Multiplex real time RT-PCR for the detection and quantitation of norovirus genogroups I and II in patients with acute gastroenteritis

Background: Conventional reverse transcription-polymerase chain reaction (Con RT-PCR) assay to detect norovirus is a complex multi-step procedure that requires gel electrophoresis as well as hybridization or sequencing to confirm the results. Objective: To develop and evaluate a multiplex real time RT-PCR (Mrt RT-PCR) assay that detect and quantify norovirus GI and GII with a single amplification and detection step. Study design: The primers and TaqMan probes for the Mrt RT-PCR were selected from the ORF1-ORF2 junction region. A total of 97 stools from 41 gastroenteritis outbreaks and 726 stools from children with sporadic diarrhoea were used for this study. Results: For the 97 outbreak samples, norovirus were detected in 61 of the 69 previously tested positive and I I of the 28 previously tested negative samples. Eight samples that tested positive for GII by Con RT-PCR but negative by the Mrt RT-PCR also tested negative by a Light Cycler RT-PCR assay. Eighty-two GII and two GI were detected in the 726 sporadic samples. Random primers were more sensitive than specific primers in the cDNA synthesis. The two-step assay using the random primers in RT reaction was 100 times more sensitive than the one-step assay. The Mrt RT-PCR had the same sensitivity as that using two real time RT-PCR for separate detection of GI and GII. A wide dynamic range was obtained with the two-step assay, detecting from 3000 to 3x10(11) of copies RNA/g stool. Very good precision was observed with no cross-reaction with other enteric viruses. The new assay is able to detect both GI and GII in one reaction and brings a cost reduction of approximately 40% compared to separate reactions for GII and GII. Conclusions: The assay has good precision, sensitivity and specificity and is cost-effective as a routine diagnostic test. (C) 2005 Elsevier B.V. All rights reserved.