Culture Collection Center, the deposit number is GDMCC 60844.
提供一株金黄色葡萄球菌，是金黄色葡萄球菌(Staphylococcus aureus)Sta1942-0，分离自冻鸡翅样品， 其分类命名为金黄色葡萄球菌(Staphylococcus aureus)，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 60845。Provide a strain of Staphylococcus aureus, which is Staphylococcus aureus Sta1942-0, isolated from frozen chicken wing samples, and its classification name is Staphylococcus aureus, which has been preserved on October 27, 2019 in Guangdong Provincial Microbial Culture Collection Center, the preservation number is GDMCC 60845.
进一步地，所述保藏编号为GDMCC60841的金黄色葡萄球菌为耐甲氧西林金黄色葡萄球菌(MRSA)，其分子型别为ST9-t899-SCCmec_type_IVb，携带如下所示的毒力基因：sec、seg、seh、sei、sem、sen、seo、sep和ser；其耐受的抗生素包括：阿莫西林/克拉维酸、氨苄西林、头孢吡肟、头孢西丁、青霉素G、头孢他啶、庆大霉素、卡那霉素、链霉素、氯霉素、克林霉素、红霉素、泰利霉素、环丙沙星、诺氟沙星、四环素和复方新诺明。Further, the staphylococcus aureus whose deposit number is GDMCC60841 is methicillin-resistant staphylococcus aureus (MRSA), and its molecular type is ST9-t899-SCCmec_type_IVb, carrying the following virulence genes: sec, seg , seh, sei, sem, sen, seo, sep, and ser; antibiotics tolerated include: amoxicillin/clavulanate, ampicillin, cefepime, cefoxitin, penicillin G, ceftazidime, gentamicin , kanamycin, streptomycin, chloramphenicol, clindamycin, erythromycin, telithromycin, ciprofloxacin, norfloxacin, tetracycline and co-trimoxazole.
进一步地，所述保藏编号为GDMCC60843的金黄色葡萄球菌为耐甲氧西林金黄色葡萄球菌(MRSA)，其分子型别为ST9-t899-SCCmec_type_IVb，携带如下所示的毒力基因：sea、seg、sei、sek、sem、seo、sep和seq；其耐受的抗生素包括：阿莫西林/克拉维酸、氨苄西林、头孢吡肟、头孢西丁、青霉素G、头孢他啶、庆大霉素、卡那霉素、链霉素、氯霉素、克林霉素、红霉素、泰利霉素、环丙沙星、诺氟沙星、四环素和喹奴普汀/达福普汀。Further, the staphylococcus aureus whose deposit number is GDMCC60843 is methicillin-resistant staphylococcus aureus (MRSA), and its molecular type is ST9-t899-SCCmec_type_IVb, carrying the following virulence genes: sea, seg , sei, sek, sem, seo, sep, and seq; antibiotics tolerated include: amoxicillin/clavulanic acid, ampicillin, cefepime, cefoxitin, penicillin G, ceftazidime, gentamicin, carbamazepine Anamycin, streptomycin, chloramphenicol, clindamycin, erythromycin, telithromycin, ciprofloxacin, norfloxacin, tetracycline, and quinupristin/dalfopristin.
进一步地，所述保藏编号为GDMCC60844的金黄色葡萄球菌为耐甲氧西林金黄色葡萄球菌(MRSA)，其分子型别为ST59-t437-SCCmec_type_IVa，携带如下所示的毒力基因：seb、sec、seg、sei、sej、sek、sel、sem、sep、seq和ser；其耐受的抗生素包括：阿莫西林/克拉维酸、氨苄西林、头孢西丁、青霉素G、头孢他啶、卡那霉素、链霉素、克林霉素、红霉素、泰利霉素和褐霉素。Further, the staphylococcus aureus whose deposit number is GDMCC60844 is methicillin-resistant staphylococcus aureus (MRSA), and its molecular type is ST59-t437-SCCmec_type_IVa, carrying the following virulence genes: seb, sec , seg, sei, sej, sek, sel, sem, sep, seq, and ser; antibiotics tolerated include: amoxicillin/clavulanate, ampicillin, cefoxitin, penicillin G, ceftazidime, kanamycin , streptomycin, clindamycin, erythromycin, telithromycin, and fenomycin.
进一步地，所述保藏编号为GDMCC60845的金黄色葡萄球菌的分子型别为ST4456-t13849，携带如下所示的毒力基因：seb、sec、seg、sei、sej、sek、sel、sem、sep、seq和ser；其耐受的抗生素包括：氨苄西林、青霉素G、链霉素、喹奴普汀/达福普汀和褐霉素。Further, the molecular type of Staphylococcus aureus with the deposit number GDMCC60845 is ST4456-t13849, carrying the following virulence genes: seb, sec, seg, sei, sej, sek, sel, sem, sep, seq and ser; antibiotics to which it is tolerated include: ampicillin, penicillin G, streptomycin, quinupristin/dalfopristin, and brownomycin.
本发明从新获得的金黄色葡萄球菌通过泛基因组分析比较，获得本发明特异性的分子靶标，能特异性的检测出本发明金黄色葡萄球菌，具有较强的特异性，所述分子靶标包括如SEQ ID NO.1～4所示的核苷酸序列。The newly obtained Staphylococcus aureus of the present invention is analyzed and compared by pan-genome to obtain a specific molecular target of the present invention, which can specifically detect the Staphylococcus aureus of the present invention, and has strong specificity, and the molecular target includes: The nucleotide sequences shown in SEQ ID NO.1～4.
进一步地，所述保藏编号为GDMCC60841的金黄色葡萄球菌含有如SEQ ID NO.1所示的核苷酸序列；所述保藏编号为GDMCC 60843的金黄色葡萄球菌含有如SEQ ID NO.2所示的核苷酸序列；所述保藏编号为GDMCC 60844的金黄色葡萄球菌含有如SEQ ID NO.3所示的核苷酸序列；所述保藏编号为GDMCC 60845的金黄色葡萄球菌含有如SEQ ID NO.4所示的核苷酸序列。Further, the staphylococcus aureus with the deposit number GDMCC60841 contains the nucleotide sequence as shown in SEQ ID NO.1; the staphylococcus aureus with the deposit number GDMCC 60843 contains as shown in SEQ ID NO.2 The nucleotide sequence of the staphylococcus aureus with the deposit number GDMCC 60844 contains the nucleotide sequence shown in SEQ ID NO.3; the staphylococcus aureus with the deposit number GDMCC 60845 contains the nucleotide sequence as shown in SEQ ID NO. .4 Nucleotide sequence shown.
本发明还提供了一组用于检测所述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引物序列如SEQ ID NO.5～6所示；针对如SEQ ID NO.2所示的核苷酸序列扩增的PCR引物序列如SEQ ID NO.7～8所示；针对如SEQ ID NO.3所示的核苷酸序列扩增的PCR引物序列如SEQ ID NO.9～10所示；针对如SEQ ID NO.4所示的核苷酸序列扩增的PCR引物序列如SEQ ID NO.11～12所示。The present invention also provides a set of primers for detecting the specific molecular target, and the PCR primer sequences for the amplification of the nucleotide sequence shown in SEQ ID NO.1 are shown in SEQ ID NO.5-6; The PCR primer sequences amplified for the nucleotide sequence shown in SEQ ID NO.2 are shown in SEQ ID NO.7-8; the PCR primers amplified for the nucleotide sequence shown in SEQ ID NO.3 The sequences are shown in SEQ ID NO.9-10; the PCR primer sequences amplified for the nucleotide sequence shown in SEQ ID NO.4 are shown in SEQ ID NO.11-12.
本发明还提供了一种用于制备定量本发明所述金黄色葡萄球菌标准菌株的冻干保护剂，其特征在于，包括0.1～10质量份的海藻酸钠，3～15质量份的脱脂奶粉，0.1-3质量份的肌醇六磷酸和0.1-3质量份还原性谷胱甘肽，0.2-4质量份的甘氨酸。The present invention also provides a freeze-drying protective agent for preparing and quantifying the standard strain of Staphylococcus aureus of the present invention, which is characterized by comprising 0.1-10 parts by mass of sodium alginate and 3-15 parts by mass of skimmed milk powder , 0.1-3 parts by mass of phytic acid and 0.1-3 parts by mass of reduced glutathione, and 0.2-4 parts by mass of glycine.
本发明提供的可长期定量保存金黄色葡萄球菌的保护剂作用原理是：其中的海藻酸钠具有多羟基结构，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。甘氨酸离子具有酸、碱两性，因此在冷冻干燥过程中抑制溶液的pH变化。还原性谷胱甘肽和肌醇六磷酸作为抗氧化剂，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。The principle of action of the protective agent for long-term quantitative preservation of Staphylococcus aureus provided by the present invention is as follows: the sodium alginate has a polyhydroxy structure, which can interact with the phosphoric acid group in the cell membrane phospholipid of the bacterial cell or with the bacterial cell during the freezing or drying process. Protein polar groups form hydrogen bonds that protect the integrity of cell membranes and protein structure and function. Skim milk powder can be wrapped in the outer layer of bacterial cells to protect the bacteria. Glycine ions are acid-base amphoteric and therefore suppress pH changes of the solution during freeze-drying. Reduced glutathione and phytate act as antioxidants, reducing the activity of cellular oxidase during freeze-drying process and long-term storage, preventing oxidative deterioration of freeze-dried products.
本发明的有益效果：Beneficial effects of the present invention:
本发明的金黄色葡萄球菌菌株，具有标准的葡萄球菌菌体显微形态和生理生化特征，可用于检验金黄色葡萄球菌显色平板的准确性。其遗传背景清晰，包括菌株分离源、采样日期及地点、生化鉴定特征、毒力基因携带情况、抗生素耐药性特征、MLST分型特征等，便于科研、生产等领域用作不同研究方向的参考及对照；此外，这些菌株携带特异性基因靶点，具有可识别性。相较于其它类型该物种的标准菌株，这些菌株具有中国地区分离到的食品源金黄色葡萄球菌最为典型的生化反应，可反映中国地区食品源流行背景的菌株，具有一定的代表性，作为参考菌株用于科学研究。The Staphylococcus aureus strain of the invention has the standard microscopic morphology and physiological and biochemical characteristics of the Staphylococcus aureus, and can be used to test the accuracy of the Staphylococcus aureus color plate. Its genetic background is clear, including the source of strain isolation, sampling date and location, biochemical identification characteristics, virulence gene carrying status, antibiotic resistance characteristics, MLST typing characteristics, etc. It is convenient for scientific research, production and other fields to be used as a reference for different research directions and controls; in addition, these strains carry specific gene targets and are identifiable. Compared with other types of standard strains of this species, these strains have the most typical biochemical reactions of the food source Staphylococcus aureus isolated in China, which can reflect the strains of the food source epidemic background in China, and are representative to a certain extent. Strains are used in scientific research.
另外，针对本发明的金黄色葡萄球菌，发明人提供了一种长期保存的冻干保护剂。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90％以上；在-20℃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。In addition, for the Staphylococcus aureus of the present invention, the inventor provides a long-term storage lyoprotectant. The protective agent has the following advantages: good molding, beautiful appearance and good water solubility, can be completely dissolved within 1-2 seconds; freeze-drying survival rate can reach more than 90%; The quantitative value does not change, and it can be used for long-term preservation of quantitative quality control strains.
生物材料保藏biological material preservation
一株金黄色葡萄球菌(Staphylococcus aureus)Sta3702B3，其分类命名为金黄色葡萄球菌(Staphylococcus aureus)，已于2020年1月8号保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC 60841。A strain of Staphylococcus aureus Sta3702B3, which is classified as Staphylococcus aureus, has been deposited in the Guangdong Provincial Microbial Culture Collection Center on January 8, 2020, and the deposit number is GDMCC 60841.
一株金黄色葡萄球菌(Staphylococcus aureus)Sta403，其分类命名为金黄色葡萄球菌(Staphylococcus aureus)，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 60843。A strain of Staphylococcus aureus Sta403, its classification name is Staphylococcus aureus, has been deposited in the Guangdong Provincial Microbial Culture Collection Center on October 27, 2019, and the preservation number is GDMCC 60843.
一株金黄色葡萄球菌(Staphylococcus aureus)Sta4127A1，其分类命名为金黄色葡萄球菌(Staphylococcus aureus)，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 60844。A strain of Staphylococcus aureus Sta4127A1, which is classified as Staphylococcus aureus, has been deposited in the Guangdong Provincial Microbial Culture Collection Center on October 27, 2019, and the deposit number is GDMCC 60844.
一株金黄色葡萄球菌(Staphylococcus aureus)Sta1942-0，其分类命名为金黄色葡萄球菌(Staphylococcus aureus)，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 60845。A strain of Staphylococcus aureus Sta1942-0, which is classified as Staphylococcus aureus, has been deposited in the Guangdong Provincial Microbial Culture Collection Center on October 27, 2019, and the deposit number is GDMCC 60845 .
附图说明Description of drawings
图1为金黄色葡萄球菌菌株标准株的菌落形态图。Fig. 1 is the colony morphology diagram of the standard strain of Staphylococcus aureus.
图2为金黄色葡萄球菌菌株标准株的镜检观察形态图。Figure 2 is a morphological view of the standard strain of Staphylococcus aureus strains observed under microscope.
图3为金黄色葡萄球菌菌株Sta3702B3、Sta403、Sta4127A1和Sta1942-0的API Staph生化鉴定示意图。Figure 3 is a schematic diagram of the biochemical identification of API Staph of Staphylococcus aureus strains Sta3702B3, Sta403, Sta4127A1 and Sta1942-0.
图4为金黄色葡萄球菌菌株Sta3702B3、Sta403、Sta4127A1和Sta1942-0的特有基因片段及其他金黄色葡萄球菌PCR扩增图；其中M：DL2000Marker；Lane 1-9：MRSA食品分离株；Lane 10-19：MSSA食品分离株；Lane 20：金黄色葡萄球菌ATCC6538；Lane 20：金黄色葡萄球菌ATCC25923；Lane 21：金黄色葡萄球菌ATCC29213；+：金黄色葡萄球菌标准株；C：空白对照。Figure 4 shows the unique gene fragments of Staphylococcus aureus strains Sta3702B3, Sta403, Sta4127A1 and Sta1942-0 and PCR amplification of other Staphylococcus aureus; M: DL2000Marker; Lane 1-9: MRSA food isolate; Lane 10- 19: MSSA food isolate; Lane 20: Staphylococcus aureus ATCC6538; Lane 20: Staphylococcus aureus ATCC25923; Lane 21: Staphylococcus aureus ATCC29213; +: Staphylococcus aureus standard strain; C: blank control.
图5为金黄色葡萄球菌菌株Sta3702B3、Sta403、Sta4127A1和Sta1942-0的特有基因片段及其他葡萄球菌PCR扩增图；其中M：DL2000Marker；Lane 1：腐生葡萄球菌；Lane 2：继发性葡萄球菌；Lane 3：松鼠葡萄球菌；Lane 4：坐骨葡萄球菌；Lane 5：施氏葡萄球菌；Lane 6：猴葡萄球菌；Lane 7：巴氏葡萄球菌；Lane8：科氏葡萄球菌；Lane 9-10：溶血葡萄球菌；Lane 11：沃氏葡萄球菌；Lane 12：人葡萄球菌；Lane 13：罗斯特里葡萄球菌；Lane 14：路邓葡萄球菌；Lane 15-16：猪葡萄球菌；Lane 17：表皮葡萄球菌；Lane 18：溶血葡萄球菌；+：金黄色葡萄球菌标准株；C：空白对照。Figure 5 shows the unique gene fragments of Staphylococcus aureus strains Sta3702B3, Sta403, Sta4127A1 and Sta1942-0 and the PCR amplification map of other Staphylococcus; M: DL2000Marker; Lane 1: Staphylococcus saprophyticus; Lane 2: Secondary Staphylococcus ; Lane 3: Staphylococcus squirrels; Lane 4: Staphylococcus ischia; Lane 5: Staphylococcus stutzeri; Lane 6: Staphylococcus simianus; Lane 7: Staphylococcus pasteureus; Staphylococcus hemolyticus; Lane 11: Staphylococcus wornerii; Lane 12: Staphylococcus hominis; Lane 13: Staphylococcus rostri; Lane 14: Staphylococcus ludens; Lane 15-16: Staphylococcus suis; Lane 17: Staphylococcus epidermidis Coccus; Lane 18: Staphylococcus hemolytica; +: Standard strain of Staphylococcus aureus; C: blank control.
图6为金黄色葡萄球菌菌株Sta3702B3、Sta403、Sta4127A1和Sta1942-0的特有基因片段及其他菌属PCR扩增图。其中，M：DL 2000Marker；Lane 1-2：副溶血性弧菌；Lane 3-6：单增李斯特菌；Lane7-9：铜绿假单胞菌；Lane 10-13：大肠杆菌；Lane 14-16：小肠耶尔森氏菌；Lane 17-19：沙门氏菌；Lane 20-22：阪崎克罗诺杆菌；Lane 23-24：空肠弯曲菌；Lane 25-27：蜡样芽孢杆菌；Lane 28：苏云金芽孢杆菌；Lane29：枯草芽孢杆菌；+：金黄色葡萄球菌标准株；C：空白对照。Figure 6 is a PCR amplification diagram of the unique gene fragments and other strains of Staphylococcus aureus strains Sta3702B3, Sta403, Sta4127A1 and Sta1942-0. Among them, M: DL 2000Marker; Lane 1-2: Vibrio parahaemolyticus; Lane 3-6: Listeria monocytogenes; Lane 7-9: Pseudomonas aeruginosa; Lane 10-13: Escherichia coli; Lane 14- 16: Yersinia enterica; Lane 17-19: Salmonella; Lane 20-22: Cronobacter sakazakii; Lane 23-24: Campylobacter jejuni; Lane 25-27: Bacillus cereus; Lane 28: Bacillus thuringiensis; Lane29: Bacillus subtilis; +: Standard strain of Staphylococcus aureus; C: blank control.
图7为金黄色葡萄球菌定量质控菌储存期内的变化曲线。Figure 7 is the change curve of the quantitative quality control bacteria of Staphylococcus aureus during the storage period.
具体实施方式Detailed ways
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。In order to express the technical solutions of the present invention more clearly, the following further description is given in conjunction with specific embodiments, but cannot be used to limit the present invention, which are only some embodiments of the present invention.
实施例1金黄色葡萄球菌菌株的分离、鉴定和培养Example 1 Isolation, identification and cultivation of Staphylococcus aureus strains
对采集的样品同时进行定性和定量检测，检测方法在国标《食品微生物检验》GB 4789.10-2010的基础上略做调整。取样25g(mL)加入225mL生理盐水的无菌均质袋中均质摇匀，制成1:10的样品液，并从中取1mL加入到装有9mL 10％氯化钠胰酪胨大豆肉汤的试管中，制成1:100的样品液，按照上述方法制成1:1000的样品液；每个梯度3个平行，将样品液放置于恒温培养箱中37℃培养48h后，将每个梯度浓度的样品液分别划线于金黄色葡萄球菌显色板培养基上，37℃中培养24-48h。其典型的金黄色葡萄球菌菌落在显色平板上为粉色球型湿润边缘平整(图1)。Qualitative and quantitative tests were carried out on the collected samples at the same time, and the test methods were slightly adjusted on the basis of the national standard "Food Microbiological Inspection" GB 4789.10-2010. Sampling 25g (mL) and adding 225mL normal saline into a sterile homogenizing bag and shaking to make a 1:10 sample solution, and taking 1mL from it and adding it to 9mL 10% sodium chloride tryptone soybean broth In the test tube of 1:100, the sample solution of 1:100 was prepared, and the sample solution of 1:1000 was prepared according to the above method; each gradient was 3 parallel. The sample solutions of gradient concentrations were streaked on the Staphylococcus aureus color plate medium, and cultured at 37°C for 24-48h. Its typical Staphylococcus aureus colonies are pink globules with smooth edges on the chromogenic plate (Figure 1).
将目标菌落从NA平板上转接到脑心浸液营养肉汤(BHI)中，于37℃过夜复苏。在无菌条件下将菌液加 入终浓度为40％甘油管中，保存于-40℃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。The target colonies were transferred from the NA plates to brain heart infusion nutrient broth (BHI) and recovered at 37°C overnight. Under sterile conditions, the bacterial solution was added to a tube with a final concentration of 40% glycerol, stored in a -40°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.
共分离得到6株金黄色葡萄球菌，分别为金黄色葡萄球菌菌株Sta3702B3、Sta403、Sta4127A1以及Sta1942-0，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号省微生物所实验楼五楼，保藏日为2019年10月27号和2020年1月8号，保藏编号分别为GDMCC 60841，GDMCC 60843，GDMCC60844，GDMCC 60845。A total of 6 strains of Staphylococcus aureus were isolated, namely Sta3702B3, Sta403, Sta4127A1 and Sta1942-0, which were stored in the Guangdong Provincial Microbial Culture Collection Center, and the address is the Provincial Institute of Microbiology, No. 100, Xianlie Middle Road, Guangzhou, China. On the fifth floor of the building, the preservation dates are October 27, 2019 and January 8, 2020, and the preservation numbers are GDMCC 60841, GDMCC 60843, GDMCC60844, and GDMCC 60845.
上述菌株分别分离自中国南京市、汕头市和西安市等城市的不同食品类型样品中，具体的分离信息如表1所示。The above strains were isolated from samples of different food types in cities such as Nanjing, Shantou and Xi'an, China. The specific isolation information is shown in Table 1.
表1菌株的分离信息Table 1 Isolation information of strains
Figure PCTCN2021086878-appb-000001
Figure PCTCN2021086878-appb-000001
实施例2金黄色葡萄球菌菌株的生理生化特征和药敏特征Example 2 Physiological and biochemical characteristics and drug susceptibility characteristics of Staphylococcus aureus strains
染色镜检：将可疑菌落涂片，进行革兰氏染色，镜检观察形态。金黄色葡萄球菌为革兰氏阳性，显微镜下成原型葡萄串状(图2)。Staining microscopy: smear suspicious colonies, carry out Gram staining, and observe the morphology by microscopy. Staphylococcus aureus was Gram-positive and formed a prototype grape cluster under the microscope (Figure 2).
血浆凝固酶实验：接种单菌落至5mLBHI培养液中，于37℃培养18-24h。吸取培养液1mL，加入血浆凝固酶中，于37℃培养。2.5小时后，每一小时观察一次是否凝结，若6h后没凝固，培养过夜再观察验证。Plasma coagulase test: inoculate a single colony into 5 mL of BHI medium and culture at 37°C for 18-24h. Aspirate 1 mL of the culture medium, add it to plasma coagulase, and incubate at 37°C. After 2.5 hours, observe once every hour whether the coagulation is coagulated. If it does not coagulate after 6 hours, incubate overnight to observe and verify.
API Staph鉴定：从显色平板上刮取粉色单个菌落，用生理盐水制备成浊度适当的细胞悬浮液，使用API Staph生化鉴定试剂条鉴定，结果如图3和表2所示。API Staph identification: scrape a single pink colony from the chromogenic plate, prepare a cell suspension with appropriate turbidity with normal saline, and use the API Staph biochemical identification reagent strip for identification. The results are shown in Figure 3 and Table 2.
药敏特征分析：利用KB法进行金黄色葡萄球菌菌株的药敏确证。经NA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：阿莫西林克拉维酸(AMC，30μg)、氨苄西林(AMP，10μg)、头孢吡肟(FEP，10μg)、头孢西丁(FOX，30μg)、青霉素(P，10U)、头孢他啶(CAZ，30μg)、阿米卡星(AK，30μg)、庆大霉素(CN，10μg)、卡那霉素(K，30μg)、链霉素(S，25μg)、氯霉素(C，30μg)、克林霉素(DA，2μg)、红霉素(E，15μg)、泰利霉素(TEL，15μg)、环丙沙星(CIP，5μg)、诺氟沙星(NOR，10μg)、四环素(TE，30μg)，利奈唑胺(LZD，30μg)、利福平(RD，5μg)、复方新诺明(SXT，25μg)、喹奴普汀/达富普汀(QD，15μg)、替拉考宁(TEC，30μg)、呋喃妥因(F，300μg)和褐霉素(FD，10μg)。Staphylococcus aureusATCC25923和Escherichia coli ATCC25922作为质控菌株。 Analysis of drug susceptibility characteristics: The KB method was used to confirm the drug susceptibility of Staphylococcus aureus strains. After activation by NA plate, add physiological saline to dilute to a final concentration of 1 × 10 7 cfu/mL and spread on MH plate. After the bacterial solution is dry, paste the antibiotic paper on the surface of the medium and cultivate 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: amoxicillin-clavulanic acid (AMC, 30 μg), ampicillin (AMP, 10 μg), cefepime (FEP, 10 μg), cefoxitin (FOX, 30 μg), penicillin (P, 10U), Ceftazidime (CAZ, 30μg), Amikacin (AK, 30μg), Gentamicin (CN, 10μg), Kanamycin (K, 30μg), Streptomycin (S, 25μg), Chloramphenicol ( C, 30 μg), clindamycin (DA, 2 μg), erythromycin (E, 15 μg), telithromycin (TEL, 15 μg), ciprofloxacin (CIP, 5 μg), norfloxacin (NOR, 10μg), tetracycline (TE, 30μg), linezolid (LZD, 30μg), rifampicin (RD, 5μg), co-trimoxazole (SXT, 25μg), quinupristine/darfupristin (QD, 15 μg), tiraconine (TEC, 30 μg), nitrofurantoin (F, 300 μg), and fulgomycin (FD, 10 μg). Staphylococcus aureus ATCC25923 and Escherichia coli ATCC25922 were used as quality control strains.
表2金黄色葡萄球菌API Staph系统鉴定结果Table 2 Identification results of Staphylococcus aureus API Staph system
Figure PCTCN2021086878-appb-000002
Figure PCTCN2021086878-appb-000002
实施例3金黄色葡萄球菌菌株的分子遗传背景分析Example 3 Molecular genetic background analysis of Staphylococcus aureus strains
分别对金黄色葡萄球菌菌株进行了多位点序列分型(MLST)、葡萄球菌A蛋白(SPA)分型、SCCmec分型及相关基因的检测。Multi-locus sequence typing (MLST), Staphylococcus protein A (SPA) typing, SCCmec typing and related gene detection were performed on Staphylococcus aureus strains respectively.
(1)实验方法(1) Experimental method
MLST分型主要是基于7个金黄色葡萄球菌管家基因(arcC、aroE、glpF、gmk、pta、tpi和yqil)的序列进行分析。引物与扩增方法参考先前文献报道。所使用的7对引物由北京六合华大基因科技有限公司合成(引物序列见表3)。PCR扩增条件如下：94℃预变性5min，94℃变性30s，55℃退火30s，72℃延伸2min，进行35个循环；最后72℃延伸10min。反应体系(25μL)包含：12.5μl 2×DreamTaq mastermix，8.5μl超纯水，80ng模板DNA，0.5μM上下游引物。DNA产物用PCR纯化试剂盒进行纯化(Qiagen，Genman)，经过纯化的PCR产物委托北京六合华大基因科技有限公司进行序列测定。每种管家基因的编号和ST型都是通过金黄色葡萄球菌MLST数据库进行比对( https://pubmlst.org/saureus/)。 MLST typing is mainly based on the sequence analysis of seven S. aureus housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi and yqil). For primers and amplification methods, refer to previous literature reports. The 7 pairs of primers used were synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd. (see Table 3 for primer sequences). PCR amplification conditions were as follows: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 2 min, and 35 cycles; the final extension was 72°C for 10 min. The reaction system (25 μL) contains: 12.5 μl 2×DreamTaq mastermix, 8.5 μl ultrapure water, 80 ng template DNA, 0.5 μM upstream and downstream primers. The DNA product was purified with a PCR purification kit (Qiagen, Genman), and the purified PCR product was entrusted to Beijing Liuhe Huada Gene Technology Co., Ltd. for sequence determination. The number and ST type of each housekeeping gene were aligned with the Staphylococcus aureus MLST database ( https://pubmlst.org/saureus/ ).
葡萄球菌A蛋白(SPA)是金黄色葡萄球菌细胞壁的组成部分，包括Fc结合区，X域和C末端3个区域，X区域是由可变数量24bp重复序列组成的，呈现出基因多态性。引物与扩增方法参考先前文献报道。所使用的引物由北京六合华大基因科技有限公司合成(引物序列见表3)。PCR扩增条件如下：80℃预变性5min，94℃变性45s，60℃退火45s，72℃延伸2min，共进行35个循环，最后继续72℃延伸10min。反应体系(25μL)包含：12.5μl 2×DreamTaq mastermix，9.5μl超纯水，40ng模板DNA，0.5μM上下游引物。扩增得到的产物进行测序，选取spa基因的重复序列(24p为一个单元)与数据库( http://spaserver2.ridom.de)中已有的型别进行比对，得到spa型别。 Staphylococcus protein A (SPA) is an integral part of the cell wall of Staphylococcus aureus, including the Fc binding region, the X domain and the C-terminal 3 regions. The X region is composed of a variable number of 24bp repeat sequences, showing gene polymorphisms . For primers and amplification methods, refer to previous literature reports. The primers used were synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd. (see Table 3 for primer sequences). PCR amplification conditions were as follows: pre-denaturation at 80 °C for 5 min, denaturation at 94 °C for 45 s, annealing at 60 °C for 45 s, extension at 72 °C for 2 min, a total of 35 cycles, and finally extended at 72 °C for 10 min. The reaction system (25 μL) contains: 12.5 μl 2×DreamTaq mastermix, 9.5 μl ultrapure water, 40 ng template DNA, 0.5 μM upstream and downstream primers. The amplified product was sequenced, and the repeat sequence of the spa gene (24p was a unit) was compared with the existing type in the database ( http://spaserver2.ridom.de ) to obtain the spa type.
表3金黄色葡萄球菌MLST引物和spa引物及扩增片段Table 3 Staphylococcus aureus MLST primers and spa primers and amplified fragments
Figure PCTCN2021086878-appb-000003
Figure PCTCN2021086878-appb-000003
由于部分菌株对所选的β-酰胺类抗生素均产生耐药，为进一步确证其是否为耐甲氧西林金黄色葡萄球菌，采用PCR的方法确认这些菌株是否含有mecA/mecC基因。mecA/mecC基因可编码青霉素结合蛋白2a(PBP2a)，使相关菌株与β-内酰胺类抗生素亲和力极低。当其他PBP被β-内酰胺类抗生素抑制时，PBP2a不被抑制而替代其他PBP，起到催化细胞壁合成的作用，使细菌得以生存。mecA和mecC基因的引物与扩增方法参考先前文献报道。采用单重PCR的方法，引物序列见表4。PCR扩增条件如下：95℃预变性5min，95℃变性45s，56℃退火45s，72℃延伸50s，进行35个循环；最后72℃延伸8min。反应体系(25μL)包含：12.5μl 2×DreamTaq mastermix，9.5μl超纯水，40ng模板DNA，0.5μM上下游引物。5μL的PCR产物上样于2.0％琼脂糖凝胶进行电泳分离(120V，25min)，使用2000bp DNA Marker。Since some strains were resistant to the selected β-amide antibiotics, in order to further confirm whether they were methicillin-resistant Staphylococcus aureus, PCR was used to confirm whether these strains contained mecA/mecC genes. The mecA/mecC gene encodes penicillin-binding protein 2a (PBP2a), which makes the related strains have a very low affinity for β-lactam antibiotics. When other PBPs are inhibited by β-lactam antibiotics, PBP2a is not inhibited and replaces other PBPs, which play a role in catalyzing cell wall synthesis, allowing bacteria to survive. The primers and amplification methods of mecA and mecC genes were reported in the previous literature. The single-plex PCR method was used, and the primer sequences were shown in Table 4. PCR amplification conditions are as follows: 95°C pre-denaturation for 5 min, 95°C denaturation for 45s, 56°C annealing for 45s, 72°C extension for 50s, for 35 cycles; and a final extension at 72°C for 8 min. The reaction system (25 μL) contains: 12.5 μl 2×DreamTaq mastermix, 9.5 μl ultrapure water, 40 ng template DNA, 0.5 μM upstream and downstream primers. 5 μL of PCR product was loaded on 2.0% agarose gel for electrophoresis separation (120V, 25min), using 2000bp DNA Marker.
表4金黄色葡萄球菌mecA/mecC引物及扩增片段Table 4 Staphylococcus aureus mecA/mecC primers and amplified fragments
Figure PCTCN2021086878-appb-000004
Figure PCTCN2021086878-appb-000004
多重PCR的方法确认MRSA菌株的SCCmec型别。引物与扩增方法参考先前文献报道。所使用的16对引物由北京六合华大基因科技有限公司合成(引物序列见表5)。这16对引物可进行TypeI、TypeII、TypeIII、TypeIVa、TypeIVb、TypeIVc、TypeIVd和TypeV型别的鉴定，反应体系(50μL)包含：23μl 2×DreamTaq mastermix，9μl超纯水，80ng模板DNA，0.5μM上下游引物。PCR扩增条件如下：94℃预变性5min，95℃变性45s，65℃退火45s，72℃延伸90s，进行10个循环；95℃变性45s，55℃退火45s，72℃延伸90s，进行 25个循环；最后72℃延伸10min。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100pb DNA ladder Marker。Multiplex PCR method to confirm the SCCmec type of MRSA strains. For primers and amplification methods, refer to previous literature reports. The 16 pairs of primers used were synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd. (see Table 5 for primer sequences). These 16 pairs of primers can be used for TypeI, TypeII, TypeIII, TypeIVa, TypeIVb, TypeIVc, TypeIVd and TypeV identification. The reaction system (50μL) contains: 23μl 2×DreamTaq mastermix, 9μl ultrapure water, 80ng template DNA, 0.5μM upstream and downstream primers. PCR amplification conditions are as follows: 94°C pre-denaturation for 5 min, 95°C denaturation for 45s, 65°C annealing for 45s, 72°C extension for 90s, for 10 cycles; 95°C denaturation for 45s, 55°C annealing for 45s, 72°C extension for 90s, 25 cycles Cycle; final extension at 72°C for 10 min. 10 μL of PCR product was loaded on 2.0% agarose gel for electrophoresis separation (120V, 40min), using 100pb DNA ladder Marker.
表5金黄色葡萄球菌SCCmec型别引物序列及扩增片段Table 5 Staphylococcus aureus SCCmec type primer sequences and amplified fragments
Figure PCTCN2021086878-appb-000005
Figure PCTCN2021086878-appb-000005
对金黄色葡萄球菌常见的18种肠毒素和类肠毒素(sea,seb,sec,sed,see,seg,seh,sei,sej,sek,sel,sem,sen,seo,sep,seq,ser,seu)、中毒休克征毒素1(tsst-1)以及杀白细胞素(pvl)编码的基因分别进行了PCR检测。引物与扩增方法参考先前文献报道。所使用的引物由北京六合华大基因科技有限公司合成(引物序列见表6)。反应体系(50μL)包含：23μl 2×DreamTaq mastermix，9μl超纯水，80ng模板DNA，0.5μM上下游引物。PCR扩增条件如下：94℃预变性5min，94℃变性45s，65℃[△T-1℃]退火45s，72℃延伸90s，进行15个循环；94℃变性45s，50℃退火45s，72℃延伸90s，进行20个循环；最后72℃延伸10min。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2000bp DNA Marker。18 enterotoxins and enterotoxoids common to Staphylococcus aureus (sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq, ser, seu), toxic shock syndrome toxin 1 (tsst-1) and genes encoding leukocidin (pvl) were detected by PCR respectively. For primers and amplification methods, refer to previous literature reports. The primers used were synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd. (see Table 6 for primer sequences). The reaction system (50 μL) contains: 23 μl 2×DreamTaq mastermix, 9 μl ultrapure water, 80 ng template DNA, 0.5 μM upstream and downstream primers. PCR amplification conditions are as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 45s, annealing at 65°C [△T-1°C] for 45s, extension at 72°C for 90s, for 15 cycles; denaturation at 94°C for 45s, annealing at 50°C for 45s, 72 90 s extension at °C for 20 cycles; a final extension at 72 °C for 10 min. 10 μL of the PCR product was loaded on a 2.0% agarose gel for electrophoresis separation (120V, 40min) using a 2000bp DNA Marker.
表6金黄色葡萄球菌相关特征性基因引物序列及扩增片段Table 6 Staphylococcus aureus-related characteristic gene primer sequences and amplified fragments
Figure PCTCN2021086878-appb-000006
Figure PCTCN2021086878-appb-000006
Figure PCTCN2021086878-appb-000007
Figure PCTCN2021086878-appb-000007
(2)实验结果(2) Experimental results
表7本发明菌株的ST型别The ST type of table 7 strains of the present invention
Figure PCTCN2021086878-appb-000008
Figure PCTCN2021086878-appb-000008
表8本发明菌株的spa型别The spa type of table 8 strain of the present invention
Figure PCTCN2021086878-appb-000009
Figure PCTCN2021086878-appb-000009
这些菌株可培养于TSA、BHI和NA培养基中。These strains can be grown in TSA, BHI and NA media.
表9本发明菌株对不同抗生素的耐药性The resistance of table 9 strains of the present invention to different antibiotics
Figure PCTCN2021086878-appb-000010
Figure PCTCN2021086878-appb-000010
Figure PCTCN2021086878-appb-000011
Figure PCTCN2021086878-appb-000011
注：*R＝耐药；I＝中度耐药；S＝敏感Note: *R=resistant; I=moderately resistant; S=susceptible
经PCR进一步确证，Sta3702B3、Sta403和Sta4127A1为耐甲氧西林金黄色葡萄球菌，相关菌株含有mecA基因，序列如SEQ ID No:13所示，SCCmec型别分别为TypeIII型、TypeIVb型和TypeIVa型。It was further confirmed by PCR that Sta3702B3, Sta403 and Sta4127A1 were methicillin-resistant Staphylococcus aureus, the related strains contained mecA gene, the sequence was shown in SEQ ID No: 13, and the SCCmec types were TypeIII, TypeIVb and TypeIVa respectively.
SEQ ID No:13SEQ ID No: 13
Figure PCTCN2021086878-appb-000012
Figure PCTCN2021086878-appb-000012
实施例4金黄色葡萄球菌菌株的特征序列分析Example 4 Characteristic sequence analysis of Staphylococcus aureus strains
主要根据金黄色葡萄球菌的泛基因组分析结果获得这四株菌株特有的非必需基因。共选取了564株代表性金黄色葡萄球菌(含分析菌株)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-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。The non-essential genes peculiar to these four strains were obtained mainly based on the results of pan-genome analysis of Staphylococcus aureus. A total of 564 representative Staphylococcus aureus (including analysis strains) genome sequences were selected for pan-genome analysis. The pan-genome was analyzed by the MP method in the prokaryotic pan-genome automated analysis software (Pan-Genomics Analysis Pipeline, PGAP), and the analysis results were processed by the local Perl script to obtain the core genes and non-core gene information of all strains.
提取这四株菌株特有的非核心基因蛋白序列，通过本地Blast分别将其比对回金黄色葡萄球菌的蛋白总库及NCBI非冗余蛋白数据库(NR)。去除能比对到已知的金黄色葡萄球菌蛋白的序列，剩下的则为标准菌株特有的基因。特有基因在相关菌株、其它金葡菌株(图4)及葡萄球菌属(图5)及其他菌属微生物(图6)中通过PCR扩增检验其特异性。The specific non-core gene protein sequences of these four strains were extracted and aligned back to the Staphylococcus aureus total protein library and the NCBI non-redundant protein database (NR) by local Blast. Sequences that align to known Staphylococcus aureus proteins were removed, and the rest were genes specific to the standard strain. The unique genes were tested for specificity by PCR amplification in related strains, other S. aureus strains (Fig. 4) and Staphylococcus (Fig. 5) and other microorganisms (Fig. 6).
结果表明，这些菌株还携带有各自特有的基因岛，序列如SEQ ID No:1～SEQ ID No:4所示，相关基因岛可通过表10中的引物SEQ ID No:5～SEQ ID No:12进行扩增检验。The results show that these strains also carry their own unique gene islands, the sequences are shown in SEQ ID No: 1 to SEQ ID No: 4, and the relevant gene islands can be obtained through primers SEQ ID No: 5 to SEQ ID No: 5 in Table 10: 12 Carry out amplification test.
本发明2株金黄色葡萄球菌分别携带的特有的基因岛(特征序列)如下：The unique gene islands (characteristic sequences) carried by 2 strains of Staphylococcus aureus of the present invention are as follows:
金黄色葡萄球菌(Staphylococcus aureus)Sta3702B3Staphylococcus aureus Sta3702B3
SEQ ID No:1SEQ ID No: 1
Figure PCTCN2021086878-appb-000013
Figure PCTCN2021086878-appb-000013
金黄色葡萄球菌(Staphylococcus aureus)Sta403Staphylococcus aureus Sta403
SEQ ID No:2SEQ ID No: 2
Figure PCTCN2021086878-appb-000014
Figure PCTCN2021086878-appb-000014
金黄色葡萄球菌(Staphylococcus aureus)Sta4127A1Staphylococcus aureus Sta4127A1
SEQ ID No:3SEQ ID No: 3
Figure PCTCN2021086878-appb-000015
Figure PCTCN2021086878-appb-000015
金黄色葡萄球菌(Staphylococcus aureus)Sta1942-0Staphylococcus aureus Sta1942-0
SEQ ID No:4SEQ ID No: 4
Figure PCTCN2021086878-appb-000016
Figure PCTCN2021086878-appb-000016
Figure PCTCN2021086878-appb-000017
Figure PCTCN2021086878-appb-000017
表10本发明所述菌株的基因岛扩增引物序列Table 10 Gene island amplification primer sequences of strains of the present invention
Figure PCTCN2021086878-appb-000018
Figure PCTCN2021086878-appb-000018
表11本发明菌株携带的部分毒力基因Table 11 Partial virulence genes carried by the strain of the present invention
序号serial number 菌株编号strain number 毒力基因携带情况 virulence gene carrier
11 Sta3702B3Sta3702B3 sec-seg-seh-sei-sem-sen-seo-sep-sersec-seg-seh-sei-sem-sen-seo-sep-ser
22 Sta403Sta403 sea-seg-sei-sek-sem-seo-sep-seqsea-seg-sei-sek-sem-seo-sep-seq
33 Sta4127A1Sta4127A1 seb-sec-seg-sei-sej-sek-sel-sem-sep-seq-serseb-sec-seg-sei-sej-sek-sel-sem-sep-seq-ser
44 Sta1942-0Sta1942-0 seb-sec-seg-sei-sej-sek-sel-sem-sep-seq-serseb-sec-seg-sei-sej-sek-sel-sem-sep-seq-ser
本发明还提供一种金黄色葡萄球菌的冻干保护剂，并提供了冻干保护剂的几个具体实施例(实施例5～7)和对比例(对比例1～3)。The present invention also provides a lyophilized protective agent for Staphylococcus aureus, and provides several specific examples (Examples 5-7) and comparative examples (Comparative Examples 1-3) of the lyophilized protective agent.
实施例5Example 5
一种金黄色葡萄球菌的冻干保护剂，含有3质量份的海藻酸钠，10质量份的脱脂奶粉，0.2质量份的肌醇六磷酸和0.1质量份还原性谷胱甘肽，0.2质量份的甘氨酸。A freeze-drying protective agent for Staphylococcus aureus, comprising 3 parts by mass of sodium alginate, 10 parts by mass of skimmed milk powder, 0.2 parts by mass of phytic acid and 0.1 part by mass of reduced glutathione, 0.2 part by mass of glycine.
实施例6Example 6
一种金黄色葡萄球菌的冻干保护剂，含有5质量份的海藻酸钠，12质量份的脱脂奶粉，0.5质量份的肌醇六磷酸和0.2质量份还原性谷胱甘肽，2质量份的甘氨酸。A lyophilized protective agent for Staphylococcus aureus, comprising 5 parts by mass of sodium alginate, 12 parts by mass of skimmed milk powder, 0.5 parts by mass of phytic acid and 0.2 parts by mass of reduced glutathione, 2 parts by mass of glycine.
实施例7Example 7
一种金黄色葡萄球菌的冻干保护剂，含有5质量份的海藻酸钠，9质量份的脱脂奶粉，1质量份的肌醇六磷酸和0.3质量份还原性谷胱甘肽，1质量份的甘氨酸。A lyophilized protective agent for Staphylococcus aureus, comprising 5 parts by mass of sodium alginate, 9 parts by mass of skimmed milk powder, 1 part by mass of phytic acid and 0.3 part by mass of reduced glutathione, 1 part by mass of glycine.
对比例1Comparative Example 1
冻干保护剂与实施例1相比，不含有还原性谷胱甘肽，仅含有3质量份的海藻酸钠，10质量份的脱脂奶粉，0.2质量份的肌醇六磷酸，0.2质量份的甘氨酸。Compared with Example 1, the lyophilized protective agent does not contain reduced glutathione, but only contains 3 parts by mass of sodium alginate, 10 parts by mass of skim milk powder, 0.2 parts by mass of phytic acid, 0.2 parts by mass of Glycine.
对比例2Comparative Example 2
冻干保护剂与实施例1相比，不含有海藻酸钠，仅含有10质量份的脱脂奶粉，0.2质量份的肌醇六磷酸和0.1质量份还原性谷胱甘肽，0.2质量份的甘氨酸。Compared with Example 1, the freeze-drying protection agent does not contain sodium alginate, but only contains 10 parts by mass of skim milk powder, 0.2 parts by mass of phytic acid, 0.1 parts by mass of reduced glutathione, and 0.2 parts by mass of glycine .
对比例3Comparative Example 3
冻干保护剂与实施例1相比，仅含有10质量份的脱脂奶粉。Compared with Example 1, the freeze-drying protective agent contained only 10 parts by mass of skimmed milk powder.
实施例8不同组成的冻干保护剂冻干存活率的比较Example 8 Comparison of freeze-drying survival rates of freeze-drying protective agents with different compositions
(1)标准菌株菌粉的制备方式如下：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。(1) The preparation method of the standard strain bacteria powder is as follows: after the bacteria are recovered, it is inserted into the shake flask and cultivated to 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. bottle, 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 the freeze dryer, perform automatic capping, ensure the complete vacuum state of the sample, and store it at 2-8 °C. Take the lyophilized samples for dilution and counting, and the counting result is the bacterial content A after lyophilization, and the lyophilized survival rate is the percentage of A and A0.
表12不同组成的冻干保护剂冻干存活率的比较Table 12 Comparison of lyophilized survival rates of lyophilized protective agents with different compositions
Figure PCTCN2021086878-appb-000019
Figure PCTCN2021086878-appb-000019
(2)分别按照实施例5～7和对比例1～3的配方制备得到不同的保护剂，对本发明的金黄色葡萄球菌GDMCC 60841、GDMCC 60843、GDMCC 60844和GDMCC 60845进行保存，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，使冻干后，菌含量均约为1000cfu/瓶。(2) different protective agents were prepared according to the formulas of Examples 5-7 and Comparative Examples 1-3 respectively, and the Staphylococcus aureus GDMCC 60841, GDMCC 60843, GDMCC 60844 and GDMCC 60845 of the present invention were stored, and placed in 2 Store at -8°C, and extract 3 tubes every month to check the bacterial content according to the aforementioned counting method. In order to better compare the effect of each protective agent in the long-term storage process, 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 freeze-drying, the bacterial content was equal to About 1000cfu/bottle.
结果如图7所示，实施例5～7的冻干保护剂在长期储存过程中活菌数变化不大，而对比例1～3的冻干保护剂在长期储存过程中活菌数显著下降。The results are shown in Figure 7, the lyophilized protective agents of Examples 5 to 7 did not change much in the number of viable bacteria during long-term storage, while the lyophilized protective agents of Comparative Examples 1 to 3 significantly decreased in the long-term storage process. .
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。Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. Although the present invention is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that, The technical solutions of the present invention may be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention.

Claims (11)

  1. 用于检测金黄色葡萄球菌的特异性分子靶标，其特征在于，所述分子靶标包括：A specific molecular target for detecting Staphylococcus aureus, characterized in that, the molecular target includes:
    (a)如SEQ ID NO:1～4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 4; 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).
  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，A primer for detecting a specific molecular target as claimed in claim 1, characterized in that,
    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:5所示的上游引物和如SEQ ID NO:6所示的下游引物；PCR primers for amplification of the nucleotide sequence shown in SEQ ID NO:1 include: an upstream primer shown in SEQ ID NO:5 and a downstream primer shown in SEQ ID NO:6;
    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:7所示的上游引物和如SEQ ID NO:8所示的下游引物；PCR primers for amplification of the nucleotide sequence shown in SEQ ID NO:2 include: an upstream primer shown in SEQ ID NO:7 and a downstream primer shown in SEQ ID NO:8;
    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:9所示的上游引物和如SEQ ID NO:10所示的下游引物；PCR primers for amplification of the nucleotide sequence set forth in SEQ ID NO:3 include an upstream primer set forth in SEQ ID NO:9 and a downstream primer set forth in SEQ ID NO:10;
    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:11所示的上游引物和如SEQ ID NO:12所示的下游引物。PCR primers for amplification of the nucleotide sequence set forth in SEQ ID NO:4 include: an upstream primer set forth in SEQ ID NO:11 and a downstream primer set forth in SEQ ID NO:12.
  3. 一种金黄色葡萄球菌(Staphylococcus aureus)，其特征在于，是(a)、(b)、(c)或(d)：A staphylococcus aureus (Staphylococcus aureus), characterized in that it is (a), (b), (c) or (d):
    (a)金黄色葡萄球菌(Staphylococcus aureus)Sta3702B3，含有如SEQ ID NO:1所示的核苷酸序列；(a) Staphylococcus aureus Sta3702B3, containing the nucleotide sequence shown in SEQ ID NO: 1;
    (b)金黄色葡萄球菌(Staphylococcus aureus)Sta403，含有如SEQ ID NO:2所示的核苷酸序列；(b) Staphylococcus aureus Sta403, containing the nucleotide sequence shown in SEQ ID NO: 2;
    (c)金黄色葡萄球菌(Staphylococcus aureus)Sta4127A1，含有如SEQ ID NO:3所示的核苷酸序列；(c) Staphylococcus aureus Sta4127A1, containing the nucleotide sequence shown in SEQ ID NO: 3;
    (d)金黄色葡萄球菌(Staphylococcus aureus)Sta1942-0，含有如SEQ ID NO:4所示的核苷酸序列。(d) Staphylococcus aureus Sta1942-0, containing the nucleotide sequence shown in SEQ ID NO:4.
  4. 如权利要求3所述的金黄色葡萄球菌(Staphylococcus aureus)，其特征在于：Staphylococcus aureus (Staphylococcus aureus) as claimed in claim 3, is characterized in that:
    所述金黄色葡萄球菌(Staphylococcus aureus)Sta3702B3，其分类命名为金黄色葡萄球菌(Staphylococcus aureus)，已于2020年1月8号保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC 60841；Described staphylococcus aureus (Staphylococcus aureus) Sta3702B3, its classification is named as staphylococcus aureus (Staphylococcus aureus), has been deposited in the Guangdong Provincial Microorganism Culture Collection Center on January 8, 2020, and the preservation number is GDMCC 60841;
    所述金黄色葡萄球菌(Staphylococcus aureus)Sta403，其分类命名为金黄色葡萄球菌(Staphylococcus aureus)，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 60843；Described staphylococcus aureus (Staphylococcus aureus) Sta403, its classification is named as staphylococcus aureus (Staphylococcus aureus), has been deposited in the Guangdong Provincial Microorganism Culture Collection Center on October 27, 2019, and the preservation number is GDMCC 60843;
    所述金黄色葡萄球菌(Staphylococcus aureus)Sta4127A1，其分类命名为金黄色葡萄球菌(Staphylococcus aureus)，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 60844；Described Staphylococcus aureus (Staphylococcus aureus) Sta4127A1, its classification is named as Staphylococcus aureus (Staphylococcus aureus), has been deposited in Guangdong Province Microorganism Culture Collection Center on October 27, 2019, and the preservation number is GDMCC 60844;
    所述金黄色葡萄球菌(Staphylococcus aureus)Sta1942-0，其分类命名为金黄色葡萄球菌(Staphylococcus aureus)，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 60845。Described Staphylococcus aureus Sta1942-0, its classification is named Staphylococcus aureus (Staphylococcus aureus), has been deposited in Guangdong Province Microorganism Culture Collection Center on October 27, 2019, and the preservation number is GDMCC 60845 .
  5. 如权利要求3所述的金黄色葡萄球菌，其特征在于：所述金黄色葡萄球菌(Staphylococcus aureus)Sta3702B3为耐甲氧西林金黄色葡萄球菌，其分子型别为ST9-t899-SCCmec_type_IVb，还包含了以下毒力基因中的至少一种：sec、seg、seh、sei、sem、sen、seo、sep和ser；或以下抗生素的抗性基因中的至少一种：阿莫西林/克拉维酸、氨苄西林、头孢吡肟、头孢西丁、青霉素G、头孢他啶、庆大霉素、卡那霉素、链霉素、氯霉素、克林霉素、红霉素、泰利霉素、环丙沙星、诺氟沙星、四环素和复方新诺明。The staphylococcus aureus of claim 3, wherein: the Staphylococcus aureus Sta3702B3 is methicillin-resistant Staphylococcus aureus, and its molecular type is ST9-t899-SCCmec_type_IVb, and further comprises at least one of the following virulence genes: sec, seg, seh, sei, sem, sen, seo, sep, and ser; or at least one of the following antibiotic resistance genes: amoxicillin/clavulanate, Ampicillin, cefepime, cefoxitin, penicillin G, ceftazidime, gentamicin, kanamycin, streptomycin, chloramphenicol, clindamycin, erythromycin, telithromycin, ciprofloxacin Star, norfloxacin, tetracycline, and co-trimoxazole.
  6. 如权利要求3所述的金黄色葡萄球菌，其特征在于，所述金黄色葡萄球菌(Staphylococcus aureus)Sta403为耐甲氧西林金黄色葡萄球菌，其分子型别为ST9-t899-SCCmec_type_IVb，还包含了以下毒力基因中的至少一种：sea、seg、sei、sek、sem、seo、sep和seq；或以下抗生素的抗性基因中的至少一种：阿莫西林/克拉维酸、氨苄西林、头孢吡肟、头孢西丁、青霉素G、头孢他啶、庆大霉素、卡那霉素、链霉素、氯霉素、克林霉素、红霉素、泰利霉素、环丙沙星、诺氟沙星、四环素和喹奴普汀/达福普汀。The staphylococcus aureus of claim 3, wherein the staphylococcus aureus (Staphylococcus aureus) Sta403 is methicillin-resistant staphylococcus aureus, and its molecular type is ST9-t899-SCCmec_type_IVb, and further comprises At least one of the following virulence genes: sea, seg, sei, sek, sem, seo, sep, and seq; or at least one of the following antibiotic resistance genes: amoxicillin/clavulanate, ampicillin , cefepime, cefoxitin, penicillin G, ceftazidime, gentamicin, kanamycin, streptomycin, chloramphenicol, clindamycin, erythromycin, telithromycin, ciprofloxacin, Norfloxacin, tetracycline, and quinupristine/dalfopristin.
  7. 如权利要求3所述的金黄色葡萄球菌，其特征在于，所述金黄色葡萄球菌(Staphylococcus aureus)Sta4127A1为耐甲氧西林金黄色葡萄球菌，其分子型别为ST59-t437-SCCmec_type_IVa，还包含了以下毒力基因中的至少一种：seb、sec、seg、sei、sej、sek、sel、sem、sep、seq和ser；或以下抗生素的抗性基因中的至少一种：阿莫西林/克拉维酸、氨苄西林、头孢西丁、青霉素G、头孢他啶、卡那霉素、链霉素、克林霉素、红霉素、泰利霉素和褐霉素。The Staphylococcus aureus of claim 3, wherein the Staphylococcus aureus Sta4127A1 is methicillin-resistant Staphylococcus aureus, and its molecular type is ST59-t437-SCCmec_type_IVa, and further comprises At least one of the following virulence genes: seb, sec, seg, sei, sej, sek, sel, sem, sep, seq, and ser; or at least one of the following antibiotic resistance genes: amoxicillin/ Clavulanic acid, ampicillin, cefoxitin, penicillin G, ceftazidime, kanamycin, streptomycin, clindamycin, erythromycin, telithromycin, and brownomycin.
  8. 如权利要求3所述的金黄色葡萄球菌，其特征在于，所述金黄色葡萄球菌(Staphylococcus aureus)Sta1942-0的分子型别为ST4456-t13849，还包含了以下毒力基因中的至少一种：seb、sec、seg、sei、sej、sek、sel、sem、sep、seq和ser；或以下抗生素的抗性基因中的至少一种：氨苄西林、青霉素G、链霉素、喹奴普汀 /达福普汀和褐霉素。The Staphylococcus aureus of claim 3, wherein the molecular type of the Staphylococcus aureus Sta1942-0 is ST4456-t13849, and further comprises at least one of the following virulence genes : seb, sec, seg, sei, sej, sek, sel, sem, sep, seq, and ser; or at least one of the following antibiotic resistance genes: ampicillin, penicillin G, streptomycin, quinupristine / dalfopristin and fucoidin.
  9. 如权利要求3所述的金黄色葡萄球菌在金黄色葡萄球菌抗生素耐药性中的应用。The application of Staphylococcus aureus as claimed in claim 3 in the antibiotic resistance of Staphylococcus aureus.
  10. 如权利要求3所述的金黄色葡萄球菌在提高检验金黄色葡萄球菌显色平板的准确性中的应用。The application of Staphylococcus aureus as claimed in claim 3 in improving the accuracy of testing Staphylococcus aureus color plates.
  11. 一种金黄色葡萄球菌冻干保护剂，其特征在于，包括以下重量份的组分：海藻酸钠0.1～10份、脱脂奶粉3～15份、肌醇六磷酸0.1-3份、还原性谷胱甘肽0.1-3份、甘氨酸0.2-4份。A freeze-drying protective agent for Staphylococcus aureus, characterized in that it comprises the following components in parts by weight: 0.1-10 parts of sodium alginate, 3-15 parts of skim milk powder, 0.1-3 parts of phytic acid, reducing 0.1-3 parts of glutathione, 0.2-4 parts of glycine.
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