Broadly reactive nested reverse transcription-PCR using an internal RNA standard control for detection of noroviruses in stool samples

Abstract

We developed a nested reverse transcription-PCR (nRT-PCR) for the detection of noroviruses in stools, using random primers for RT, the JV12/JV13 primer pair in the first round of nPCR, and a set of nine inner primers for the second, comprising the reverse sequences of primers SR46, SR48, SR50, and SR52, and five novel oligonucleotide sequences (113-1, 113-2, 115-1, 115-2, and 115-3). The specificity of the nRT-PCR was confirmed by testing 61 stools containing enteric viruses other than noroviruses. In comparative assays on either stools or RNA dilutions from two genogroup I and three genogroup II (GII) norovirus-positive samples, nRT-PCR was always at least as sensitive as RT-PCR and Southern hybridization. With some of the samples tested, the increase in sensitivity was 10-fold or higher. For GII viruses, the detectable range of nRT-PCR was estimated to be 8.4 x 10(4) to 2 RNA viral particles. When used on 85 stools from pediatric patients with acute gastroenteritis negative for viruses by electron microscopy and cell culture, the nRT-PCR detected norovirus in 19 samples (22.3%), while it failed to detect one reference RT-PCR-positive sample containing a Desert Shield strain. Sixteen of the 19 nRT-PCR-positive samples gave concordant results with reference RT-PCR and Southern hybridization, and all with sequence analysis. Partial sequencing of the polymerase region revealed that from January to April 2000 all GII strains except two (Rotterdam- and Leeds-like viruses) formed a tight cluster related to Hawaii virus. The nRT-PCR described could prove suitable for large epidemiological studies and for specialized clinical laboratories performing routine molecular testing.

FIG. 1.

Representative norovirus detection by nRT-PCR compared to RT-PCR and Southern hybridization in a 10-fold dilution series from a positive stool sample containing a Bristol genotype at a concentration of 3.9 × 10⁸ virus particles/ml and the internal RNA standard control. (A) Agarose gel electrophoresis of nRT-PCR products from 10⁻³ to 10⁻⁸ dilutions. (B) Results of ethidium bromide staining (top) and corresponding Southern hybridization (bottom) of conventional RT-PCR products from 10⁻³ to 10⁻⁶ dilutions. N, zero RNA; M, DNA molecular size markers.