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

Young Kwang Kim; Kyoung Hoon Lee; Ki Wook Yun; Mi Kyung Lee; In Seok Lim

doi:10.14776/piv.2016.23.3.194

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Kim, Lee, Yun, Lee, and Lim: Real-time Reverse Transcription Polymerase Chain Reaction Using Total RNA Extracted from Nasopharyngeal Aspirates for Detection of Pneumococcal Carriage in Children

Original Article

Pediatric Infection & Vaccine 2016; 23(3): 194-201.

Published online: 20 December 2016

DOI: https://doi.org/10.14776/piv.2016.23.3.194

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

Young Kwang Kim¹, Kyoung Hoon Lee¹, Ki Wook Yun², Mi Kyung Lee³, 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 (pedwilly@snu.ac.kr)

Received 11 June 2016 Revised 7 August 2016 Accepted 21 September 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 and Tables

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.

Table 1

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

^*2.4 ng/µL (sample 1), 2.1 (sample 2), and 0.6 (sample 3).

^†Fisher exact test.

Abbreviations: gDNA, genomic DNA; PCR, polymerase chain reaction; NA, not available.

Table 2

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

Abbreviations: PCR, polymerase chain reaction; gDNA, genomic DNA; cDNA, complementary DNA.

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