Rapid and Sensitive Detection of Noroviruses by Using TaqMan-Based One-Step Reverse Transcription-PCR Assays and Application to Naturally Contaminated Shellfish Samples

Abstract

Noroviruses (NoV), which are members of the family Caliciviridae, are the most important cause of outbreaks of acute gastroenteritis worldwide and are commonly found in shellfish grown in polluted waters. In the present study, we developed broadly reactive one-step TaqMan reverse transcription (RT)-PCR assays for the detection of genogroup I (GI) and GII NoV in fecal samples, as well as shellfish samples. The specificity and sensitivity of all steps of the assays were systematically evaluated, and in the final format, the monoplex assays were validated by using RNA extracted from a panel of 84 stool specimens, which included NoV strains representing 19 different genotypes (7 GI, 11 GII, and 1 GIV strains). The assays were further validated with 38 shellfish cDNA extracts previously tested by nested PCR. Comparison with a recently described real-time assay showed that our assay had significantly higher sensitivity and was at least as sensitive as the nested PCR. For stool specimens, a one-step duplex TaqMan RT-PCR assay performed as well as individual genogroup-specific monoplex assays. All other enteric viruses examined were negative, and no cross-reaction between genogroups was observed. These TaqMan RT-PCR assays provide rapid (less than 90 min), sensitive, and reliable detection of NoV and should prove to be useful for routine monitoring of both clinical and shellfish samples.

Analytical Methods for Virus Detection in Water and Food

“…Direct lysis of virus particles is used more and more frequently. For example, proteinase K or Trizol and lysis of shellfish tissues using Zirconia beads and a denaturing buffer have all been used for virus elution (Jothikumar et al 2005;LodderVerschoor et al 2005). …”

pilF polymorphism-based real-time PCR to distinguish Vibrio vulnificus strains of human health relevance

“…The digestive glands were subsequently pooled together, and then finely chopped using a sterile razor blade. Homogenates were then prepared by treating the chopped digestive glands with 100 mg/ml Proteinase K solution (30 U/mg; Promega) as previously described (Jothikumar et al, 2005;Baker-Austin et al, 2010b). Homogenates were stored at 4 C prior to testing.…”

Detection of GI and GII noroviruses in drinking water and vegetables using filtration and real-time RT-PCR

“…The primers and probes (Table 1) were designed [11] using primer express 3.0 software for spanning ORF1 and ORF2 of norovirus. The oligonucleotides were synthesized, and the probes were labeled with FAM for GI NoV and VIC for GII NoV in TAKARA Corporation, Dalian.…”