Broadly reactive and highly sensitive assay for Norwalk-like viruses based on real-time quantitative reverse transcription-PCR

Abstract

We have developed an assay for the detection of Norwalk-like viruses (NLVs) based on reverse transcription-PCR (RT-PCR) that is highly sensitive to a broad range of NLVs. We isolated virus from 71 NLV-positive stool specimens from 37 outbreaks of nonbacterial acute gastroenteritis and sequenced the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the NLV genome. The data were subjected to multiple-sequence alignment analysis and similarity plot analysis. We used the most conserved sequences that react with diverse NLVs to design primers and TaqMan probes for the respective genogroups of NLV, GI and GII, for use in a real-time quantitative RT-PCR assay. Our method detected NLV in 99% (80 of 81) of the stool specimens that were positive by electron microscopy, a better detection rate than with the two available RT-PCR methods. Furthermore, our new method also detected NLV in 20 of 28 stool specimens from the same NLV-related outbreaks that were negative for virus by electron microscopy. Our new assay is free from carryover DNA contamination and detects low copy numbers of NLV RNA. It can be used as a routine assay for diagnosis as well as for elucidation of the epidemiology of NLV infections.

FIG. 1.

Nucleotide sequences of full-length NLV genomes were analyzed with the PlotSimilarity program of Genetics Computer Group software. Similarity scores of a 150-nt sliding window were plotted. Four NLV GI strains (SzUGI, Norwalk/68, Southampton, and BS5) (A) and 10 NLV GII strains (U1, U3, U4, U16, U17, U18, U201, U25, Lordsdale, and Camberwell) (B) were compared. The locations of the ORFs are noted. Thick lines depict the most conserved regions. The average similarity score within each genogroup is represented as a dotted line.

FIG. 2.

Alignment of nucleotide sequences and partial predicted amino acid sequences of the ORF1-ORF2 junctions of NLV GI (A) and GII (B). The nucleotide sequences of NLV GI were aligned from nt 5254 to 5425 in Norwalk/68, and those of NLV GII were aligned from nt 4981 to 5152 in Camberwell. The strain name and accession number are shown beside its sequence; a lowercase letter in the strain name indicates a different clone from the same stool specimen. Some strains with identical sequence are omitted from the figure. The conserved amino acid sequences of NLV GI ORF1 are indicated above the sequences between nt 5279 and 5371 in Norwalk/68, and those of ORF2 are indicated between nt 5358 and 5381. The conserved amino acid sequences of NLV GII ORF1 are indicated above the nucleotide sequences between nt 4988 and 5101 in Camberwell, and those of ORF2 are indicated between nt 5085 and 5108. The asterisks below the alignment show consensus nucleotide sequences. Arrows and double lines show locations of newly designed primers and probes, respectively, used in this study.

FIG. 3.

Real-time RT-PCR quantification of NLV GI and GII standard plasmids. (A and B) Amplification plots of fluorescence intensities (ΔRn) versus the PCR cycle numbers are displayed for serial 10-fold dilutions of standard plasmids (10⁷ to 10¹ copy equivalents per reaction) for NLV GI (A) and GII (B). Each plot corresponds to a particular input target quantity marked by a corresponding symbol. Results are the average of those from three reactions. (C and D) Relationship of known numbers of NLV GI (C) and GII (D) standard plasmids to the threshold cycle (Ct). The Ct is directly proportional to the log of the input copy equivalents, as demonstrated by the standard curve. Results are the average of those from three reactions, and error bars indicate standard deviations.