title: Development and application of one-step multiplex reverse transcription PCR for simultaneous detection of five diarrheal viruses in adult cattle.
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 10(2), 10(0), 10(1), and 10(2) TCID(50)/ml, respectively, and that for GBR was 10(6) 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.
authors.affiliation: Saitama Prefectural Chuo Livestock Hygiene Service Center, Kita-ku, Japan.
authors.forename: Masaharu
authors.initials: M
authors.lastname: Fukuda
authors.forename: Kazufumi
authors.initials: K
authors.lastname: Kuga
authors.forename: Ayako
authors.initials: A
authors.lastname: Miyazaki
authors.forename: Tohru
authors.initials: T
authors.lastname: Suzuki
authors.forename: Keito
authors.initials: K
authors.lastname: Tasei
authors.forename: Tsunehiko
authors.initials: T
authors.lastname: Aita
authors.forename: Masaji
authors.initials: M
authors.lastname: Mase
authors.forename: Makoto
authors.initials: M
authors.lastname: Sugiyama
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Tsunemitsu
mesh_terms: D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis* / Q000821:virology; D017938:Coronavirus, Bovine / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D003967:Diarrhea / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D005243:Feces / Q000821:virology; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012401:Rotavirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D017003:Torovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers