Development and Evaluation of a 'Real-Time' RT-PCR for the Detection of Enterovirus and Parechovirus RNA in CSF and Throat Swab Samples

J Med Virol. 2002 Aug;67(4):555-62. doi: 10.1002/jmv.10138.

Abstract

A two-step reverse transcriptase TaqMantrade mark duplex PCR (RT-PCR) assay was developed using the ABI 7700 Sequence Detection System for the detection of enterovirus (EV) and parechovirus type 1 and 2 (PEV) RNA from samples of cerebrospinal fluid (CSF) and throat swabs. Using sequence-specific fluorescent dye labeled probes and continuous 'real-time' monitoring, PCR amplified product accumulation was measured. Based on limiting dilutions, the TaqMantrade mark enterovirus and parechovirus RT-PCR showed an increase of two orders of magnitude compared to cell culture with sensitivity of 100% (7/7) when assessed using enterovirus cell culture positive samples (CSF, TS). The assays were specific for enterovirus and parechovirus and did not amplify a wide selection of virus and bacterial isolates. RNA was amplified from 22 enterovirus serotypes: coxsackie A7, A9, A21; coxsackie B2, B3, B4, B5; echovirus 2, 4, 6, 7, 9, 11, 13, 17, 18, 19, 30, 31; poliovirus types 1, 2, and 3, and parechovirus types 1 and 2. The assay was used to assess the incidence of enterovirus and parechovirus RNA in cell culture negative CSF and throat swab samples (n = 200). An additional 33 (15.9%) enterovirus and 2 (1%) parechovirus were identified as positive by RT-PCR. Also, of 100 CSF samples from suspected cases of meningococcal meningitis submitted for meningococcal PCR testing, 59 (59%) were enterovirus and 2 (2%) parechovirus 1 and 2 were positive by RT-PCR. The TaqMantrade mark duplex assay offers a more rapid and sensitive alternative to conventional cell culture for the diagnosis of enterovirus and parechovirus infection. Closed tube real-time detection using the ABI Sequence Detection System obviates the need for post-PCR manipulation, which reduces hands on time and eliminates the risk of contamination from amplified PCR product.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Enterovirus / classification
  • Enterovirus / genetics*
  • Enterovirus / isolation & purification*
  • Enterovirus Infections / diagnosis*
  • Enterovirus Infections / virology
  • Humans
  • Meningitis, Viral / diagnosis
  • Meningitis, Viral / virology
  • Pharynx / virology*
  • Polymerase Chain Reaction / methods*
  • RNA, Viral / analysis*
  • RNA, Viral / cerebrospinal fluid*
  • RNA, Viral / isolation & purification
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sensitivity and Specificity
  • Taq Polymerase / metabolism

Substances

  • RNA, Viral
  • Taq Polymerase