WO2022141936A1 - 含有特异性分子靶标的银白色葡萄球菌标准参考菌株及其检测和应用 - Google Patents
含有特异性分子靶标的银白色葡萄球菌标准参考菌株及其检测和应用 Download PDFInfo
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- argenteus
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Definitions
- the invention relates to the technical field of bioengineering, in particular to a standard reference strain of Staphylococcus albicans containing a specific molecular target and its detection and application.
- Staphylococcus argenteus is a new species discovered in the genus Staphylococcus in recent years. Previously, it has been considered as a species of Staphylococcus aureus due to its very similar biochemical reactions with Staphylococcus aureus. Subtype. Through whole genome sequencing, it was found that although the 16S rRNA nucleic acid sequence of these two species is the same, the average nucleotide identity is only 87%, and the DNA-DNA hybridization is only 34% homologous. In addition, S. aureus lacks aurein and is phenotypically distinct from S. aureus.
- this bacteria can also cause bacterial food poisoning, and can also lead to various infections such as toxic shock syndrome, osteomyelitis, suppurative endocarditis, bacteremia and fatal pneumonia, threatening human health. . Understanding the transmission rules and pathogenic evolution of the bacteria is the basis for effective prevention and control of diseases caused by the bacteria, and the use of appropriate standard strains determines the reliability of the research results.
- this bacterium Since this bacterium has not been completely differentiated from Staphylococcus aureus, it has only been reported sporadically in my country, and the standard strains used to study the evolutionary laws and pathogenicity of this bacterium are from foreign clinical sources, which cannot reflect well. In the actual situation of the bacteria in my country, there is a lack of representative isolates to study the genetic structure and transmission rules of the bacteria in Chinese food and environment, which brings difficulties to related scientific research work.
- the currently reported protective agent is basically used for qualitative storage of Staphylococcus aureus, but not in qualitative storage of Staphylococcus aureus. Therefore, it is of great significance to provide a freeze-drying protective agent that can store quantitative Staphylococcus albicans for a long time.
- the purpose of the present invention is to overcome the deficiencies of the prior art, and to provide a standard reference strain of Staphylococcus albicans and a specific molecular target and application for detecting the strain.
- the three isolates of food-borne Staphylococcus albicans provided by the present invention have typical characteristics of Staphylococcus albicans and can better reflect the genetic background of the bacteria in China.
- the technical scheme adopted in the present invention is:
- Staphylococcus argenteus which is Staphylococcus argenteus Sta3513A1, isolated from tomato samples, and its classification name is Staphylococcus argenteus, which has been preserved in Guangdong City on January 8, 2020 Microbial Culture Collection Center, Address: Building 59, Yard, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong province, China, Postal Code: 510075, preservation number: GDMCC 60949.
- the molecular type of Staphylococcus aureus with the deposit number GDMCC60854 is ST2250-t701, and the antibiotic resistant is telithromycin.
- the molecular type of the Staphylococcus aureus with the deposit number GDMCC60948 is ST2250-t127, and the antibiotics tolerated include: ampicillin, penicillin G, chloramphenicol and tetracycline.
- the molecular type of the Staphylococcus aureus with the deposit number GDMCC60949 is ST2250-t3092, and the antibiotics tolerated include: ampicillin, penicillin G, streptomycin, chloramphenicol, tetracycline, and brownomycin.
- the present invention also provides a set of specific molecular targets for detecting standard strains of Staphylococcus aureus Staphylococcus, characterized in that, the molecular targets include the nucleotide sequences shown in SEQ ID NO. 1-2.
- the staphylococcus aeruginosa with the deposit number of GDMCC60854 contains the nucleotide sequence shown in SEQ ID NO.1; the staphylococcus aureus with the deposit number of GDMCC 60948 and GDMCC 60949 contains the nucleotide sequence as shown in SEQ ID NO.1. 2 shows the nucleotide sequence.
- 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.3-4; The PCR primer sequences amplified for the nucleotide sequence shown in SEQ ID NO.2 are shown in SEQ ID NO.5-6.
- the present invention also provides a freeze-drying protective agent for preparing the quantitative Staphylococcus albicans of the present invention, which is characterized in that it comprises 0.1-10 parts by mass of sodium alginate, 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, 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 standard reference strain of Staphylococcus albicans of the present invention has the standard microscopic morphology and physiological and biochemical characteristics of Staphylococcus aureus, its genetic background is clear, and it carries specific gene targets and is identifiable. Compared with other types of standard strains of this species, these strains have the most typical characteristics of the food source Staphylococcus albicans isolated in China, which can reflect the strains of the food source epidemic background in China, and have a certain representativeness, which can be used as a reference. Strains are used in scientific research.
- the inventor provides a freeze-drying protective agent.
- 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 a colony morphology diagram of a standard reference strain of Staphylococcus aureus strains.
- Figure 2 shows the phylogenetic tree analysis of Staphylococcus aureus strains and Staphylococcus aureus MLST.
- Figure 3 shows the results of PCR detection of Staphylococcus aureus NRPS gene.
- Figure 4 shows the comparison between Staphylococcus aureus and Staphylococcus aureus after culturing on nutrient agar (NA) plate for 48 hours; the left picture is Staphylococcus aureus ATCC6538, and the right picture is Staphylococcus aureus Sta180-0.
- Figure 5 is a schematic diagram of the biochemical identification of API Staph of Staphylococcus aureus strains Sta180-0, Sta226-0 and Sta3513A1.
- Figure 6 shows the unique gene fragments of Staphylococcus aeruginosa strains Sta226-0 and Sta3513A1 and PCR amplification of other Staphylococcus aureus; M: DL 2000Marker; Lane 1-118: Staphylococcus aureus food isolates.
- Figure 7 shows the unique gene fragment of Staphylococcus aeruginosa strain Sta180-0 and PCR amplification of other Staphylococcus aureus; M: DL 2000Marker; Lane 1-118: Staphylococcus aeruginosa food isolate; +: Silver grape Coccus strain Sta180-0; C: blank control.
- Fig. 8 Changes in bacterial content of the standard strains of Staphylococcus albicans of the present invention prepared by each protective agent after 12 months of storage.
- the Staphylococcus aureus discovered this time was collected from the previously isolated Staphylococcus aureus. A total of 1581 of these strains were isolated from 4300 food samples of different food types in 39 cities in my country.
- the specific separation method is as follows: qualitative and quantitative detection of the collected samples are carried out at the same time, and the detection method is slightly adjusted on the basis of the national standard "Food Microbiological Inspection" GB 4789.10-2010.
- 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.
- the three strains were isolated from food samples in Liwan District of Guangzhou City, Panyu District of Guangzhou City and Hong Kong, China.
- the specific isolation information is shown in Table 1.
- Embodiment 2 Identification of Staphylococcus albicans
- Staphylococcus aureus can be completely distinguished from Staphylococcus aureus.
- Multi-locus sequence analysis is mainly for sequence analysis of 7 housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi and yqil), among which arcC site numbers are 36, 151, 207 and 272 and pta site numbers are 39, 107, 145, 175, 198, 256, 268 and 287. , identified as Staphylococcus aureus.
- NRPS gene detection was performed ( Figure 3). For primers and amplification methods, refer to previous literature reports.
- the 8 pairs of primers used were synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd. (see Table 2 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/ ).
- Staining microscopy smear suspicious colonies, carry out Gram staining, and observe the morphology by microscopy.
- Staphylococcus aureus like Staphylococcus aureus, is Gram-positive and forms a prototype grape bunch under the microscope.
- 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.
- NA plate identification The standard strain of Staphylococcus aureus and Staphylococcus aureus were streaked on the NA plate at the same time, and it could be seen that Staphylococcus aureus was significantly whiter than Staphylococcus aureus ( Figure 4).
- KB method was used to confirm drug susceptibility. 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
- Staphylococcus protein A is an integral part of the Staphylococcus cell wall, 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.
- 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.
- the non-essential genes peculiar to these three strains were obtained mainly according to the results of pan-genome analysis of Staphylococcus aureus.
- a total of 245 representative strains of Staphylococcus aureus (including the analyzed strains) were selected for pan-genome analysis.
- the pan-genome was analyzed by the MP method in the prokaryotic Pan-Genomics Analysis Pipeline (PGAP), and the analysis results were processed by the local Perl script to obtain the core gene and non-core gene information of all strains.
- PEP Pan-Genomics Analysis Pipeline
- strains can be grown in TSA, BHI and NA media.
- Sta180-0 carries SEQ ID No: 1
- Sta226-0 and Sta3513A1 carry the same gene island sequence SEQ ID No: 2.
- Relevant gene islands can be amplified and tested by primers SEQ ID No: 3 to SEQ ID No: 6 in Table 7, and the verification results are shown in Figure 4 and Figure 5.
- the peculiar gene islands (characteristic sequences) carried respectively by 3 strains of Staphylococcus aeruginosa of the present invention are as follows:
- the present invention also provides a freeze-drying protective agent for Staphylococcus albicans, and provides several specific examples (Examples 3-5) and comparative examples (Comparative Examples 1-3) of the freeze-drying protective agent.
- a freeze-drying protective agent for Staphylococcus albicans comprising 3 parts by mass of sodium alginate, 10 parts by mass of skim 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 freeze-drying protective agent for Staphylococcus albicans 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 freeze-drying protective agent for Staphylococcus albicans comprising 5 parts by mass of sodium alginate, 9 parts by mass of skim 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 bacterial powder is as follows: after the bacterial strain is revived, it is inserted into a 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. In a vial, and sampling for dilution and counting, it 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
本发明公开了保藏编号分别为GDMCC 60854、GDMCC 60948和GDMCC 60949的银白色葡萄球菌标准参考菌株，其可用作食品、药品、临床检验等不同领域。本发明还涉及一组用于检测、鉴别上述3株银白色葡萄球菌标准菌株的特异性靶标基因以及对应的PCR引物。本发明还提供了一种银白色葡萄球菌冻干保护剂，其可用于本发明标准菌株的长期储存。
Description
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银白色葡萄球菌标准参考菌株及其检测和应用。
银白色葡萄球菌(Staphylococcus argenteus,S.argenteus)是近年来在葡萄球菌属中发现的一个新种，此前，由于其生化反应与金黄色葡萄球菌非常相似，一直被认为是金黄色葡萄球菌的一个亚型。通过全基因组测序，发现这两个物种虽然16S rRNA的核酸序列一样，但平均核苷酸一致性仅87％，而DNA-DNA杂交仅34％同源性。此外，银白色葡萄球菌缺乏金黄色素，在表型上与金黄色葡萄球菌有着明显的区别。与金葡菌类似，该菌同样会引起细菌性食物中毒，同样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。
由于该菌一直与金黄色葡萄球菌未进行完全区别，在我国也仅零星报告，而目前研究该菌的进化规律和致病性使用的标准菌株为国外临床来源的菌株，并不能很好地反映该菌在我国的实际情况，缺乏代表性的分离株来研究该菌在中国食品和环境中的遗传结构和传播规律，为相关科研工作带来困难。
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，未见于定性存储银白色葡萄球菌。因此，提供一种可长期保存定量化银白色葡萄球菌的冻干保护剂具有重要意义。
发明内容
本发明的目的在于克服现有技术的不足，提供一种银白色葡萄球菌标准参考菌株及检测该菌株的特异性分子靶标和应用。本发明提供的3株中国地区食源性银白色葡萄球菌分离菌株，这些菌株具有典型的银白色葡萄球菌特性，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：
[根据细则26改正08.06.2021]
提供一株银白色葡萄球菌，是银白色葡萄球菌(Staphylococcus argenteus)Sta180-0，分离自大头鱼样品中，其分类命名为银白色葡萄球菌(Staphylococcus argenteus)，已于2020年1月8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 60854。
提供一株银白色葡萄球菌，是银白色葡萄球菌(Staphylococcus argenteus)Sta180-0，分离自大头鱼样品中，其分类命名为银白色葡萄球菌(Staphylococcus argenteus)，已于2020年1月8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 60854。
[根据细则26改正08.06.2021]
提供一株银白色葡萄球菌，是银白色葡萄球菌(Staphylococcus argenteus)Sta226-0，分离自鱿鱼样品中，其分类命名为银白色葡萄球菌(Staphylococcus argenteus)，已于2020年1月8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 60948。
提供一株银白色葡萄球菌，是银白色葡萄球菌(Staphylococcus argenteus)Sta226-0，分离自鱿鱼样品中，其分类命名为银白色葡萄球菌(Staphylococcus argenteus)，已于2020年1月8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 60948。
[根据细则26改正08.06.2021]
提供一株银白色葡萄球菌，是银白色葡萄球菌(Staphylococcus argenteus)Sta3513A1，分离自西红柿样品中，其分类命名为银白色葡萄球菌(Staphylococcus argenteus)，已于2020年1月8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 60949。
提供一株银白色葡萄球菌，是银白色葡萄球菌(Staphylococcus argenteus)Sta3513A1，分离自西红柿样品中，其分类命名为银白色葡萄球菌(Staphylococcus argenteus)，已于2020年1月8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 60949。
进一步地，所述保藏编号为GDMCC60854的银白色葡萄球菌的分子型别为ST2250-t701，耐受的抗生素为泰利霉素。
进一步地，所述保藏编号为GDMCC60948的银白色葡萄球菌的分子型别为ST2250-t127，耐受的抗生素包括：氨苄西林、青霉素G、氯霉素和四环素。
进一步地，所述保藏编号为GDMCC60949的银白色葡萄球菌的分子型别为ST2250-t3092，耐受的抗生素包括：氨苄西林、青霉素G、链霉素、氯霉素、四环素和褐霉素。
本发明还提供了一组用于检测银白色葡萄球菌葡萄球菌标准菌株的特异性分子靶标，其特征在于，所述分子靶标包括如SEQ ID NO.1～2所示的核苷酸序列。
进一步地，所述保藏编号为GDMCC60854的银白色葡萄球菌含有如SEQ ID NO.1所示的核苷酸序列；所述保藏编号为GDMCC 60948和GDMCC 60949的银白色葡萄球菌含有如SEQ ID NO.2所示的核苷酸序列。
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.3～4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.5～6所示。
本发明还提供了一种用于制备定量本发明所述银白色葡萄球菌的冻干保护剂，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。
本发明提供的可长期定量保存银白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。
本发明的有益效果：
(1)本发明的银白色葡萄球菌标准参考菌株，具有标准的葡萄球菌菌体显微形态和生理生化特征，其遗传背景清晰，且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银白色葡萄球菌最为典型的特征，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，可作为参考菌株用于科学研究。
(2)针对本发明的银白色葡萄球菌，发明人提供了一种冻干保护剂。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。
生物材料保藏
[根据细则26改正08.06.2021]
一株银白色葡萄球菌(Staphylococcus argenteus)Sta180-0，其分类命名为银白色葡萄球菌(Staphylococcus argenteus)，已于2020年1月8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GDMCC60854。
一株银白色葡萄球菌(Staphylococcus argenteus)Sta180-0，其分类命名为银白色葡萄球菌(Staphylococcus argenteus)，已于2020年1月8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GDMCC60854。
[根据细则26改正08.06.2021]
一株银白色葡萄球菌(Staphylococcus argenteus)Sta226-0，其分类命名为银白色葡萄球菌(Staphylococcus argenteus)，已于2020年1月8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 60948。
一株银白色葡萄球菌(Staphylococcus argenteus)Sta226-0，其分类命名为银白色葡萄球菌(Staphylococcus argenteus)，已于2020年1月8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 60948。
[根据细则26改正08.06.2021]
一株银白色葡萄球菌(Staphylococcus argenteus)Sta3513A1，其分类命名为银白色葡萄 球菌(Staphylococcus argenteus)，已于2020年1月8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 60949。
一株银白色葡萄球菌(Staphylococcus argenteus)Sta3513A1，其分类命名为银白色葡萄 球菌(Staphylococcus argenteus)，已于2020年1月8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 60949。
图1为银白色葡萄球菌菌株标准参考菌株的菌落形态图。
图2为银白色葡萄球菌菌株与金黄色葡萄球菌MLST进化树分析。
图3为银白色葡萄球菌NRPS基因PCR检测结果。
图4为银白色葡萄球菌与金黄色葡萄球菌在在营养琼脂(NA)平板上培养48h后比较；左图为金黄色葡萄球菌ATCC6538，右图为银白色葡萄球菌Sta180-0。
图5为银白色葡萄球菌菌株Sta180-0、Sta226-0和Sta3513A1的API Staph生化鉴定示意图。
图6为银白色葡萄球菌菌株Sta226-0和Sta3513A1的特有基因片段及其他银白色葡萄球菌PCR扩增图；其中M：DL 2000Marker；Lane 1-118：银白色葡萄球菌食品分离株。
图7为银白色葡萄球菌菌株Sta180-0的特有基因片段及其他银白色葡萄球菌PCR扩增图；其中M：DL 2000Marker；Lane 1-118：银白色葡萄球菌食品分离株；+：银白色葡萄球菌菌株Sta180-0；C：空白对照。
图8各保护剂制备的本发明所述的银白色葡萄球菌标准菌株经12个月储存后含菌量变化。
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银白色葡萄球菌的分离
本次发现的银白色葡萄球菌收集自此前分离的金黄色葡萄球菌。这些菌株共1581株，分离自我国39个城市4300份不同食品类型的食品样品中。具体分离方法如下：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)。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。
共分离得到3株银白色葡萄球菌菌株Sta180-0、Sta226-0以及Sta3513A1，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12月30号，保藏编号分别为GDMCC 60854，GDMCC 60948，GDMCC 60949。
3株菌株分别是分离自广州市荔湾区、广州市番禺区和中国香港的食品样品中，具体的分离信息如表1所示。
表1 菌株分离信息
实施例2银白色葡萄球菌的鉴定
1、鉴定方法
1.1多位点序列(MLST)分型分析
利用多位点序列分型(MLST)和NRPS基因检测，可将银白色葡萄球菌与金黄色葡萄球菌完全区分。多位点序列分析主要是对7种管家基因(arcC,aroE,glpF,gmk,pta,tpi and yqil)进行序列分析，其中arcC位点号为36,151,207和272以及pta位点号为39,107,145,175,198,256,268和287时，鉴定为银白色葡萄球菌。同时进行NRPS基因检测(图3)。引物与 扩增方法参考先前文献报道。所使用的8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2)。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/)。
表2 MLST引物和NRPS引物及扩增片段
1.2银白色葡萄球菌菌株的生理生化特征和药敏特征分析
染色镜检：将可疑菌落涂片，进行革兰氏染色，镜检观察形态。银白色葡萄球菌跟金黄色葡萄球菌一样，为革兰氏阳性，显微镜下成原型葡萄串状。
血浆凝固酶实验：接种单菌落至5mL BHI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。
NA板鉴定：将金黄色葡萄球菌标准菌株同银白色葡萄球菌同时划板NA平板，可见银白色葡萄球菌要明显白于金黄色葡萄球菌(图4)。
药敏特征分析：利用KB法进行药敏确证。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 coliATCC25922作为质控菌株。
1.3葡萄球菌A蛋白(SPA)分型
葡萄球菌A蛋白(SPA)是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。
表3 spa引物及扩增片段
1.4银白色葡萄球菌菌株的特征序列分析
主要根据银白色葡萄球菌的泛基因组分析结果获得这三株菌株特有的非必需基因。共选取了245株代表性银白色葡萄球菌(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。
提取这三株菌株特有的非核心基因蛋白序列，通过本地Blast分别将其比对回银白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银白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、银葡分离株中通过PCR扩增检验其特异性。
2、鉴定结果
表4 本发明所述菌株的ST型别
表5 本发明所述菌株的spa型别
这些菌株可培养于TSA、BHI和NA培养基中。
表6 本发明所述菌株对不同抗生素的耐药性
注：*R＝耐药；I＝中度耐药；S＝敏感
这些菌株还携带有特有的基因岛，其中Sta180-0携带SEQ ID No:1，Sta226-0和Sta3513A1携带同一个基因岛序列SEQ ID No:2。相关基因岛可通过表7中引物SEQ ID No:3～SEQ ID No:6进行扩增检验，验证结果如图4和图5所示。
本发明3株银白色葡萄球菌分别携带的特有的基因岛(特征序列)如下：
银白色葡萄球菌(Staphylococcus argenteus)Sta180-0
SEQ ID No:1
>180-0_02558group_23936
银白色葡萄球菌(Staphylococcus argenteus)Sta226-0和Sta3513A1
SEQ ID No:2
>226-0_03040group_24153
表7 本发明所述菌株的基因岛验证引物
本发明还提供一种银白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3～5)和对比例(对比例1～3)。
实施例3
一种银白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。
实施例4
一种银白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。
实施例5
一种银白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。
对比例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。
对比例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。
对比例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。
实施例6定量质控菌的冻干存活率及稳定性比较
在本实施例中，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。
表8 不同组成的冻干保护剂冻干存活率的比较
实施例7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3～5和对比例1～3的配方制备得到不同的保护剂，并对本发明的银白色葡萄球菌菌株Sta180-0、Sta226-0和Sta3513A1进行保存，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，在制备本发明所述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，本发明所述菌含量均约为1000cfu/瓶。
结果如图8所示，实施例3～5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。
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 (10)
- 用于检测银白色葡萄球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:1～2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。
- 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:3所示的上游引物和如SEQ ID NO:4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:5所示的上游引物和如SEQ ID NO:6所示的下游引物。
- 一种银白色葡萄球菌(Staphylococcus argenteus)，其特征在于，是(a)、(b)或(c)：(a)是银白色葡萄球菌(Staphylococcus argenteus)Sta180-0，含有如SEQ ID NO:1所示的核苷酸序列；(b)是银白色葡萄球菌(Staphylococcus argenteus)Sta226-0，含有如SEQ ID NO:2所示的核苷酸序列；(c)是银白色葡萄球菌(Staphylococcus argenteus)Sta3513A1，含有如SEQ ID NO:2所示的核苷酸序列。
- 如权利要求3所述的银白色葡萄球菌，其特征在于，所述银白色葡萄球菌(Staphylococcus argenteus)Sta180-0的分子型别为ST2250-t701，还包含了以下抗生素的抗性基因：泰利霉素。
- 如权利要求3所述的银白色葡萄球菌，其特征在于，所述银白色葡萄球菌(Staphylococcus argenteus)Sta226-03的分子型别为ST2250-t127，还包含了以下抗生素的抗性基因中的至少一种：氨苄西林、青霉素G、氯霉素和四环素。
- 如权利要求3所述的银白色葡萄球菌，其特征在于，所述银白色葡萄球菌(Staphylococcus argenteus)Sta3513A1的分子型别为ST2250-t3092，还包含了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、氯霉素、四环素和褐霉素。
- 如权利要求3所述的银白色葡萄球菌，其特征在于：所述银白色葡萄球菌(Staphylococcus argenteus)Sta180-0，其分类命名为银白色葡萄球菌(Staphylococcus argenteus)，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 60854；所述银白色葡萄球菌(Staphylococcus argenteus)Sta226-0，其分类命名为银白色葡萄球菌(Staphylococcus argenteus)，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 60948；所述银白色葡萄球菌(Staphylococcus argenteus)Sta3513A1，其分类命名为银白色葡萄球菌(Staphylococcus argenteus)，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 60949。
- 如权利要求3所述的银白色葡萄球菌在银白色葡萄球菌抗生素耐药性中的应用。
- 如权利要求3所述的银白色葡萄球菌在提高检验银白色葡萄球菌显色平板的准确性中的应用。
- 一种银白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。
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