A Comparison of a Nested Two-Step qPCR and a Non-Nested One-Step RT-qPCR for Detection of Genogroup II Noroviruses in Diluted Clinical Fecal Samples

Introduction: Human Noroviruses (NoV) are a major source of foodborne illness in the United States. Molecular assays such as reverse transcription real time PCR (RT-qPCR) are considered the gold standard for detection of NoV. Broadly reactive RT-qPCR assays are generally used to detect NoV in samples where the virus load is expected to be high. When the virus load is expected to be low (as is the case in food and environmental samples), nested PCRs have been used. Unfortunately, a major disadvantage of nested methods is their propensity for cross-contamination.

Purpose: We conducted a controlled comparison of two widely used NoV genogroup II detection assays: a broadly reactive one-step RT-qPCR assay and a nested two-step qPCR assay.

Methods: A human fecal specimen containing GII.2 NoV was serially diluted in DEPC treated water. Total nucleic acids were extracted from each dilution, and a one-step RT-qPCR assay targeting conserved region at the ORF1-ORF2 junction of the genome was performed. In parallel, a nested two-step qPCR targeting the viral RNA-dependent RNA polymerase gene was also performed. For both assay designs, confirmation of sample positivity was performed using a dot blot hybridization. PCR assays were repeated four times, while hybridization assays were repeated eight times. Detection limits and PCR efficiencies of the assays were compared.

Results: Both assays displayed similar standard curves, however, the nested assay consistently detected one log₁₀ lower virus titer. Dot blot hybridization increased the sensitivity of the nested qPCR by one log₁₀ virus titer, but decreased sensitivity of the one-step RT-qPCR by two log₁₀ virus titer.

Significance: These results confirm that nested two-step qPCR assay for NoV detection is  best suited for situations where sensitivity is valued over rapidity, for example with naturally contaminated samples, such as shellfish. However, despite increased analytical sensitivity, the nested two-step qPCR assays suffers from delayed time to results as well as increased chances for cross-contamination.