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.
Bibliographical noteFunding Information:
This work was supported by grants from the National Institute of Allergy and Infectious Diseases, National Institutes of Health (Grant R01 AI 49742) and NRI, US Department of Agriculture (CGP Grant 1999 02009) and by the Ohio Agricultural Research and Development Center (OARDC), The Ohio State University (Graduate Student Research Enhancement Grant project 2002-114). Salaries and research support were provided by state and federal funds provided to the OARDC.
Copyright 2008 Elsevier B.V., All rights reserved.
- Internal control RNA
- Microwell hybridization