Pediatr Infect Vaccine.  2016 Dec;23(3):194-201. 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

• Kim YK
• Lee KH
• Yun KW
• Lee MK
• Lim IS

• KMID: 2362478
• DOI: http://doi.org/10.14776/piv.2016.23.3.194

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.

Keyword

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

MeSH Terms

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

Figure

• 

  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.

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