Enhanced Reverse Transcription Polymerase Chain Reaction Enzyme-linked Immunosorbent Assay (RT-PCR-ELISA) for Simultaneous Detection of Genogroup I and Genogroup II Noroviruses

Introduction: Noroviruses (NoVs) were identified as one of the major foodborne viruses which cause acute viral gastroenteritis. NoVs are transmitted via fecal-oral route and mainly infected through contaminated foods and water. Although various PCR assays to detect NoVs were developed not only in contaminated water and foods but also in stools, RT-PCR-ELISA which combined RT-PCR and ELISA technique does not applied for NoVs detection.

Purpose: The purpose of this study was to develop a specific and sensitive RT-PCR-ELISA for simultaneous detection of Genogroup I (GI) and Genogroup II (GII) noroviruses (NoVs) using universal probe.

Methods: Primers and probes were designed at ORF1 and ORF2 region of GI and GII NoVs. Universal probe was combined with each GI and GII NoVs probe. RT-PCR-ELISA for detection of NoVs was performed following semi-nested reverse transcription-polymerase chain reaction (semi-nested RT-PCR). DIG-labeled DNA fragments were hybridized with biotinylated universal probe at 37°C. After the conjugation with anti-digoxigenin-peroxidase antibody, ABTS colorized the positive products. Optical absorbance was measured at 405 nm.

Results: RT-PCR-ELISA using universal probe for the detection of NoVs could simultaneously detect GI and GII NoVs. RT-PCR-ELISA could detect by at least 100 copy number/μl GI and GII NoVs from human stool specimens. Compared with semi-nested RT-PCR and real-time RT-PCR, the sensitivity of RT-PCR-ELISA was comparable with real-time RT-PCR. In specificity test, RT-PCR-ELISA detected only GI and GII NoVs except hepatitis A virus, hepatitis E virus, rotavirus, adenovirus and murine norovirus.

Significance: With high sensitivity and specificity, RT-PCR-ELISA with universal probes could detect different genogroups of NoVs, simultaneously.