title: Development of a new microwell hybridization assay and an internal control RNA for the detection of porcine noroviruses and sapoviruses by reverse transcription-PCR.
abstract: Recently, genetically diverse porcine noroviruses (NoV) and sapoviruses (SaV) were identified from field pig fecal samples. Reverse transcription (RT)-PCR is the primary method used for detection of human NoVs and SaVs. However, RT-PCR inhibitors frequently cause false-negative results. In this study, a competitive internal control (IC) RNA, specific for use in the SaV RT-PCR assay, was developed to monitor inhibition of RT-PCR; primers for detection of genetically diverse porcine NoVs and SaVs were designed; and microwell hybridization assays to confirm the specific RT-PCR products were developed. The primer pairs and the RT-PCR-hybridization combinations were compared using representative porcine NoV and SaV strains, positive pig fecal samples and a panel of 30 field pig fecal samples. Extracted RNA from 3 of 30 samples failed to amplify the IC RNA. However, this inhibition was not present after a 10-fold dilution of the extracted RNA. The five different RT-PCR-hybridization combinations developed specifically detected all three genotypes of porcine NoVs, all GIII porcine SaVs, unclassified JJ681-like, QW19 and LL26-like porcine SaVs, respectively. These RT-PCR-hybridization assays are specific, less time consuming and economical and particularly applicable to testing large number of samples for porcine NoVs and SaVs.
authors.affiliation: Food Animal Health Research Program, Ohio Agricultural Research and Development Center, Department of Veterinary Preventive Medicine, The Ohio State University, 1680 Madison Avenue, Wooster, OH 44691, USA.
authors.forename: Qiu-Hong
authors.initials: QH
authors.lastname: Wang
authors.forename: Kyeong-Ok
authors.initials: KO
authors.lastname: Chang
authors.forename: Myung Guk
authors.initials: MG
authors.lastname: Han
authors.forename: Srinand
authors.initials: S
authors.lastname: Sreevatsan
authors.forename: Linda J
authors.initials: LJ
authors.lastname: Saif
mesh_terms: D000818:Animals; D001483:Base Sequence; D017250:Caliciviridae Infections / Q000662:veterinary* / Q000821:virology; D017931:DNA Primers; D005243:Feces / Q000821:virology; D008969:Molecular Sequence Data; D029322:Norovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D029341:Sapovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D013553:Swine Diseases / Q000821:virology
chemical_list: D017931:DNA Primers; D012367:RNA, Viral