CN112575100B - Staphylococcus albus standard reference strain containing specific molecular target and detection and application thereof - Google Patents
Staphylococcus albus standard reference strain containing specific molecular target and detection and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- 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
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- 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
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Abstract
The invention relates to the technical field of bioengineering, and relates to 3 standard reference strains of silver white staphylococcus which have independent intellectual property rights in China and accord with the domestic propagation rule, and the reference strains can be used as standard reference strains in different fields of food, medicine, clinical examination and the like. The preservation numbers are respectively as follows: GDMCC60854, GDMCC60948, and GDMCC 60949. The 3 silver white staphylococcus strains have clear genetic background, clear information such as sample sources, drug resistance, molecular detection targets and the like, and completely meet the requirements of food, medicines and clinical examination on standard strains. The invention also relates to a group of specific target genes and corresponding PCR primers for detecting and identifying the 3 silver white staphylococcus standard strains. Finally, the invention also provides a silver white staphylococcus freeze-drying protective agent with high survival rate and specificity, which can be used for long-term storage of the standard strain.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a standard reference strain of staphylococcus albus containing a specific molecular target and detection and application thereof.
Background
S. argentia is a new species discovered in Staphylococcus in recent years, and has been considered a subtype of Staphylococcus aureus since its biochemical reaction is very similar to that of Staphylococcus aureus. Through whole genome sequencing, the nucleic acid sequences of 16S rRNA of the two species are identical, but the average nucleotide identity is only 87%, and DNA-DNA hybridization is only 34% homologous. In addition, Staphylococcus albus lacks the golden yellow pigment, and is phenotypically clearly different from Staphylococcus aureus. Similar to staphylococcus aureus, the bacterium can cause bacterial food poisoning, and can also cause various infections such as toxic shock syndrome, osteomyelitis, suppurative endocarditis, bacteremia, lethal pneumonia and the like, thereby threatening human health. The understanding of the propagation rule and pathogenic evolution of the strain is the basis for effectively preventing and controlling the diseases caused by the strain, and whether a proper standard strain is used determines the reliability of the research result.
The bacterium is not completely different from staphylococcus aureus all the time, and is only sporadically reported in China, but the current standard strains for researching the evolution law and pathogenicity of the bacterium are foreign clinical strains, so the actual situation of the bacterium in China cannot be well reflected, and a representative isolate is lacked for researching the genetic structure and the propagation law of the bacterium in Chinese foods and environments, thereby bringing difficulties for related scientific research work.
The adopted protective agent reported at present is basically used for qualitatively storing staphylococcus aureus, and is not used for qualitatively storing staphylococcus albus. Therefore, the freeze-drying protective agent capable of preserving the quantified silver white staphylococcus for a long time is of great significance.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a standard reference strain of staphylococcus albus, a specific molecular target for detecting the strain and application. The 3 food-borne staphylococcus albus isolated strains in the China provided by the invention have typical staphylococcus albus characteristics and can better reflect the genetic background of the strains in the China.
In order to achieve the purpose, the invention adopts the technical scheme that:
a strain of Staphylococcus albus is provided, is Staphylococcus albus (Staphylococcus argenteus) Sta180-0, is separated from a big head fish sample, is classified and named as Staphylococcus albus (Staphylococcus argenteus), is preserved in Guangdong province microorganism culture collection No. 1-8 in 2020, and has a preservation number of GDMCC 60854.
A strain of Staphylococcus albus is provided, is Staphylococcus albus (Staphylococcus argenteus) Sta226-0, is separated from a squid sample, is classified and named as Staphylococcus albus (Staphylococcus albus), is preserved in Guangdong province microorganism culture collection No.1 and No. 8 in 2020, and has a preservation number of GDMCC 60948.
A strain of Staphylococcus albus is provided, which is Staphylococcus albus (Staphylococcus argenteus) Sta3513A1, is separated from a tomato sample, is classified and named as Staphylococcus albus (Staphylococcus albus) and is preserved in Guangdong province microorganism culture collection No.1 and No. 8 in 2020, and the preservation number is GDMCC 60949.
Further, the molecular type of the staphylococcus albus with the collection number of GDMCC60854 is ST2250-t701, and the resistant antibiotic is telithromycin.
Further, the staphylococcus albus with deposit number GDMCC60948 has molecular type ST2250-t127, and the antibiotics that are tolerated include: ampicillin, penicillin G, chloramphenicol, and tetracycline.
Further, the staphylococcus albus with deposit number GDMCC60949 has a molecular type ST2250-t3092, and the antibiotics that are tolerated include: ampicillin, penicillin G, streptomycin, chloramphenicol, tetracycline, and fuscin.
The invention also provides a group of specific molecular targets for detecting the staphylococcus albus standard strain, which is characterized in that the molecular targets comprise nucleotide sequences shown as SEQ ID NO. 1-2.
Further, the staphylococcus albus with the preservation number being GDMCC60854 contains a nucleotide sequence shown as SEQ ID NO. 1; the staphylococcus albus with the preservation numbers of GDMCC60948 and GDMCC60949 contains a nucleotide sequence shown in SEQ ID NO. 2.
The invention also provides a group of primers for detecting the specific molecular target, and the PCR primer sequence for the amplification of the nucleotide sequence shown as SEQ ID NO.1 is shown as SEQ ID NO. 3-4; the PCR primer sequence for the nucleotide sequence amplification shown as SEQ ID NO.2 is shown as SEQ ID NO. 5-6.
The invention also provides a freeze-drying protective agent for preparing quantitative silver white staphylococcus, which is characterized by comprising 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, 0.1-3 parts by mass of reductive glutathione and 0.2-4 parts by mass of glycine.
The action principle of the protective agent capable of quantitatively preserving the silver white staphylococcus for a long time provided by the invention is as follows: the sodium alginate has a polyhydroxy structure, and can form hydrogen bonds with phosphate groups in thallus cell membrane phospholipid or with thallus protein polar groups in the freezing or drying process to protect the structural and functional integrity of cell membranes and proteins. The skim milk powder can be wrapped on the outer layer of the bacterial cells to protect the bacteria. The glycine ions have both acid and base amphoteric properties, and thus inhibit the pH change of the solution during the freeze-drying process. The reduced glutathione and phytic acid act as antioxidants to reduce cellular oxidase activity during lyophilization and long term storage, preventing oxidative deterioration of lyophilized products.
The invention has the beneficial effects that:
(1) the silver white staphylococcus standard reference strain has the advantages of standard staphylococcus thalli microscopic morphology and physiological and biochemical characteristics, clear genetic background, specific gene targets, and identifiability. Compared with other standard strains of the species, the strains have the most typical characteristics of food source staphylococcus albus isolated in China, can reflect the background of food source flow in China, have certain representativeness, and can be used as reference strains for scientific research.
(2) Aiming at the staphylococcus albus of the invention, the inventor provides a freeze-drying protective agent. The protective agent has the following advantages: the molding is good, the appearance is beautiful, the water solubility is good, and the solution can be completely dissolved within 1 to 2 seconds; the freeze-drying survival rate can reach more than 90 percent; can be stored for at least more than one year at the temperature of minus 20 ℃, has no change in quantity value, and can be used for long-term storage of quantitative quality control strains.
Biological material preservation
A strain of Staphylococcus albus (Staphylococcus argenteus) Sta180-0 is classified and named Staphylococcus albus (Staphylococcus argenteus), is deposited in Guangdong province microorganism culture collection center in No.1 and No. 8 in 2020, and has a deposit number of GDMCC 60854.
Staphylococcus argentis (Staphylococcus argenteus) Sta226-0, which is classified and named Staphylococcus argentis (Staphylococcus argenteus), has been deposited in Guangdong province microorganism culture collection center in No. 1/8 of 2020, with the deposit number of GDMCC 60948.
A strain of Staphylococcus albus (Staphylococcus argenteus) Sta3513A1, which is classified and named Staphylococcus albus (Staphylococcus argenteus), is deposited in Guangdong province collection center of microorganism strains in No.1 and No. 8 in 2020, and has a deposit number of GDMCC 60949.
Drawings
FIG. 1 is a colony morphology of a standard reference strain of Staphylococcus albus strain.
FIG. 2 shows the MLST clade analysis of Staphylococcus albus strains and Staphylococcus aureus.
FIG. 3 shows the PCR detection result of NRPS gene of Staphylococcus albus.
FIG. 4 is a comparison of Staphylococcus albus and Staphylococcus aureus after 48h incubation on Nutrient Agar (NA) plates; the left panel is Staphylococcus aureus ATCC6538, and the right panel is Staphylococcus albus Sta 180-0.
FIG. 5 is a schematic diagram of the biochemical identification of API Staph of Sta180-0, Sta226-0 and Sta3513A1 strains of Staphylococcus argentatus.
FIG. 6 is PCR amplification chart of specific gene segments of Sta226-0 and Sta3513A1 of Staphylococcus albus strain and other Staphylococcus albus; wherein M: DL 2000 Marker; lane 1-118: a food isolate of staphylococcus albus.
FIG. 7 is PCR amplification chart of specific gene segment of Staphylococcus argentis strain Sta180-0 and other Staphylococcus argentis; wherein M: DL 2000 Marker; lane 1-118: a food isolate of staphylococcus albus; +: 180-0 parts of staphylococcus albus strain Sta; c: blank control.
FIG. 8 shows the change of bacterial content of standard Staphylococcus albus strains of the present invention prepared by each protective agent after 12 months of storage.
Detailed Description
To more clearly illustrate the technical solutions of the present invention, the following embodiments are further described, but the present invention is not limited thereto, and these embodiments are only some examples of the present invention.
Example 1 isolation of Staphylococcus albus
The silver white staphylococcus found this time was collected from staphylococcus aureus isolated previously. The strains are 1581 strains in total and are separated from 39 cities in China by 4300 parts of food samples of different food types. The specific separation method is as follows: qualitative and quantitative detection are carried out on the collected samples 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. Sampling 25g (mL), adding 225mL of physiological saline into a sterile homogenizing bag, homogenizing and shaking uniformly to prepare a sample solution in a ratio of 1:10, adding 1mL of the sample solution into a test tube filled with 9mL of 10% sodium chloride trypticase soybean broth to prepare a sample solution in a ratio of 1:100, and preparing a sample solution in a ratio of 1:1000 according to the method; 3 gradients are parallel, the sample solution is placed in a constant temperature incubator at 37 ℃ for culturing for 48h, the sample solution with each gradient concentration is respectively streaked on a staphylococcus aureus chromogenic plate culture medium, and the culture is carried out for 24-48h at 37 ℃. Its typical staphylococcus aureus colonies were pink ball-shaped with smooth wet edges on the chromogenic plate (fig. 1). Target colonies were transferred from NA plates into brain heart infusion Broth (BHI) and allowed to resuscitate overnight at 37 ℃. Adding the bacterial liquid into a glycerin tube with the final concentration of 40% under the aseptic condition, storing in a refrigerator at the temperature of minus 40 ℃, and storing in a freeze-drying tube. The purified colony can be identified in aspects of morphological characteristics, physiological biochemistry, serotype, molecular biology and the like.
3 silver white staphylococcus strains Sta180-0, Sta226-0 and Sta3513A1 are obtained by co-separation, and are preserved in Guangdong province microbial strain preservation center, the address is microbial institute laboratory building of Michelia Toxico 100, Guangzhou, China, the preservation date is No. 12/30 in 2019, and the preservation numbers are GDMCC60854, GDMCC60948 and GDMCC60949 respectively.
The 3 strains were isolated from food samples from Guangzhou gulf of litchi, Guangzhou Caryu and hong Kong, respectively, and the specific isolation information is shown in Table 1.
TABLE 1 isolation information of the strains
Example 2 identification of Staphylococcus albus
1. Identification method
1.1 Multi-site sequence (MLST) typing analysis
Staphylococcus albus can be completely distinguished from staphylococcus aureus by multi-locus sequence typing (MLST) and NRPS gene detection. Multi-site sequence analysis was mainly performed on 7 housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi and yqil), which were identified as Staphylococcus albus when the arcC site numbers were 36,151,207 and 272 and the pta site numbers were 39,107,145,175,198,256,268 and 287. NRPS gene detection was performed simultaneously (fig. 3). The primers and amplification method are described in the literature. The 8 primers used were synthesized by Beijing Liuhe Dagenetechnology GmbH (see Table 2 for primer sequences). The PCR amplification conditions were as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 2min, and performing 35 cycles; finally, extension is carried out for 10min at 72 ℃. The reaction system (25. mu.L) contained: 12.5. mu.l of 2 XDreamtaq mastermix, 8.5. mu.l of ultrapure water, 80ng of template DNA, 0.5. mu.M of upstream and downstream primers. The DNA product was purified using a PCR purification kit (Qiagen, Genman), and the purified PCR product was subjected to sequence determination by the agency of Beijing Liuhe Dageno science and technology Co., Ltd. The numbering and ST type of each housekeeping gene was compared by the Staphylococcus aureus MLST database: (https://pubmlst.org/saureus/)。
TABLE 2 MLST and NRPS primers and amplified fragments
1.2 analysis of physiological and biochemical characteristics and drug sensitivity characteristics of Staphylococcus albus strains
Dyeing and microscopic examination: and smearing the suspicious colonies, performing gram staining, and observing the morphology by microscopic examination. The silver white staphylococcus is gram positive like staphylococcus aureus and forms a prototype staphylococcus string under a microscope.
Plasma coagulase assay: a single colony was inoculated into 5mL of BHI medium and cultured at 37 ℃ for 18-24 h. The culture medium was aspirated by 1mL, added to plasma coagulase, and cultured at 37 ℃. After 2.5 hours, whether coagulation occurs or not is observed every hour, and if coagulation does not occur after 6 hours, the culture is incubated overnight and then observed and verified.
And (3) NA plate identification: the standard strain of staphylococcus aureus and the staphylococcus albus were scribed into NA plates at the same time, and the staphylococcus albus was clearly seen (fig. 4).
And (3) drug sensitivity characteristic analysis: the KB method is used for verifying drug sensitivity. Activating by NA plate, adding physiological saline, and diluting to final concentration of 1 × 107cfu/mL was spread on MH plates, and after the bacterial solution had dried, antibiotic paper sheets were attached to the surface of the medium and cultured at 37 ℃ for 24 h. And measuring the size of the bacteriostatic zone by using a vernier caliper to be accurate to 0.01 mm. The antibiotics selected were as follows: ammoxiflorin clavulanic acid (AMC, 30 μ g), ampicillin (AMP, 10 μ g), cefepime (FEP, 10 μ g), cefoxitin (FOX, 30 μ g), penicillin (P, 10U), ceftazidime (CAZ, 30 μ g), amikacin (AK, 30 μ g), gentamycin (CN, 10 μ g), kanamycin (K, 30 μ g), streptomycin (S, 25 μ g), chloramphenicol (C, 30 μ g), clindamycin (DA, 2 μ g), erythromycin (E, 15 μ g), telithromycin (TEL, 15 μ g), ciprofloxacin (CIP, 5 μ g), norfloxacin (NOR, 10 μ g), tetracycline (TE, 30 μ g), linezolid (LZD, 30 μ g), rifampicin (RD, 5 μ g), neonomycin (T, 25 μ g), quinupristin/daltepine (QD, 15 μ g), ziram (QD, 15 μ g), Teicoplanin (TEC, 30 μ g), nitrofurantoin (F, 300 μ g) and fuscin (FD, 10 μ g). Staphylococcus aureus ATCC25923 and Escherichia coli ATCC25922 are used as quality control strains.
1.3 Staphylococcus Protein A (SPA) typing
Staphylococcal Protein A (SPA) is a constituent of the staphylococcal cell wall and comprises an Fc binding region, an X domain consisting of a variable number of 24bp repeats and 3C-terminal regions,exhibiting genetic polymorphisms. The primers and amplification method are described in the literature. The primers used were synthesized by Beijing Liuhe Dagen science and technology Co., Ltd (see Table 3 for the sequence of the primers). The PCR amplification conditions were as follows: pre-denaturation at 80 deg.C for 5min, denaturation at 94 deg.C for 45s, annealing at 60 deg.C for 45s, and extension at 72 deg.C for 2min, performing 35 cycles, and extending at 72 deg.C for 10 min. The reaction system (25. mu.L) contained: 12.5. mu.l of 2 XDreamTAq mastermix, 9.5. mu.l of ultrapure water, 40ng of template DNA, 0.5. mu.M of upstream and downstream primers. Sequencing the amplified product, selecting the repeat sequence of spa gene (24p is a unit) and databasehttp://spaserver2.ridom.de) Comparing the existing types to obtain the spa type.
TABLE 3 spa primers and amplified fragments
1.4 characteristic sequence analysis of Staphylococcus albus strains
The non-essential genes specific to the three strains are mainly obtained according to the pan-genomic analysis result of the staphylococcus albus. The genome sequence of a representative strain of Staphylococcus albus (containing the analyzed strain) was co-selected 245 for pan-genomic analysis. The Pan-genome is analyzed by using an MP method in prokaryotic Pan-genome automated Analysis software (PGAP), and the Analysis result is processed by using a local Perl script to obtain the information of the core genes and non-core genes of all strains.
Extracting the non-core gene protein sequences specific to the three strains, and respectively comparing the sequences back to a total protein library of staphylococcus albus and a NCBI non-redundant protein database (NR) by local Blast. The sequences that align to the known silver white staphylococcal protein were removed, leaving genes unique to the standard strain. The specificity of the specific gene is tested in related strains and silver grape isolates through PCR amplification.
2. Identification results
TABLE 4 ST type of the strains according to the invention
TABLE 5 spa type of the strains according to the invention
These strains can be cultured in TSA, BHI and NA media.
TABLE 6 resistance of the strains of the invention to different antibiotics
Note: r ═ drug resistance; i is moderate drug resistance; s-sensitive
These strains also carry a unique gene island, in which Sta180-0 carries SEQ ID No.1, Sta226-0 and Sta3513A1 carry the same gene island sequence SEQ ID No. 2. The relevant gene islands can be subjected to amplification test through primers SEQ ID No. 3-SEQ ID No. 6 in Table 7, and the verification results are shown in FIG. 4 and FIG. 5.
The 3 silver white staphylococcus strains respectively carry the following specific gene islands (characteristic sequences):
sta180-0 of Staphylococcus argentis (Staphylococcus argenteus)
SEQ ID No:1
>180-0_02558group_23936
TTGCTGAATTTCTGTATCTATATTTGTAACTATATTCCATTATTTGTTTTAATATTTTTGAATGAAATTAGAAAAGCTGATGATAATCATAAAAGTATACGCTTTGTAATTTTAGAATATAAAACATATTGGATTATACTGTTATTAGCTAGTTTAATAAGTATTTGTGTACTTATTTATAAATGGCTTAAAAATGCACAAAGAACTTATGAAGTTCCAGGTAAGCTAAAAATAATAAACACTGACATTTTAAATTATTTTGTTACATATTTAGTCCCATTATTATCATTAGACTTATCAAGCATTTACAGTATAATAATTAACTTTATAATTTTTGCAATAATAGGTACATTTCACACAAAGAGTGATATGCTACATTATAATTTATTATTAGTTTTAATGGGCTATAATATATATTCAAATGAAAAAAAATAA
Staphylococcus argentiacus (Staphylococcus argenteus) Sta226-0 and Sta3513A1
SEQ ID No:2
>226-0_03040group_24153
ATGAGAGAGAAAGAACAAGAAGCAATCACATCAAAATTGAAAGAGACGACGAAGGACTTTCGAGATAATAGCATAAAAACACATAATGATTTTGTAAGATTACTTCAAGATAATTTAAAAAAAAGTGAACACAGAAGAAATTCAAGCAGAATTAAGAAGAGATGTACGTAA
TABLE 7 Gene island verification primers for strains of the invention
The invention also provides a freeze-drying protective agent for the staphylococcus albus, and provides a plurality of specific examples (examples 3-5) and comparative examples (comparative examples 1-3) of the freeze-drying protective agent.
Example 3
A freeze-drying protective agent for silver white staphylococcus comprises 3 parts by mass of sodium alginate, 10 parts by mass of skimmed milk powder, 0.2 part by mass of phytic acid, 0.1 part by mass of reductive glutathione and 0.2 part by mass of glycine.
Example 4
A freeze-drying protective agent for silver white staphylococcus comprises 5 parts by mass of sodium alginate, 12 parts by mass of skimmed milk powder, 0.5 part by mass of phytic acid, 0.2 part by mass of reductive glutathione and 2 parts by mass of glycine.
Example 5
A freeze-drying protective agent for silver white staphylococcus comprises 5 parts by mass of sodium alginate, 9 parts by mass of skimmed milk powder, 1 part by mass of phytic acid, 0.3 part by mass of reductive glutathione and 1 part by mass of glycine.
Comparative example 1
Compared with the freeze-drying protective agent in the embodiment 1, the freeze-drying protective agent does not contain reducing glutathione, and only contains 3 parts by mass of sodium alginate, 10 parts by mass of skimmed milk powder, 0.2 part by mass of phytic acid and 0.2 part by mass of glycine.
Comparative example 2
Compared with the freeze-drying protective agent in the example 1, the freeze-drying protective agent does not contain sodium alginate, and only contains 10 parts by mass of skimmed milk powder, 0.2 part by mass of phytic acid, 0.1 part by mass of reducing glutathione and 0.2 part by mass of glycine.
Comparative example 3
The lyoprotectant contained 10 parts by mass of skimmed milk powder only, compared to example 1.
Example 6 comparison of lyophilized survival rate and stability of quantitative quality control bacteria
In this example, the bacterial powder was prepared as follows: inoculating the recovered strain into a shake flask, culturing until a proper bacterial quantity is selected and added into a protective agent from the late logarithmic phase to the early stationary phase, uniformly mixing, subpackaging into penicillin bottles, sampling, diluting and counting to obtain the bacterial content A0 before freeze-drying. Transferring the half-stoppered split penicillin bottles into a freeze dryer for pre-freezing at the temperature of minus 40 ℃ for 3 hours, starting main drying for 20-25 hours, then entering an analysis drying stage for 6-8 hours, finishing drying, pressing the stoppered split penicillin bottles in a vacuum state, moving the split penicillin bottles out of the freeze dryer, automatically capping the split penicillin bottles, ensuring the complete vacuum state of the samples, and storing the split penicillin bottles at the low temperature of 2-8 ℃. And (4) taking the freeze-dried sample for dilution counting, wherein the counting result is the bacterial content A after freeze-drying, and the freeze-drying survival rate is the percentage of A to A0.
TABLE 8 comparison of the freeze-drying survival rates of lyoprotectants of different compositions
Example 7 variation of bacterial content over shelf-life of lyoprotectants of different compositions
Different protective agents are prepared according to the formulas of the examples 3-5 and the comparative examples 1-3, the silver white staphylococcus strains Sta180-0, Sta226-0 and Sta3513A1 are stored, the silver white staphylococcus strains are stored at the temperature of 2-8 ℃, and 3 samples are extracted every month to check the bacteria content according to the counting method. In order to better compare the effect of each protective agent in the long-term storage process, the number of live bacteria before freeze-drying is calculated according to the freeze-drying survival rate of each protective agent when the quantitative quality control bacteria is prepared, so that the bacteria content is about 1000 cfu/bottle after freeze-drying.
As shown in fig. 8, the number of viable bacteria of the lyoprotectants of examples 3 to 5 was not greatly changed during long-term storage, while the number of viable bacteria of the lyoprotectants of comparative examples 1 to 3 was significantly decreased during long-term storage.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
SEQUENCE LISTING
<110> Guangdong province institute for microbiology (Guangdong province center for microbiological analysis and detection)
GUANGDONG HUANKAI BIOTECHNOLOGY Co.,Ltd.
<120> Staphylococcus albus standard reference strain containing specific molecular target, and detection and application thereof
<130> 2020.12.28
<160> 6
<170> PatentIn version 3.3
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Claims (2)
1. The application of the primer for detecting the specific molecular target in preparing the silver white staphylococcus detection reagent is characterized in that the specific molecular target is as follows:
1, as shown in SEQ ID NO;
primer for detecting specific molecular target shown in SEQ ID NO.1 for preparing silver white staphylococcusStaphylococcus argenteus) Sta180-0, accession number GDMCC60854 detection reagent.
2. The use according to claim 1,
the primer aiming at detecting the specific molecular target shown as SEQ ID NO.1 comprises: the upstream primer shown as SEQ ID NO.3 and the downstream primer shown as SEQ ID NO. 4.
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| PCT/CN2021/086921 WO2022141936A1 (en) | 2020-12-30 | 2021-04-13 | Standard reference strains of staphylococcus argenteus containing specific molecular target and detection and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2017163969A (en) * | 2016-03-14 | 2017-09-21 | 東洋紡株式会社 | Oligonucleotide for detecting staphylococcus |
| JP2017163970A (en) * | 2016-03-14 | 2017-09-21 | 東洋紡株式会社 | Oligonucleotide for detecting staphylococcus aureus |
| CN111154899A (en) * | 2020-01-19 | 2020-05-15 | 广东省微生物研究所（广东省微生物分析检测中心） | 4 common pathogenic staphylococcus specific new molecular targets and rapid detection method thereof |
| CN111893198A (en) * | 2020-07-16 | 2020-11-06 | 广东省微生物研究所（广东省微生物分析检测中心） | Specific molecular target for identifying staphylococcus albus, detection primer group and rapid detection method thereof |
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| US9279160B2 (en) * | 2009-06-22 | 2016-03-08 | Statens Serum Institut | DNA-based methods for clone-specific identification of staphylococcus aureus |
| CN112575100B (en) * | 2020-12-30 | 2022-06-14 | 广东省微生物研究所（广东省微生物分析检测中心） | Staphylococcus albus standard reference strain containing specific molecular target and detection and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2017163969A (en) * | 2016-03-14 | 2017-09-21 | 東洋紡株式会社 | Oligonucleotide for detecting staphylococcus |
| JP2017163970A (en) * | 2016-03-14 | 2017-09-21 | 東洋紡株式会社 | Oligonucleotide for detecting staphylococcus aureus |
| CN111154899A (en) * | 2020-01-19 | 2020-05-15 | 广东省微生物研究所（广东省微生物分析检测中心） | 4 common pathogenic staphylococcus specific new molecular targets and rapid detection method thereof |
| CN111893198A (en) * | 2020-07-16 | 2020-11-06 | 广东省微生物研究所（广东省微生物分析检测中心） | Specific molecular target for identifying staphylococcus albus, detection primer group and rapid detection method thereof |
Non-Patent Citations (4)
| Title |
|---|
| Staphylococcus argenteus isolated from retail foods in China: Incidence, antibiotic resistance, biofilm formation and toxin gene profile;Shi Wu等;《Food Microbiology》;20200426;第91卷;第1-10页 * |
| 葡萄球菌属不同靶基因序列种水平鉴定能力的比较评价研究;宋明辉等;《药物分析杂志》;20180430;第38卷(第4期);第672-679页 * |
| 银白色葡萄球菌实时荧光PCR快速检测方法的建立;郭正洋等;《食品工业科技》;20171025;第39卷(第6期);第222-226页 * |
| 银白色葡萄球菌的发现、分布与致病性;张道锋等;《微生物学报》;20200617;第61卷(第2期);第263-278页 * |
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