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Archives of Virology
Published in: Archives of Virology 6/2012

01-06-2012 | Original Article

Development and application of one-step multiplex reverse transcription PCR for simultaneous detection of five diarrheal viruses in adult cattle

Authors: Masaharu Fukuda, Kazufumi Kuga, Ayako Miyazaki, Tohru Suzuki, Keito Tasei, Tsunehiko Aita, Masaji Mase, Makoto Sugiyama, Hiroshi Tsunemitsu

Published in: Archives of Virology | Issue 6/2012

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Abstract

A one-step multiplex reverse transcription (RT)-PCR method was developed for the simultaneous detection of five viruses causing diarrhea in adult cattle: bovine group A rotavirus (GAR), bovine group B rotavirus (GBR), bovine group C rotavirus (GCR), bovine coronavirus (BCV), and bovine torovirus (BToV). The detection limit of the one-step multiplex RT-PCR for GAR, GCR, BCV, and BToV was 102, 100, 101, and 102 TCID50/ml, respectively, and that for GBR was 106 copies/ml. The one-step multiplex RT-PCR with newly designed primers to detect GAR had higher sensitivity than a single RT-PCR with conventional primers, with no false-positive reactions observed for ten other kinds of bovine RNA viruses To assess its field applicability, 59 of 60 fecal samples containing one of these five viruses from all 25 epidemic diarrhea outbreaks in adult cattle were positive in the one-step multiplex RT-PCR assay. Furthermore, using four additional fecal samples containing two viruses (GBR and BCV or BToV), two amplified products of the expected sizes were obtained simultaneously. In contrast, all 80 fecal samples lacking the five target viruses from normal adult cattle were negative in the multiplex assay. Taken together, our results indicate that the one-step multiplex RT-PCR developed here for the detection of GAR, GBR, GCR, BCV, and BToV can be expected to be a useful tool for the rapid and cost-effective diagnosis and surveillance of viral diarrhea in adult cattle.
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Metadata
Title
Development and application of one-step multiplex reverse transcription PCR for simultaneous detection of five diarrheal viruses in adult cattle
Authors
Masaharu Fukuda
Kazufumi Kuga
Ayako Miyazaki
Tohru Suzuki
Keito Tasei
Tsunehiko Aita
Masaji Mase
Makoto Sugiyama
Hiroshi Tsunemitsu
Publication date
01-06-2012
Publisher
Springer Vienna
Published in
Archives of Virology / Issue 6/2012
Print ISSN: 0304-8608
Electronic ISSN: 1432-8798
DOI
https://doi.org/10.1007/s00705-012-1271-5

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