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

M. K. Yadav, Seong Keun Kwon, H. J. Huh, S. W. Chae, J. J. Song

Research output: Contribution to journalArticle

2 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)37-45
Number of pages9
JournalFolia Microbiologica
Volume57
Issue number1
DOIs
StatePublished - 1 Jan 2012

Fingerprint

Thymine
Multiplex Polymerase Chain Reaction
Adenine
Methicillin-Resistant Staphylococcus aureus
Nose
Real-Time Polymerase Chain Reaction
Freezing
Sensitivity and Specificity
Contagious Ecthyma
DNA
Fluorescence
Genome
Polymerase Chain Reaction

Cite this

@article{16896f0f4fd948798829e109c355d772,
title = "Detection of methicillin-resistant Staphylococcus aureus (MRSA) from nasal samples by multiplex real-time PCR based on dual priming AT-rich primers",
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.",
author = "Yadav, {M. K.} and Kwon, {Seong Keun} and Huh, {H. J.} and Chae, {S. W.} and Song, {J. J.}",
year = "2012",
month = "1",
day = "1",
doi = "10.1007/s12223-011-0085-2",
language = "English",
volume = "57",
pages = "37--45",
journal = "Folia Microbiologica",
issn = "0015-5632",
publisher = "Springer Netherlands",
number = "1",

}

Detection of methicillin-resistant Staphylococcus aureus (MRSA) from nasal samples by multiplex real-time PCR based on dual priming AT-rich primers. / Yadav, M. K.; Kwon, Seong Keun; Huh, H. J.; Chae, S. W.; Song, J. J.

In: Folia Microbiologica, Vol. 57, No. 1, 01.01.2012, p. 37-45.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Detection of methicillin-resistant Staphylococcus aureus (MRSA) from nasal samples by multiplex real-time PCR based on dual priming AT-rich primers

AU - Yadav, M. K.

AU - Kwon, Seong Keun

AU - Huh, H. J.

AU - Chae, S. W.

AU - Song, J. J.

PY - 2012/1/1

Y1 - 2012/1/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84857996028&partnerID=8YFLogxK

U2 - 10.1007/s12223-011-0085-2

DO - 10.1007/s12223-011-0085-2

M3 - Article

C2 - 22187362

AN - SCOPUS:84857996028

VL - 57

SP - 37

EP - 45

JO - Folia Microbiologica

JF - Folia Microbiologica

SN - 0015-5632

IS - 1

ER -