Open Access, Peer-reviewed
pISSN 2384-1079
eISSN 2384-1087
    Pediatr Infect Vaccine. 2016 Dec;23(3):194-201. Korean.
    Published online Dec 20, 2016.
    https://doi.org/10.14776/piv.2016.23.3.194
    Copyright © 2016 The Korean Society of Pediatric Infectious Diseases
    Original Article

    Real-time Reverse Transcription Polymerase Chain Reaction Using Total RNA Extracted from Nasopharyngeal Aspirates for Detection of Pneumococcal Carriage in Children

    Young Kwang Kim,1 Kyoung Hoon Lee,1 Ki Wook Yun,2 Mi Kyung Lee,3 and In Seok Lim1
      • 1Department of Pediatrics, Chung-Ang University Hospital, Chung-Ang University College of Medicine, Seoul, Korea.
      • 2Department of Pediatrics, Seoul National University Children's Hospital, Seoul National University College of Medicine, Seoul, Korea.
      • 3Department of Laboratory Medicine, Chung-Ang University Hospital, Chung-Ang University College of Medicine, Seoul, Korea.
    • Corresponding author (Email: pedwilly@snu.ac.kr )
    Received June 11, 2016; Revised August 07, 2016; Accepted September 21, 2016.

    Abstract

    Purpose

    Monitoring pneumococcal carriage rates is important. We developed and evaluated the accuracy of a real-time reverse transcription polymerase chain reaction (RT-PCR) protocol for the detection of Streptococcus pneumoniae.

    Methods

    In October 2014, 157 nasopharyngeal aspirates were collected from patients aged <18 years admitted to Chung-Ang University Hospital. We developed and evaluated a real-time PCR method for detecting S. pneumoniae by comparing culture findings with the results of the real-time PCR using genomic DNA (gDNA). Of 157 samples, 20 specimens were analyzed in order to compare the results of cultures, realtime PCR, and real-time RT-PCR.

    Results

    The concordance rate between culture findings and the results of real-time PCR was 0.922 (P <0.01, Fisher exact test). The 133 culture-negative samples were confirmed to be negative for S. pneumoniae using real-time PCR. Of the remaining 24 culture-positive samples, 21 were identified as S. pneumonia-positive using real-time PCR. The results of real-time RT-PCR and real-time PCR from 20 specimens were consistent with culture findings for all S. pneumoniae-positive samples except one. Culture and real-time RT-PCR required 26.5 and 4.5 hours to perform, respectively.

    Conclusions

    This study established a real-time RT-PCR method for the detection of pneumococcal carriage in the nasopharynx. Real-time RT-PCR is an accurate, convenient, and time-saving method; therefore, it may be useful for collecting epidemiologic data regarding pneumococcal carriage in children.

    Keywords
    Streptococcus pneumoniae; Nasopharynx; RNA; Real-time polymerase chain reaction

    Figures

    Fig. 1
    (A) Amplification curves and (B) standard curve of real-time reverse transcription polymerase chain reaction for Streptococcus pneumoniae. Abbreviations: PnA, Streptococcus pneumoniae (ATCC49619); RFU, relative fluorescence unit; Cq, quantification cycle.

    Fig. 2
    Amplification curves of real-time reverse transcription polymerase chain reaction using (A) complementary DNA and (B) genomic DNA. Abbreviations: RFU, relative fluorescence units; PnA, Streptococcus pneumoniae (ATCC49619); PPn, Streptococcus pseudopneumoniae (KCTC5765); E. coli, Escherichia coli.

    Tables

    Table 1
    Number of Detected Pneumococcus and Mean Concentration of gDNA (n=157)

    Table 2
    Results of Conventional Culture Method and Real-Time PCR with gDNA or cDNA for Detecting Streptococcus pneumoniae Carriage Isolates (n=20)

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    MeSH Terms
    Child*
    DNA
    Humans
    Methods
    Nasopharynx
    Pneumonia
    Polymerase Chain Reaction*
    RNA*
    Real-Time Polymerase Chain Reaction
    Reverse Transcription*
    Streptococcus pneumoniae
    Metrics
    Share
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