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Development and evaluation of a ‘real-time’ RT-PCR for the detection of enterovirus and parechovirus RNA in CSF and throat swab samples

Corless, Caroline E. and Guiver, Malcolm and Borrow, Ray and Edwards-Jones, Valerie and Fox, Andrew J. and Kaczmarski, Edward B. and Mutton, Kenneth J. (2002) Development and evaluation of a ‘real-time’ RT-PCR for the detection of enterovirus and parechovirus RNA in CSF and throat swab samples. ISSN 0146-6615

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Abstract

A two-step reverse transcriptase TaqMan™ 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 TaqMan™ 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 TaqManTM 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. J. Med. Virol. 67:555-562, 2002. © 2002 Wiley-Liss, Inc.

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