title: Method for detection of extraneous active bovine viral diarrhoea virus and classical swine fever virus in animal viral vaccines by RT-PCR, which amplify negative-strand viral RNA in infected cells.
abstract: An oligonucleotide sense primer, Pst324alpha, was designed and used for synthesizing cDNA from negative-strand viral RNA in infected cells and used for rapid detection of active extraneous bovine viral diarrhoea virus (BVDV) and classical swine fever virus (CSFV) in animal viral vaccines by culturing a sample in cells followed by reverse transcriptase-polymerase chain reaction (RT-PCR). Active and inactivated viruses of BVDV No. 12-43 strain and CSFV GPE(-)strain were inoculated to bovine testicle and swine testicle cells for incubation. After the complete extraction of RNA from these cells, cDNA was synthesized using Pst324alpha, and PCR was carried out using primers 324 and 326 (novel RT-PCR). Amplification of novel RT-PCR products was observed in cells infected with active viruses but not in cells inoculated with inactivated viruses, inoculums and cultured media after incubation. This novel RT-PCR was able to amplify viral sequences from cells infected with only a small number of infectious particles (less than 10 TCID50) at three days postinoculation and was as sensitive as the general RT-PCR using a random primer and the interference and immunofluorescent antibody (FA) methods. The results of experiments on detection of BVDV RNA from vaccines contaminated with active and inactivated BVDV showed that the sensitivity of the novel RT-PCR was almost the same as the sensitivities of the interference and FA methods. These results suggest that the novel RT-PCR is easier and more rapid than the interference method for detection of active BVDV and that the novel RT-PCR is a reliable means for detection of active extraneous BVDV for quality control of animal vaccines.
authors.affiliation: Assay Division I, National Veterinary Assay Laboratory, 1-15-1 Tokura Kokubunji, Tokyo, 185-8511.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Aoki
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Shimazaki
authors.forename: Chikako
authors.initials: C
authors.lastname: Takahashi
authors.forename: Yoshimasa
authors.initials: Y
authors.lastname: Sasaki
authors.forename: Shoko
authors.initials: S
authors.lastname: Suzuki
authors.forename: Akio
authors.initials: A
authors.lastname: Fukusho
mesh_terms: D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D006692:Classical Swine Fever Virus / Q000737:chemistry* / Q000235:genetics; D030241:Diarrhea Virus 1, Bovine Viral / Q000737:chemistry* / Q000235:genetics; D008297:Male; D008856:Microscopy, Fluorescence; D010576:Pestivirus / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011786:Quality Control; D012367:RNA, Viral / Q000032:analysis* / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D013737:Testis / Q000378:metabolism; D014612:Vaccines / Q000737:chemistry*; D014773:Virology / Q000379:methods*
chemical_list: D012367:RNA, Viral; D014612:Vaccines