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Published on: October 23, 2011
Development and Evaluation of Duplex MIRA-qPCR Assay for Simultaneous Detection of Staphylococcus aureus and
Jiulian Lai1,2,3, Zhenzhou Huang2,3, Yue Xiao2,3
1Institution of Health Statistics and Epidemiology, School of Public Health, Lanzhou University, Lanzhou 730000, China.
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.
Insights
A new duplex MIRA-qPCR method rapidly detects Staphylococcus aureus (S. aureus) and other Staphylococcus species. This assay offers high specificity and a low detection threshold for quicker infection diagnosis.
Area of Science:
- Microbiology
- Molecular Biology
- Infectious Diseases
Background:
- Staphylococcus species, particularly Staphylococcus aureus (S. aureus), are significant causes of hospital-acquired infections and foodborne illnesses.
- Accurate and rapid detection of S. aureus is crucial for effective patient management and public health.
Purpose of the Study:
- To develop and validate a novel multienzyme isothermal rapid amplification combined with duplex quantitative PCR (duplex MIRA-qPCR) method.
- To simultaneously detect the S. aureus-specific gene FMN-bgsfp and the Staphylococcus genus-specific gene tuf.
Main Methods:
- Developed a duplex MIRA-qPCR assay for simultaneous detection of S. aureus and Staphylococcus genus.
- Performed amplification at a constant temperature of 39 °C within 20 minutes.
- Evaluated specificity using common Staphylococcus species and non-Staphylococcus species, and assessed sensitivity and repeatability.
Main Results:
- The duplex MIRA-qPCR assay demonstrated high specificity, correctly identifying S. aureus and differentiating it from other Staphylococci.
- The limit of detection (LoD) for the assay was as low as 3 × 10^2 CFU/mL.
- The assay showed good repeatability with coefficient variation values ranging from 0.13% to 2.09% and successfully detected S. aureus in simulated samples.
Conclusions:
- The duplex MIRA-qPCR method is a highly specific, sensitive, and rapid tool for detecting S. aureus and non-aureus Staphylococci species.
- This assay offers advantages in terms of shorter detection time, lower detection threshold, and simpler operation compared to existing methods.
- The duplex MIRA-qPCR is an effective tool for the rapid diagnosis of S. aureus and Staphylococci infections.
