International collaborative study to compare reverse transcriptase PCR assays for detection and genotyping of noroviruses

Abstract

To allow more rapid and internationally standardized assessment of the spread of noroviruses (previously called Norwalk-like viruses [NLVs]) as important food-borne pathogens, harmonization of methods for their detection is needed. Diagnosis of NLVs in clinical diagnostic laboratories is usually performed by reverse transciptase PCR (RT-PCR) assays. In the present study, the performance of five different RT-PCR assays for the detection of NLVs was evaluated in an international collaborative study by five laboratories in five countries with a coded panel of 91 fecal specimens. The assays were tested for their sensitivity, detection limit, and ease of standardization. In total, NLVs could be detected by at least one RT-PCR assay in 69 (84%) of the samples that originally tested positive. Sensitivity ranged from 52 to 73% overall and from 54 to 100% and 58 to 85% for genogroup I and II viruses, respectively. In all, 64% of the false-negative results were obtained with a set of diluted stools (n = 20) that may have lost quality upon storage. Sensitivity was improved when these samples were excluded from analysis. No one single assay stood out as the best, although the p1 assay demonstrated the most satisfactory overall performance. To promote comparability of data, this assay will be recommended for newly starting groups in future collaborative studies.

FIG. 1.

Phylogenetic relationships based on a 145-bp region of the RNA POL gene showing the relationships among 66 panel strains from which a sequence was obtained by p1 or p6, as well as NLV strains representing the individual genotypes or clusters. Full capsid numbers are shown in parentheses as follows: Bristol (GII.4), Wortley (GII.1b), Hawaii (GII.1), GII Finland, Rotterdam, Melksham (GII.2), Hillingdon (GII.5), Toronto (GII.3), Amsterdam (GII.8), Leeds (GII.7), Stavanger (GI.3b), Winchester (GI.7), Chiba (GI.4), Southampton (GI.2), Sindlesham (GI.6), and Alphatron (GIV.1). Several other NLV strains (Lordsdale, Snow Mountain virus, Girlington, Desert Shield virus, Hesse, Queens Arms, and Musgrove) were included as a reference. Bootstrap values of >50 of the internal nodes are indicated.

FIG. 2.

Comparison of detection limit of the five different NLV detection assays with RNA from nine different NLV genotypes. For more information about the partner laboratories (p), see Table 1 and Materials and Methods.

FIG. 3.

Detection and simultaneous typing of NLVs from 30 panel strains (strains 31 to 60). Probes specific for the detection of genogroup I (GIa and b) and genogroup II (GII) and for 15 ORF1 clusters are indicated (53). The name of each probe is shown with the representative full capsid number (e.g., GII.4) in parentheses. ✽, The Rotterdam probe detects strains that are GII.3 in the capsid region, but that group separately in the POL region; p, positive Bristol (GII.4) control; n, negative control (water). Note that probes specific for strains of the clusters Seacroft (GII.6), Amsterdam (GII.8), and Alphatron (GIV.1) were not included on the membrane.