Detection of methicillin-resistant Staphylococcus aureus (MRSA) from nasal samples by multiplex real-time PCR based on dual priming AT-rich primers.
Folia Microbiol (Praha)
; 57(1): 37-45, 2012 Jan.
Article
en En
| MEDLINE
| ID: mdl-22187362
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Infecciones Estafilocócicas
/
Nariz
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Staphylococcus aureus Resistente a Meticilina
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Reacción en Cadena de la Polimerasa Multiplex
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Reacción en Cadena en Tiempo Real de la Polimerasa
Tipo de estudio:
Diagnostic_studies
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Evaluation_studies
/
Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
Folia Microbiol (Praha)
Año:
2012
Tipo del documento:
Article
País de afiliación:
Corea del Sur