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Development and Evaluation of Duplex MIRA-qPCR Assay for Simultaneous Detection of Staphylococcus aureus and non-aureus Staphylococci

Authors :
Jiulian Lai
Zhenzhou Huang
Yue Xiao
Keyi Yu
Xuemei Bai
He Gao
Hang Dai
Xiaoning Liu
Duochun Wang
Source :
Microorganisms, Vol 10, Iss 9, p 1734 (2022)
Publication Year :
2022
Publisher :
MDPI AG, 2022.

Abstract

Staphylococcus spp., especially Staphylococcus aureus (S. aureus), is an important pathogen in hospital-acquired infection and food poisoning. Here, we developed a multienzyme isothermal rapid amplification combined with duplex quantitative PCR (duplex MIRA-qPCR) method, which can simultaneously detect the S. aureus species-specific conserved gene FMN-bgsfp and the Staphylococcus genus-specific conserved gene tuf. This assay enabled the amplification of DNA within 20 min at a constant temperature of 39 °C. Specificity analysis indicated that all nine common Staphylococcus species were positive and non-Staphylococcus spp. were negative for tuf gene, whereas S. aureus was positive, non-aureus Staphylococci species and non-Staphylococcus spp. were negative for FMN-bgsfp gene, suggesting that duplex MIRA-qPCR exhibited high specificity. Meanwhile, the sensitivity was tested and the limit of detection (LoD) was 3 × 102 CFU/mL. The coefficient variation values ranged from 0.13% to 2.09%, indicating that the assay had good repeatability. Furthermore, all the nine common Staphylococcus species (including S. aureus) could be detected from four kinds of simulated samples and the LoD of S. aureus was 8.56 × 103 CFU/mL. In conclusion, the duplex MIRA-qPCR has advantages of stronger specificity, lower detection threshold, shorter detection time, and simpler operation, which is an effective tool to detect S. aureus and non-aureus Staphylococci spp. infections rapidly.

Details

Language :
English
ISSN :
10091734 and 20762607
Volume :
10
Issue :
9
Database :
Directory of Open Access Journals
Journal :
Microorganisms
Publication Type :
Academic Journal
Accession number :
edsdoj.f2ee41314ff4ddca3bd8901d1d6a1da
Document Type :
article
Full Text :
https://doi.org/10.3390/microorganisms10091734