Detection of methicillin-resistant <Emphasis Type="BoldItalic">Staphylococcus aureus</Emphasis> (MRSA) from nasal samples by multiplex real-time PCR based on dual priming AT-rich primers |
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Authors: | M?K?Yadav S?K?Kwon H?J?Huh S-W?Chae Email author" target="_blank">J-J?SongEmail author |
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Institution: | (1) Department of Otorhinolaryngology-Head and Neck Surgery, Dongguk University Ilsan Hospital, 814 Siksa-Dong, Goyang, Gyeonggi, South Korea, 410-773;(2) Department of Laboratory Medicine, Dongguk University Ilsan Hospital, Goyang, Gyeonggi, South Korea;(3) Department of Otorhinolaryngology-Head and Neck Surgery, Korea University College of Medicine, Seoul, South Korea; |
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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. |
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