RPB0161

Pathogen Description

Target Pathogen Pathogen Name NCBI Taxonomy ID Order Family Genus Species Pathogen type
Staphylococcus aureus Staphylococcus aureus, Micrococcus aureus, Staphylococcus pyogenes aureus 1280 Bacillales Staphylococcaceae Staphylococcus Staphylococcus aureus Bacterium

Primer Description

Primer Name Sequence(5'-3') Length(bp) Primer Final Concentration(μM） GC Content（%） Predicted Melting Temperature(℃） Molecular Weight(g/moles) Positions in GenBank accession number
ermA-F TCAGTAAACAAGACAACGTAATAGAAATCGGATC 34 0.425 35.3 57.3 10484.93 \
ermA-R TATCCGTTTGAATCACTTTTATATTCTCAG 30 0.425 30 52.16 9112 \
ermA-Probe AGAGCTAGTCAAAATGAGTCGATCAGTTACT(HEX-dT)GC(THF)TAT(BHQ1)AGAAATTGATGGAGGC(C3-Spacer) 52 0.125 40.4 65.63 16163.57 \

Gene Description

Target Gene GenBank ID
ermA DQ836131.1

Assay Description

Application Assay Primer Designing Software Reaction Time(min) Assay Temperature(℃) Readout System(s) Limit of Detection(LoD) Sensitivity(%) Specificity(%)
detection of two resistance genes in drug-resistant Staphylococcus aureus RPA \ 10 39 exo probe 2*10¹ copies per reaction \ \

Publication Description

Year of Publication Title Author(s) Journal PMID DOI
2022 Duplex real-time fluorescent recombinase polymerase amplification for the rapid and sensitive detection of two resistance genes in drug-resistant Staphylococcus aureus Zhonglin Shi,Yanan Li,Anzhong Hu,Junsheng Cui,Min Shao,Ling Zhu,Ke Yang,Yong Liu,Guoqing Deng,Cancan Zhu Journal of microbiological methods 36195263 10.1016/j.mimet.2022.106590

Duplex real-time fluorescent recombinase polymerase amplification for the rapid and sensitive detection of two resistance genes in drug-resistant Staphylococcus aureus

Author(s):

Zhonglin Shi,Yanan Li,Anzhong Hu,Junsheng Cui,Min Shao,Ling Zhu,Ke Yang,Yong Liu,Guoqing Deng,Cancan Zhu

Journal:

Journal of microbiological methods

Year:

2022

Abstract:

In the clinic, drug-resistant Staphylococcus aureus (S. aureus) is the most common suppurative infection pathogen in humans. It can cause local infections in humans and animals, such as pneumonia, mastitis, and other systemic illnesses. At present, the detection of drug-resistant S. aureus includes traditional isolation by culture and antimicrobial susceptibility tests. However, these methods are complicated in experimental design, specialized in operation and time consuming. Therefore, a rapid and accurate drug-resistant S. aureus detection technology is urgently needed. In this study, we combined duplex pairs of fluorescent probes with recombinase polymerase amplification (RPA) to realize the simultaneous detection of two resistance genes in drug-resistant S. aureus. The method shows low detection limit, detecting 20 copies within 10 min. The analytical specificity of this method was evaluated with several related drug-resistant bacterial strains (Non-resistant S. aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae), and the positive signal was only observed with drug-resistant S. aureus. In addition, the clinical suitability of this method was verified by 30 clinical isolates. Compared with qPCR, the coincidence rate of drug resistance genes were 100% (mecA) and 96.7% (ermA), respectively. These results show that the duplex real-time fluorescent RPA assay is a rapid, low detection limit and specific detection of mecA and ermA genes in isolates of drug-resistant S. aureus.