Development of a new microwell hybridization assay and an internal control RNA for the detection of porcine noroviruses and sapoviruses by reverse transcription-PCR.
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.
Wang
Qiu-Hong
QH
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.
Chang
Kyeong-Ok
KO
Han
Myung Guk
MG
Sreevatsan
Srinand
S
Saif
Linda J
LJ
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
D017931:DNA Primers; D012367:RNA, Viral