CN116622865A - Staphylococcus aureus drug resistance RPA detection composition and application thereof - Google Patents

Staphylococcus aureus drug resistance RPA detection composition and application thereof Download PDF

Info

Publication number
CN116622865A
CN116622865A CN202310236032.0A CN202310236032A CN116622865A CN 116622865 A CN116622865 A CN 116622865A CN 202310236032 A CN202310236032 A CN 202310236032A CN 116622865 A CN116622865 A CN 116622865A
Authority
CN
China
Prior art keywords
nfo
exo
gene
seq
probe
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202310236032.0A
Other languages
Chinese (zh)
Inventor
林创兴
曾咏梅
廖佳宇
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Second Affiliated Hospital of Shantou University Medical College
Original Assignee
Second Affiliated Hospital of Shantou University Medical College
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Second Affiliated Hospital of Shantou University Medical College filed Critical Second Affiliated Hospital of Shantou University Medical College
Priority to CN202310236032.0A priority Critical patent/CN116622865A/en
Publication of CN116622865A publication Critical patent/CN116622865A/en
Pending legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/44—Staphylococcus
    • C12R2001/445—Staphylococcus aureus
    • 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

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a staphylococcus aureus drug resistance RPA detection composition and application thereof. The staphylococcus aureus drug resistance RPA detection composition comprises at least one of 10 primer pair probe combinations for respectively detecting 10 genes such as nuc genes and the like. By adopting the primer probe combination to carry out RPA, whether staphylococcus aureus is infected or not can be obtained within 40 minutes, and if so, the drug resistance condition of staphylococcus aureus can be obtained. The RPA amplification of the primer probe composition has higher sensitivity and specificity, and the detection accuracy rate of penicillin, oxacillin sodium, erythromycin, clindamycin, tetracycline and gentamicin in a sample reaches 92-97%. Primer probe compositions for RPA-LFD and RPA-FSM are suitable for areas of resource starvation and lack of professionals, respectively.

Description

Staphylococcus aureus drug resistance RPA detection composition and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to a staphylococcus aureus drug resistance RPA detection composition and application thereof.
Background
Antibiotic resistance (AMR) refers to the ability of microorganisms (including bacteria, viruses, parasites and fungi) to block the antibiotic, antiviral, antimalarial or antifungal agent from bacteria. In recent years, the misuse and misuse of antibiotics has led to an increasing number and type of resistant microorganisms, which has become one of the greatest threats to global public health safety. Microbial resistance is mainly due to abuse and overuse of antibacterial drugs, which may develop AMR if under-dosed or incorrect antibiotic species are used.
Staphylococcus aureus (s. Aureus) is an important pathogen that causes a variety of diseases with high morbidity and mortality, and is environmentally friendly. Staphylococcus aureus strains produce a mechanism of resistance to almost all antibacterial drugs used in therapy, including-lactams, glycopeptides, tetracyclines, and the like. Therefore, it is important to find a method for detecting staphylococcus aureus resistance, which has an important role in providing targeted antimicrobial treatment and reducing the risk of mortality for clinical diagnosis.
Currently, the method of detecting staphylococcus aureus resistance is to conduct a bacterial growth test in the presence of antibiotics to determine the susceptibility or resistance of a pathogen to a particular antibiotic. These tests typically take 20-72 hours to obtain results, and thus tend to delay the optimal time for treatment. Furthermore, strains carrying AMR genes may exhibit antibiotic susceptibility due to lack of gene expression or dysplasia, which may lead to misleading negative results. Molecular diagnostic tests, such as techniques based on polymerase chain reaction, matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and next generation sequencing, can be performed directly on clinical samples, providing early information about the resistance profile of clinical strains. However, these techniques are complex, require specialized training by the operator, or rely on expensive equipment, and are therefore unsuitable for use in areas of scarce resources or areas lacking specialized technicians.
Multiple resistant staphylococcus aureus strains are the primary source of acquired disease, and therefore, their accurate and early identification is a key goal of clinical microbiology. Point of care testing (POCT) is a portable mobile testing system that approaches a sample under test in a non-laboratory environment and reports the results immediately. POCT has become a key detection technology for a variety of specific pathogens. In recent years, several studies have demonstrated the extremely high ability of RPA to selectively identify microorganisms and genes of interest. This capability makes RPA a valuable POCT technique in clinical microbiology research. Some researchers have demonstrated the feasibility of RPA or RPA binding to various aptamer technologies, such as binding to multicomponent nuclease (MNAzymes) based technologies and CRISPR/Cas based technologies. However, almost all RPA-based genotyping assays have focused on determining two genes (nuc and mecA) associated with methicillin-resistant streptococcus, while our goal is to identify staphylococcus aureus and analyze genes representing four families of common antibiotics.
Disclosure of Invention
The primary object of the present invention is to overcome the disadvantages and shortcomings of the prior art and to provide a staphylococcus aureus resistance RPA detection composition.
The aim of the invention is achieved by the following technical scheme: the staphylococcus aureus drug resistance RPA detection composition comprises at least one combination of 10 primer pair probe combinations for respectively detecting nuc gene, blaZ gene, mecA gene, ermA gene, ermB gene, ermC gene, msrA gene, tetK gene, tetM gene and aadD1 gene.
Detecting a primer pair nuc-exo-F4, nuc-exo-R1 and a probe nuc-exo-P01 of the nuc gene; the nucleotide sequences of nuc-exo-F4, nuc-exo-R1 and nuc-exo-P01 are shown as SEQ ID NO.1-SEQ ID NO. 3;
detecting primer pairs blaZ-exo-F4, blaZ-exo-R4 and a probe blaZ-exo-P01 of the blaZ gene; the nucleotide sequences of the blaZ-exo-F4, the blaZ-exo-R4 and the blaZ-exo-P01 are shown as SEQ ID NO.4-SEQ ID NO. 6;
detecting primer pairs mecA-exo-F1, mecA-exo-R2 and a probe mecA-exo-P01 of the mecA gene; the nucleotide sequences of mecA-exo-F1, mecA-exo-R2 and mecA-exo-P01 are shown as SEQ ID NO.7-SEQ ID NO. 9;
detecting a primer pair ermA-exo-F3, ermA-exo-R1 and a probe ermA-exo-P01 of the ermA gene; the nucleotide sequences of the ermA-exo-F3, the ermA-exo-R1 and the ermA-exo-P01 are shown as SEQ ID NO.10-SEQ ID NO. 12;
detecting a primer pair ermB-exo-F3, ermB-exo-R3 and a probe ermB-exo-P01 of the ermB gene; the nucleotide sequences of ermB-exo-F3, ermB-exo-R3 and ermB-exo-P01 are shown as SEQ ID NO.13-SEQ ID NO. 15;
detecting a primer pair ermC-exo-F5, ermC-exo-R5 and a probe ermC-exo-P02 of the ermC gene; the nucleotide sequences of ermC-exo-F5, ermC-exo-R5 and ermC-exo-P02 are shown as SEQ ID NO.16-SEQ ID NO. 18;
detecting a primer pair msrA-exo-F2, msrA-exo-R5 and a probe msrA-exo-P01 of the msrA gene; the nucleotide sequences of the msrA-exo-F2, the msrA-exo-R5 and the msrA-exo-P01 are shown as SEQ ID NO.19-SEQ ID NO. 21;
detecting a primer pair tetK-exo-F2, tetK-exo-R2 and a probe tetK-exo-P03 of the tetK gene; the nucleotide sequences of the tetK-exo-F2, the tetK-exo-R2 and the tetK-exo-P03 are shown as SEQ ID NO.22-SEQ ID NO. 24;
detecting a primer pair tetM-exo-F2, tetM-exo-R3 and a probe tetM-exo-P03 of the tetM gene; the nucleotide sequences of tetM-exo-F2, tetM-exo-R3 and tetM-exo-P03 are shown as SEQ ID NO.25-SEQ ID NO. 27;
detecting a primer pair aadD1-exo-F2, aadD1-exo-R1 and a probe aadD1-exo-P01 of the aadD1 gene; the nucleotide sequences of aadD1-exo-F2, aadD1-exo-R1 and aadD1-exo-P01 are shown as SEQ ID NO.28-SEQ ID NO. 30.
Preferably, in the staphylococcus aureus resistance RPA detection composition, the probe 46nt comprises flanking 6-carboxyfluorescein deoxythymine (Fam-dT), the corresponding quenching group 1-deoxythymine (BHQ 1-dT), tetrahydrofuran (THF) substituted at nt 31, a C3 blocking group carried at the 3' end of the probe and an oligonucleotide 15nt downstream adjacent; the internal label of the probe is only thymine, and the number of inserted nucleotides is less than 5.
An RPA-FSM detection reagent for rapidly detecting the drug resistance of staphylococcus aureus, which comprises the RPA detection composition.
A method for detecting staphylococcus aureus resistance for non-disease diagnostic purposes comprising the steps of: taking DNA of a sample to be detected as a template, and adopting the RPA detection composition to carry out RPA detection to obtain a fluorescence value; and determining whether the sample to be detected contains staphylococcus aureus according to the fluorescence value.
Preferably, the reaction conditions for the RPA assay are 39℃for 20min.
Preferably, the RPA assay is performed using 2. Mu.L template, 2.1. Mu.L primer at 10. Mu.M, 0.6. Mu.L probe at 10. Mu.M, 2.5. Mu.L magnesium acetate at 280mM, and reaction buffer 29.5. Mu. L, ddH 2 O was added to 50. Mu.L. The staphylococcus aureus drug resistance RPA detection composition comprises at least one combination of 10 primer pair probe combinations for respectively detecting nuc gene, blaZ gene, mecA gene, ermA gene, ermB gene, ermC gene, msrA gene, tetK gene, tetM gene and aadD1 gene.
Detecting a primer pair nuc-nfo-F4, nuc-nfo-R1 and a probe nuc-nfo-P01 of the nuc gene; the nucleotide sequences of nuc-nfo-F4, nuc-nfo-R1 and nuc-nfo-P01 are shown as SEQ ID NO.31-SEQ ID NO. 33;
detecting primer pairs blaZ-nfo-F4, blaZ-nfo-R4 and a probe blaZ-nfo-P01 of the blaZ gene; the nucleotide sequences of the blaZ-nfo-F4, the blaZ-nfo-R4 and the blaZ-nfo-P01 are shown as SEQ ID NO.34-SEQ ID NO. 36;
detecting primer pairs mecA-nfo-F1, mecA-nfo-R2 and a probe mecA-nfo-P01 of the mecA gene; the nucleotide sequences of mecA-nfo-F1, mecA-nfo-R2 and mecA-nfo-P01 are shown as SEQ ID NO.37-SEQ ID NO. 39;
detecting primer pairs of the ermA gene, namely ermA-nfo-F3, ermA-nfo-R1 and a probe ermA-nfo-P01; the nucleotide sequences of the ermA-nfo-F3, the ermA-nfo-R1 and the ermA-nfo-P01 are shown as SEQ ID NO.40-SEQ ID NO. 42;
detecting a primer pair ermB-nfo-F3, ermB-nfo-R3 and a probe ermB-nfo-P01 of the ermB gene; the nucleotide sequences of ermB-nfo-F3, ermB-nfo-R3 and ermB-nfo-P01 are shown as SEQ ID NO.43-SEQ ID NO. 45;
detecting a primer pair ermC-nfo-F5, ermC-nfo-R5 and a probe ermC-nfo-P02 of the ermC gene; the nucleotide sequences of ermC-nfo-F5, ermC-nfo-R5 and ermC-nfo-P02 are shown as SEQ ID NO.46-SEQ ID NO. 48;
detecting a primer pair msrA-nfo-F2, msrA-nfo-R5 and a probe msrA-nfo-P01 of the msrA gene; the nucleotide sequences of the msrA-nfo-F2, the msrA-nfo-R5 and the msrA-nfo-P01 are shown as SEQ ID NO.49-SEQ ID NO. 51;
detecting a primer pair tetK-nfo-F2, tetK-nfo-R2 and a probe tetK-nfo-P03 of the tetK gene; the nucleotide sequences of the tetK-nfo-F2, the tetK-nfo-R2 and the tetK-nfo-P03 are shown as SEQ ID NO.52-SEQ ID NO. 54;
detecting a primer pair tetM-nfo-F2, tetM-nfo-R3 and a probe tetM-nfo-P03 of the tetM gene; the nucleotide sequences of tetM-nfo-F2, tetM-nfo-R3 and tetM-nfo-P03 are shown as SEQ ID NO.55-SEQ ID NO. 57;
detecting primer pairs aadD1-nfo-F2, aadD1-nfo-R1 and a probe aadD1-nfo-P01 of the aadD1 gene; the nucleotide sequences of aadD1-nfo-F2, aadD1-nfo-R1 and aadD1-nfo-P01 are shown as SEQ ID NO.58-SEQ ID NO. 60.
Preferably, in the staphylococcus aureus resistance RPA detection composition, the reverse primer of the primer pair is labeled with Fluorescein Isothiocyanate (FITC).
Preferably, in the staphylococcus aureus resistance RPA detection composition, the probe 46nt comprises a 5 '-biotin tag, a THF spacer substituted at nt 31 and an adjacent downstream 15nt oligonucleotide carrying a C3 blocking group at the 3' end of the probe.
An RPA-LFD detection reagent for rapidly detecting the drug resistance of staphylococcus aureus, which comprises the RPA detection composition.
A method for detecting staphylococcus aureus resistance for non-disease diagnostic purposes comprising the steps of: taking DNA of a sample to be detected as a template, and adopting the RPA detection composition to carry out RPA detection to obtain a fluorescence value; and determining whether the sample to be detected contains staphylococcus aureus according to the fluorescence value.
Preferably, the reaction condition detected by the RPA-LFD detection reagent is that the reaction is carried out for 15min at 37 ℃.
Preferably, the reaction system for detection using the RPA-LFD detection reagent is 2. Mu.L of template, 2.1. Mu.L of primer at a concentration of 10. Mu.M, 0.6. Mu.L of probe at a concentration of 10. Mu.M, 2.5. Mu.L of magnesium acetate at a concentration of 280mM, and 29.5. Mu. L, ddH of reaction buffer 2 O was added to 50. Mu.L.
The primer pair probe combinations designed aiming at the RPA-LFD and the RPA-FSM can respectively run under the conventional and unified conditions. While RPA-LFD is popular because of its fast visualization, RPA-FSM can significantly reduce test costs.
In clinical practice, monitoring detection in combination with source control is an effective method for preventing the spread of multidrug-resistant staphylococcus aureus. Nasal screening is performed, for example, on patients at high risk (ICU patients and patients with CVC or midline catheters outside the ICU) or patients entering a high risk setting (such as an intensive care unit). Results should be obtained as soon as possible after the sample is collected with the nasal swab. Patients who are colonized or infected with multiple resistant staphylococcus aureus (especially methicillin resistant staphylococcus aureus, MRSA) are then placed in the private ward and contact precautions are taken in the hospitalized acute care setting. These effective measures can greatly reduce the transmission of MRSA in patients. The primer probe combination is adopted to carry out RPA, detect the drug resistance of staphylococcus aureus, and can obtain a result within 40 minutes, thereby being beneficial to implementation of the intervention measures.
Compared with the prior art, the invention has the following beneficial effects:
by adopting the primer probe composition to carry out RPA, whether staphylococcus aureus is infected or not can be obtained within 40 minutes, and if so, 9 AMR genes aiming at 4 antibiotic families can be obtained, so that the drug resistance condition of staphylococcus aureus can be obtained, and the traditional difficult problem caused by drug resistance staphylococcus aureus infection can be solved.
The RPA amplification using the primer probe composition of the invention has higher sensitivity and specificity, and the detection lower limit is 10 1 -10 2 copy/. Mu.L, only Staphylococcus aureus was detected, and the primers did not crossAnd (3) carrying out a fork reaction. The clinical detection result is basically consistent with the PCR result. The detection accuracy rates of penicillin, oxacillin sodium, erythromycin, clindamycin, tetracycline and gentamicin in the sample respectively reach 92.59%, 94.44%, 96.30%, 94.44% and 96.30%.
For areas with lack of resources, the primer probe composition for the RPA-LFD is more suitable, and high-cost equipment is not needed; for areas lacking professionals, the primer probe composition for RPA-FSM of the invention is more suitable, and quantitative analysis can be simply realized.
Drawings
Fig. 1 is a flowchart of the operation of the RPA-LFD system.
FIG. 2 is a graph of the results of the RPA detection sensitivity test.
FIG. 3 is a graph of RPA detection specificity test results.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Staphylococcus aureus (ATCC-25923), klebsiella pneumoniae (ATCC-13883), proteus mirabilis (ATCC-35659), pseudomonas aeruginosa (ATCC-700829), escherichia coli (ATCC-25922), bacillus cereus (ATCC-14579), streptococcus pneumoniae (ATCC-49619), staphylococcus human (ATCC-700237), salmonella enterica (ATCC-14028) and Listeria monocytogenes (ATCC-7644) reference strains were all purchased from BIOBW (China).
Culture-based biochemical and drug sensitive assays, whole genome sequencing (NGS) and PCR-Sanger sequencing were used to identify all strains.
Genomic DNA (gDNA) was isolated from bacterial cells collected from bacterial culture medium using bacterial genomic DNA extraction kit (TIANamp Biotechnology co., ltd., beijin, china) according to the manufacturer's instructions.
The RPA kit was purchased from Twitdx corporation, UK.
All extracted gDNA were quantified using a NanoDrop2000 spectrophotometer (Thermo Fisher UK, no longer available) and kept at-20 ℃ until tested.
Example 1 design of target sequences, primers and probes
Isothermal amplification (Recombinase polymerase isothermal amplification, RPA) of recombinant enzymes is a technique for isothermal amplification of genes of interest using recombinant enzymes and polymerases as core components, single-stranded binding proteins, etc. as auxiliary components. When the impurity content in the detected sample is high, the RPA is less prone to error, which makes it possible to achieve the diagnostic requirements of rapid, immediate, sensitive and portable detection. The RPA amplification products can be detected by Agarose Gel Electrophoresis (AGE), fluorescent Signal Monitoring (FSM), and lateral flow assay (LFD). The RPA-LFD system combines the isothermal amplification technology of recombinase polymerase with the rapid detection technology of transverse flow detection test strips, can be used for rapidly, accurately and visually detecting drug-resistant staphylococcus aureus, and has the principle shown in figure 1.
The present study uses a DNA boiling lysis method to extract staphylococcus aureus whole genome DNA from a patient's blood culture specimen. The A260/280 and A260/230 ratios were measured using Nanodrop2000 c to determine sample purity, and DNA samples met the following criteria: 260/280=1.8; a260/230=2.0-2.2, and the quality is checked and stored at-80 ℃ for standby.
The whole genome DNA is used for recombinase polymerase amplification. First, a unique genomic region of the nuc gene was amplified to identify staphylococcus aureus infection. Secondly, the drug resistance genotype of staphylococcus aureus is detected by targeted amplification of the severe AMR genes of 4 different antibiotic families, such as blaZ and mecA. The study selected a high temperature resistant nuclease (nuc) gene and 9 different AMR genes, as shown in fig. 1. nuc gene and AMR gene sequences were obtained from the antibiotic resistance gene database (https:// ardbcbcb. Umd. Edu) and the national center for biotechnology information (NCBI, https:// www.ncbi.nlm.nih.gov), respectively. All bacterial whole genome sequences were from NCBI. Using basic local alignment search toolsBLAST, https:// BLAST. Ncbi. Nlm. Nih. Gov/BLAST. Cgi) finds the most conserved region. According toThe test design manual (https:// www.twistdx.co.uk/support/RPA-assay-design /) was used to design and screen for RPA primers and probes using Primer Premier 5.0 software (Premier Biosoft International, CA, USA) and Primer3Plus (https:// www.primer3plus.com /), and the Primer and probe combinations screened are shown in tables 1 and 2. Screening use->Basic kit was screened in combination with 2% AGE.
The study designed two versions of RPA-FSM and RPA-LFD. For resource starved areas, the RPA-Nfo primer and probe set in combination with LFD can provide a visual result quickly and without the need for costly equipment. Quantitative analysis can be achieved simply for areas lacking professionals using FSM versions of RPA-Exo probes. For the RPA-FSM version, the Exo probe (46 nt) included flanking 6-carboxyfluorescein deoxythymine (6-Fam-dT), the corresponding black hole quencher-1 deoxythymine (BHQ 1-dT) quencher group, a Tetrahydrofuran (THF) spacer substituted at nt 31, and an adjacent downstream oligonucleotide (15 nt) carrying a C3 spacer (polymerase extension blocking group) at its 3' end. The internal labels used in the probes are available only on thymine, with fewer than about 5 inserted nucleotides. For the RPA-LFD type, the reverse primer was labeled with Fluorescein Isothiocyanate (FITC). Nfo probe (46 nt) comprises a 5 '-biotin tag, a THF spacer substituted at nt 31 and an adjacent downstream oligonucleotide (15 nt), carrying a c3 spacer at its 3' end. All primers and probes were tested for cross-reactivity using an OligoAnalyzer3.1 (https:// www.idtdna.com/calc/Analyzer) from Integrated DNA technologies. DNA oligonucleotides were synthesized by GENEWIZ Biotechnology Inc. of Suzhou, china.
Taking nuc gene as an example, the above primer and probe screening strategy is explained: as a result of matching all the reverse primers using the forward primer F3, it was found that the F3R1 band (250 bp) was the most clear, and therefore, the R1 primer was determined to be the best reverse primer. The R1 primer was used to match all forward primers, indicating F4R1 is the best primer pair. In addition, 3 paired probes were paired with F4R1, respectively, and the probe P1-F4R1 combination amplified 2 positive clear bands. Based on the above results, it was determined that the P1-F4R1 combination was useful in subsequent experiments. Based on the same strategy, the optimal probe primer combinations for 9 AMR genes were also confirmed.
For RPA-FSM primer, use is made ofexo kit (twist dx ltd., maidenhead, UK). The final volume of all reaction mixtures was 50. Mu.L, including 2. Mu.L template, 2.1. Mu.L primer (10. Mu.M), 0.6. Mu.L Exo probe (10. Mu.M) and other standard reaction components, and 2.5. Mu.L magnesium acetate (280 mM) was added first and stirred well. The amplification visualization was performed using a real-time PCR detection system (product code: SLAN-96S, shanghai Marble medical science and technology Co., shanghai, china) at 39deg.C for 20min, with fluorescence data collected every 30S for 20min.
For RPA-LFD primer, use is made ofThe nfo kit was subjected to experiments (twist dx ltd., maidenhead, UK). To each 50. Mu.L of the reaction mixture were added 2. Mu.L of a template, 0.6. Mu.L of each 2.1. Mu. L, nfo probe (10 mM) of a primer (10 mM), and the mixture was then mixed with 2.5. Mu.L of magnesium acetate (280 mM), followed by mixing. Then, the reaction mixture was reacted at 37℃for 15min. The amplification results were diluted to 1/50 and tested on commercial LFD (beijing bao yingtong biotechnology limited, beijing, china) and the results were visually checked within 2-4 min. Commercial LFD consisted of sample pad, gold-labeled antibody pad (soaked with aunp-labeled anti-fitc antibody of mouse origin), streptavidin-coated detection line (T), anti-mouse antibody-coated control line (C) and absorbent pad, organized according to solvent migration pathway.
TABLE 1 primers and probes for RPA-FSM
TABLE 2 primers and probes for RPA-LFD
Example 2 optimization of the RPA System
In order to study the influence of each important parameter on experimental results, the concentration of the primer, the probe and magnesium acetate (280 mM), the reaction time and the reaction temperature are adjusted to obtain ideal RPA reaction conditions. The result shows that the optimal condition of the RPA-LFD is that the reaction is carried out for 15min at 37 ℃; the optimal reaction conditions for RPA-FSM were 39℃for 20min. The optimal reaction system for RPA-LFD was 2. Mu.L template, 2.1. Mu.L primer at 10. Mu.M, 0.6. Mu.L probe at 10. Mu.M, 2.5. Mu.L magnesium acetate at 280mM, and reaction buffer 29.5. Mu. L, ddH 2 O was made up to 50. Mu.L; the optimal reaction system for RPA-FSM was 2. Mu.L template, 2.1. Mu.L primer at 10. Mu.M, 0.6. Mu.L probe at 10. Mu.M, 2.5. Mu.L magnesium acetate at 280mM, reaction buffer 29.5. Mu. L, ddH 2 O was added to 50. Mu.L.
Example 3 sensitivity of RPA detection
To evaluate the sensitivity of the RPA detection primer probe combinations, the sequences of the 10 genes described above were cloned intoIn T Easy Vector (GENEWIZ Biotechnology Co., st. Of China) and using different copies of the recombinant plasmid (10) 1 copies/μL、10 2 copies/μL、10 3 copies/μL、10 4 copies/μL、10 5 copies/μL、10 6 The RPA reaction was carried out using copies/. Mu.l as a template (the reaction conditions were the optimal reaction conditions obtained in example 2; the reaction system was the best reaction system obtained in example 2). Based on the size of the plasmid (3015 bp) and insert (300-400 bp), the copy number was estimated and the plasmid was diluted to 1X 10 1 To 1X 10 5 The copies/. Mu.L to determine the precise sensitivity of the RPA-LFD and RPA-FSM assays. Each synthetic gene plasmid standard was used to determine Staphylococcus aureusThe lower detection Limit (LOD) of the AMR diagnostic platform. As a result, see FIG. 2, the LOD of the RPA-FSM and the LOD range of the RPA-LFD are both 10 1 -10 2 The probes/. Mu.L has high detection sensitivity.
EXAMPLE 4 specificity of RPA detection
To evaluate the specificity of the RPA detection primer probe combinations, DNA was extracted from strains such as Staphylococcus aureus, klebsiella pneumoniae, pseudomonas aeruginosa, proteus mirabilis, escherichia coli, streptococcus pneumoniae, bacillus cereus, staphylococcus human, salmonella enterica and Listeria monocytogenes, and the like, and the evaluation was performed using the specificity of the 10-gene RPA-FSM and RPA-LFD primer-probe combinations (using the optimal reaction conditions, reaction system obtained in example 2).
The presence of nuc gene was detected by RPA reaction of staphylococcus aureus with DNA of other bacteria, respectively. The results indicate that the RPA-FSM, RPA-FSM primer-probe combinations were not effective in amplifying other microorganisms, such as Klebsiella pneumoniae and Streptococcus pneumoniae (FIG. 3).
EXAMPLE 5 antibiotic susceptibility testing
The susceptibility of staphylococcus aureus to 6 antibiotics (penicillin, gentamicin, oxacillin sodium, clindamycin, erythromycin, tetracycline) was detected using a VITEK 2Compact fully automated microbiological analysis system (bioMerieux China, inc.). The drug susceptibility test was performed using staphylococcus aureus ATCC 29213 as a quality control strain.
54 clinical samples (6 blood samples, 3 throat swabs, 19 urine, 26 other samples) of patients suspected of having staphylococcus aureus infection in a second affiliated hospital of the university of Shanzhi medical school were collected and pretreated. DNA extraction was performed using a bacterial genomic DNA extraction kit (TIANamp Biotechnology co., ltd., beijin, china). Using the above extracted DNA as a template, performing RPA amplification by using RPA-LFD and RPA-FSM primer-probe combinations, respectively, performing PCR amplification by using RPA primer (reaction system (25. Mu.L): 2 XPCR Mix 12.5. Mu.L, 1. Mu.L each of the upstream and downstream primers (10. Mu.M), 8.5. Mu.L of ultrapure water, 2. Mu.L of template DNA; the reaction conditions were 95℃for 3min, 95℃for 30s,55℃for 30s,72℃for 1min for 30 cycles, and 72℃for 10min for detection of the amplified product by 1.2% agarose gel electrophoresis under the conditions of 110V voltage, 300mA current for 25min, and the results observed on a gel imaging system after the electrophoresis was completed.
Compared with the detection results of the hospital diagnosis facilities, the detection accuracy rates of penicillin, oxacillin sodium, erythromycin, clindamycin, tetracycline and gentamicin by adopting the RPA-LFD and RPA-FSM primer-probe combination to carry out RPA amplification reach 92.59%, 94.44%, 96.30%, 94.44% and 96.30%, and the detection accuracy rates are consistent with the PCR detection results.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.

Claims (10)

1. The staphylococcus aureus drug resistance RPA detection composition is characterized by comprising at least one combination of 10 primer pair probe combinations for respectively detecting nuc gene, blaZ gene, mecA gene, ermA gene, ermB gene, ermC gene, msrA gene, tetK gene, tetM gene and aad1 gene;
detecting a primer pair nuc-exo-F4, nuc-exo-R1 and a probe nuc-exo-P01 of the nuc gene; the nucleotide sequences of nuc-exo-F4, nuc-exo-R1 and nuc-exo-P01 are shown as SEQ ID NO.1-SEQ ID NO. 3;
detecting primer pairs blaZ-exo-F4, blaZ-exo-R4 and a probe blaZ-exo-P01 of the blaZ gene; the nucleotide sequences of the blaZ-exo-F4, the blaZ-exo-R4 and the blaZ-exo-P01 are shown as SEQ ID NO.4-SEQ ID NO. 6;
detecting primer pairs mecA-exo-F1, mecA-exo-R2 and a probe mecA-exo-P01 of the mecA gene; the nucleotide sequences of mecA-exo-F1, mecA-exo-R2 and mecA-exo-P01 are shown as SEQ ID NO.7-SEQ ID NO. 9;
detecting a primer pair ermA-exo-F3, ermA-exo-R1 and a probe ermA-exo-P01 of the ermA gene; the nucleotide sequences of the ermA-exo-F3, the ermA-exo-R1 and the ermA-exo-P01 are shown as SEQ ID NO.10-SEQ ID NO. 12;
detecting a primer pair ermB-exo-F3, ermB-exo-R3 and a probe ermB-exo-P01 of the ermB gene; the nucleotide sequences of ermB-exo-F3, ermB-exo-R3 and ermB-exo-P01 are shown as SEQ ID NO.13-SEQ ID NO. 15;
detecting a primer pair ermC-exo-F5, ermC-exo-R5 and a probe ermC-exo-P02 of the ermC gene; the nucleotide sequences of ermC-exo-F5, ermC-exo-R5 and ermC-exo-P02 are shown as SEQ ID NO.16-SEQ ID NO. 18;
detecting a primer pair msrA-exo-F2, msrA-exo-R5 and a probe msrA-exo-P01 of the msrA gene; the nucleotide sequences of the msrA-exo-F2, the msrA-exo-R5 and the msrA-exo-P01 are shown as SEQ ID NO.19-SEQ ID NO. 21;
detecting a primer pair tetK-exo-F2, tetK-exo-R2 and a probe tetK-exo-P03 of the tetK gene; the nucleotide sequences of the tetK-exo-F2, the tetK-exo-R2 and the tetK-exo-P03 are shown as SEQ ID NO.22-SEQ ID NO. 24;
detecting a primer pair tetM-exo-F2, tetM-exo-R3 and a probe tetM-exo-P03 of the tetM gene; the nucleotide sequences of tetM-exo-F2, tetM-exo-R3 and tetM-exo-P03 are shown as SEQ ID NO.25-SEQ ID NO. 27;
detecting a primer pair aadD1-exo-F2, aadD1-exo-R1 and a probe aadD1-exo-P01 of the aadD1 gene; the nucleotide sequences of aadD1-exo-F2, aadD1-exo-R1 and aadD1-exo-P01 are shown as SEQ ID NO.28-SEQ ID NO. 30.
2. The staphylococcus aureus resistant RPA assay composition of claim 1, wherein in the staphylococcus aureus resistant RPA assay composition, the probe 46nt comprises flanking 6-carboxyfluorescein deoxythymine (Fam-dT), the corresponding quenching group 1-deoxythymine (BHQ 1-dT), tetrahydrofuran (THF) substituted at nt 31, a C3 blocking group carried at the 3' end of the probe, and an oligonucleotide 15nt downstream adjacent; the internal label of the probe is only thymine, and the number of inserted nucleotides is less than 5.
3. An RPA-FSM assay reagent for rapid detection of staphylococcus aureus resistance comprising an RPA assay composition according to claim 1 or 2.
4. A method for detecting staphylococcus aureus resistance for non-disease diagnostic purposes comprising the steps of: taking DNA of a sample to be detected as a template, and adopting the RPA detection composition of claim 1 or 2 to carry out RPA detection to obtain a fluorescence value; and determining whether the sample to be detected contains staphylococcus aureus according to the fluorescence value.
5. The method for detecting resistance to staphylococcus aureus of claim 4, wherein at least one of the following:
the reaction condition of the RPA detection is 39 ℃ for 20min;
the RPA detection reaction system comprises 2 mu L of template, 2.1 mu L of primer with the concentration of 10 mu M, 0.6 mu L of probe with the concentration of 10 mu M, 2.5 mu L of magnesium acetate with the concentration of 280mM and 29.5 mu L, ddH of reaction buffer 2 O was added to 50. Mu.L.
6. The staphylococcus aureus drug resistance RPA detection composition is characterized by comprising at least one combination of 10 primer pair probe combinations for respectively detecting nuc gene, blaZ gene, mecA gene, ermA gene, ermB gene, ermC gene, msrA gene, tetK gene, tetM gene and aad1 gene;
detecting a primer pair nuc-nfo-F4, nuc-nfo-R1 and a probe nuc-nfo-P01 of the nuc gene; the nucleotide sequences of nuc-nfo-F4, nuc-nfo-R1 and nuc-nfo-P01 are shown as SEQ ID NO.31-SEQ ID NO. 33;
detecting primer pairs blaZ-nfo-F4, blaZ-nfo-R4 and a probe blaZ-nfo-P01 of the blaZ gene; the nucleotide sequences of the blaZ-nfo-F4, the blaZ-nfo-R4 and the blaZ-nfo-P01 are shown as SEQ ID NO.34-SEQ ID NO. 36;
detecting primer pairs mecA-nfo-F1, mecA-nfo-R2 and a probe mecA-nfo-P01 of the mecA gene; the nucleotide sequences of mecA-nfo-F1, mecA-nfo-R2 and mecA-nfo-P01 are shown as SEQ ID NO.37-SEQ ID NO. 39;
detecting primer pairs of the ermA gene, namely ermA-nfo-F3, ermA-nfo-R1 and a probe ermA-nfo-P01; the nucleotide sequences of the ermA-nfo-F3, the ermA-nfo-R1 and the ermA-nfo-P01 are shown as SEQ ID NO.40-SEQ ID NO. 42;
detecting a primer pair ermB-nfo-F3, ermB-nfo-R3 and a probe ermB-nfo-P01 of the ermB gene; the nucleotide sequences of ermB-nfo-F3, ermB-nfo-R3 and ermB-nfo-P01 are shown as SEQ ID NO.43-SEQ ID NO. 45;
detecting a primer pair ermC-nfo-F5, ermC-nfo-R5 and a probe ermC-nfo-P02 of the ermC gene; the nucleotide sequences of ermC-nfo-F5, ermC-nfo-R5 and ermC-nfo-P02 are shown as SEQ ID NO.46-SEQ ID NO. 48;
detecting a primer pair msrA-nfo-F2, msrA-nfo-R5 and a probe msrA-nfo-P01 of the msrA gene; the nucleotide sequences of the msrA-nfo-F2, the msrA-nfo-R5 and the msrA-nfo-P01 are shown as SEQ ID NO.49-SEQ ID NO. 51;
detecting a primer pair tetK-nfo-F2, tetK-nfo-R2 and a probe tetK-nfo-P03 of the tetK gene; the nucleotide sequences of the tetK-nfo-F2, the tetK-nfo-R2 and the tetK-nfo-P03 are shown as SEQ ID NO.52-SEQ ID NO. 54;
detecting a primer pair tetM-nfo-F2, tetM-nfo-R3 and a probe tetM-nfo-P03 of the tetM gene; the nucleotide sequences of tetM-nfo-F2, tetM-nfo-R3 and tetM-nfo-P03 are shown as SEQ ID NO.55-SEQ ID NO. 57;
detecting primer pairs aadD1-nfo-F2, aadD1-nfo-R1 and a probe aadD1-nfo-P01 of the aadD1 gene; the nucleotide sequences of aadD1-nfo-F2, aadD1-nfo-R1 and aadD1-nfo-P01 are shown as SEQ ID NO.58-SEQ ID NO. 60.
7. The staphylococcus aureus resistance RPA detection composition of claim 6, comprising at least one of:
the reverse primer of the primer pair is marked by fluorescein isothiocyanate FITC;
in the staphylococcus aureus resistance RPA detection composition, the probe 46nt, comprising a 5 '-biotin tag, a THF spacer substituted at nt 31 and an adjacent downstream 15nt oligonucleotide, carries a C3 blocking group at the 3' end of the probe.
8. An RPA-LFD assay reagent for rapid detection of staphylococcus aureus resistance comprising the RPA assay composition of claim 6 or 7.
9. A method for detecting staphylococcus aureus resistance for non-disease diagnostic purposes comprising the steps of: using DNA of a sample to be detected as a template, and adopting the RPA detection composition of claim 6 or 7 to carry out RPA detection to obtain a fluorescence value; and determining whether the sample to be detected contains staphylococcus aureus according to the fluorescence value.
10. The method for detecting resistance to staphylococcus aureus for the purpose of non-disease diagnosis according to claim 9, comprising the steps of: comprising at least one of the following:
the reaction condition detected by the RPA-LFD detection reagent is that the reaction is carried out for 15min at 37 ℃;
the reaction system detected by adopting the RPA-LFD detection reagent comprises 2 mu L of template, 2.1 mu L of primer with the concentration of 10 mu M, 0.6 mu L of probe with the concentration of 10 mu M, 2.5 mu L of magnesium acetate with the concentration of 280mM and 29.5 mu L, ddH of reaction buffer solution 2 O was added to 50. Mu.L.
CN202310236032.0A 2023-03-13 2023-03-13 Staphylococcus aureus drug resistance RPA detection composition and application thereof Pending CN116622865A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310236032.0A CN116622865A (en) 2023-03-13 2023-03-13 Staphylococcus aureus drug resistance RPA detection composition and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310236032.0A CN116622865A (en) 2023-03-13 2023-03-13 Staphylococcus aureus drug resistance RPA detection composition and application thereof

Publications (1)

Publication Number Publication Date
CN116622865A true CN116622865A (en) 2023-08-22

Family

ID=87640529

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310236032.0A Pending CN116622865A (en) 2023-03-13 2023-03-13 Staphylococcus aureus drug resistance RPA detection composition and application thereof

Country Status (1)

Country Link
CN (1) CN116622865A (en)

Similar Documents

Publication Publication Date Title
US20100255474A1 (en) Method for Detecting Bacteria and Fungi
Wang et al. Rapid and sensitive recombinase polymerase amplification combined with lateral flow strips for detecting Candida albicans
He et al. Real-time PCR for the rapid detection of vanA, vanB and vanM genes
Sun et al. Development of real-time PCR systems based on SYBR Green for the specific detection and quantification of Klebsiella pneumoniae in infant formula
CN117363767B (en) Probe combination, primer set and kit for real-time fluorescence PCR detection of target genes and application of probe combination and primer set and kit
Mu et al. The fluorescent probe-based recombinase-aided amplification for rapid detection of Escherichia coli O157: H7
Htun et al. Loop-mediated isothermal amplification (LAMP) for identification of Pythium insidiosum
CN116516032B (en) Primer probe combination for rapidly detecting mycoplasma in broad spectrum, kit and application thereof
Silva Zatti et al. Isothermal nucleic acid amplification techniques for detection and identification of pathogenic fungi: A review
Sunagar et al. Differentiation of Staphylococcus aureus and Staphylococcus epidermidis by PCR for the fibrinogen binding protein gene
US20110287965A1 (en) Methods and compositions to detect clostridium difficile
Xiao et al. Evaluation of real-time PCR coupled with multiplex probe melting curve analysis for pathogen detection in patients with suspected bloodstream infections
KR102467600B1 (en) Primer sets for detecting toxin agents, and method for detecting toxin agents using the same
US20160177378A1 (en) Method for simultaneous detection of bacteria and fungi in a biological preparation by pcr, primers as well as bacteria and fungi detection kit
CN116622865A (en) Staphylococcus aureus drug resistance RPA detection composition and application thereof
Weiss et al. Evaluation of the NanoCHIP® Infection Control Panel test for direct detection and screening of methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella pneumoniae carbapenemase (KPC)-producing bacteria and vancomycin-resistant Enterococcus (VRE)
Buddhachat et al. Rapid detection of two pathogenically important Xanthomonas in rice using a loop-mediated isothermal amplification with lateral flow dipstick (LAMP-LFD)
Werckenthin et al. Rapid identification of the animal pathogens Streptococcus uberis and Arcanobacterium pyogenes by fluorescence in situ hybridization (FISH)
Chen et al. Development of a closed-tube isothermal multiple self-matching-initiated amplification assay for visual detection of Staphylococcus aureus in milk samples
Wang et al. An improved recombinase polymerase amplification assay for the visual detection of Staphylococcus epidermidis with lateral flow strips
Wang et al. A CRISPR-Cas12a-based platform facilitates the detection and serotyping of Streptococcus suis serotype 2
Lin et al. A Rapid Antimicrobial Resistance Diagnostic Platform for Staphylococcus aureus Using Recombinase Polymerase Amplification
CN114196767B (en) Specific molecular target and method for detecting staphylococcus aureus ST type by using same
Wang et al. A visualized isothermal amplification method for rapid and specific detection of emetic and non-emetic Bacillus cereus in dairy products
Fadhil et al. Multiple–loci number analyses of variable-number tandem repeat of molecular detection of Staphylococcus aureus isolates

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination