NDLI: Detection of methicillin-resistant Staphylococcus aureus (MRSA) from nasal samples by multiplex real-time PCR based on dual priming AT-rich primers

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Detection of methicillin-resistant Staphylococcus aureus (MRSA) from nasal samples by multiplex real-time PCR based on dual priming AT-rich primers

Content Provider	Springer Nature Link
Author	Yadav, M. K. Kwon, S. K. Huh, H. J. Chae, S W. Song, J J.
Copyright Year	2011
Abstract	In this study, we reported on the design of a multiplex real-time PCR assay based on SYBR Green I, incorporating dual priming adenine-thymine (AT)-rich primers for direct detection of MRSA from nasal samples. The multiplex real-time polymerase chain reaction (RT-PCR) assay reported in this study is based on SYBR Green I with incorporation of six dual priming AT-rich primers designed from the SCCmec/orf junction. A string (4–6 bp) of low-melting bases, such as adenine and thymine, was incorporated into the primers, which virtually divided a single primer in two functional regions, thus decreasing non-specific PCR products. The analytical sensitivity and specificity of the RT-PCR assay was determined with genomic DNA of reference strains (MRSA, MSSA, and MRCoNS). RT-PCR assay was performed for analysis of 72 nasal swab specimens, and the results were confirmed by use of a culture method. Furthermore, the results of RT-PCR were compared with LightCycler MRSA advance test. The multiplex RT-PCR assay reproducibly detected a minimum of 1 pg genomic DNA (31.5 copy of genome) of MRSA reference strains and clinical isolates, with a specific melting peak at 83.5 ± 1.5°C, and neither fluorescence nor a melting peak was detected in non-target isolates. The concordance rate between RT-PCR assay and culture method was 87.5% with Cohen's kappa value (κ) 0.75, which showed good agreement between the two assays. The sensitivity, specificity, positive predictive value, and negative predictive value of the assay were 93.5%, 82.9%, 80.5%, and 94.4%, respectively. In a comparative study for the detection of 72 nasal samples, the sensitivity, specificity, positive predictive value, and negative predictive value of the multiplex RT-PCR assay with respect to LightCycler MRSA advance test was 84.2%, 88.2%, 89%, and, 83.3%, respectively. The results of RT-PCR assay demonstrated high specificity (88.2%) and positive predictive value (89%) for the direct detection of MRSA from nasal samples.
Starting Page	37
Ending Page	45
Page Count	9
File Format	PDF
ISSN	00155632
Journal	Folia Microbiologica
Volume Number	57
Issue Number	1
e-ISSN	18749356
Language	English
Publisher	Springer Netherlands
Publisher Date	2011-12-22
Publisher Place	Dordrecht
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Environmental Engineering/Biotechnology Immunology Applied Microbiology Life Sciences Microbiology
Content Type	Text
Resource Type	Article
Subject	Medicine Microbiology

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