Korean J Clin Microbiol.  2010 Jun;13(2):53-58. 10.5145/KJCM.2010.13.2.53.

Detection of Enterovirus using Real-Time Nucleic Acid Sequence-based Amplification

• Jun SH
• Sung KH
• Song SH
• Park KU
• Kim HB
• Song J
• Choi EH
• Park SS
• Kim EC

• KMID: 1461633
• DOI: http://doi.org/10.5145/KJCM.2010.13.2.53

Abstract

BACKGROUND
Enteroviruses are the most frequent etiologic agents of aseptic meningitis and are estimated to be the cause of 70% to 90% of viral meningitis cases. Enterovirus diagnosis can be difficult because clinical features vary according to patient immunity and age. The purpose of this study was to evaluate the performance of the real-time nucleic acid sequence-based amplification (NASBA) assay compared to that of the real-time nested RT-PCR assay for enterovirus detection.
METHODS
This study was performed on 96 patients suspected of aseptic meningitis based on clinical features. RNA was extracted using NucliSENS EasyMAG and real-time NASBA assay was performed using NucliSENS EasyQ Enterovirus and NucliSENS EasyQ Basic 2. We also executed in-house real-time nested RT-PCR assay for RNA extracted via QIAamp Viral RNA Mini.
RESULTS
The positive rate of real-time NASBA assay was 45.8% for enterovirus detection. The positive rate of first real-time reverse transcription PCR was 22.9% and the second real-time PCR was 57.3%. The concordant rate of the real-time NASBA assay and first real-time reverse transcription PCR was 75.0%. The concordant rate of the real-time NASBA assay and second real-time PCR was 86.5%.
CONCLUSION
The detection of enteroviruses using the real-time NASBA assay is less prone to cross-contamination and is simple, without the need for reverse transcription. We conclude that the NASBA assay is an effective method for the rapid diagnosis of aseptic meningitis.

Keyword

Enterovirus; Real-time PCR; Nucleic acid sequence-based amplification; Aseptic meningitis

MeSH Terms

Enterovirus
Humans
Meningitis, Aseptic
Meningitis, Viral
Polymerase Chain Reaction
Real-Time Polymerase Chain Reaction
Reverse Transcription
RNA
RNA, Viral
Self-Sustained Sequence Replication
RNA
RNA, Viral

Figure

• 

  Fig. 1. Detection of enterovirus by nucleic acid sequence-based amplification and realtime nested reverse transcription PCR. (A) Nucleic acid sequence-based amplification. ①∼ ② and ④∼⑥, samples with a internal control signal (red) and no enterovirus signal (blue); ③ and ⑦, samples with a internal control signal and a strong enterovirus signal. (B) The first realtime reverse transcription PCR. ③, positive for enterovirus. (C) The nested realtime PCR. ③ and ⑦, positive for enterovirus; PC, positive control.

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