Development and evaluation of a 'real-time' RT-PCR for the detection of enterovirus and parechovirus RNA in CSF and throat swab samples.
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.
Meningococcal Reference Unit, Manchester Public Health Laboratory, Withington Hospital, United Kingdom.
Caroline E
CE
Corless
Malcolm
M
Guiver
Raymond
R
Borrow
Valerie
V
Edwards-Jones
Andrew J
AJ
Fox
Edward B
EB
Kaczmarski
Kenneth J
KJ
Mutton
D004770:Enterovirus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D004769:Enterovirus Infections / Q000175:diagnosis* / Q000821:virology; D006801:Humans; D008587:Meningitis, Viral / Q000175:diagnosis / Q000821:virology; D010614:Pharynx / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis* / Q000134:cerebrospinal fluid* / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000378:metabolism
D012367:RNA, Viral; D019914:Taq Polymerase