Background: Conventional reverse transcription-polymerase chain reaction (RT-PCR) amplification of the RNA-dependent RNA polymerase (RdRp) gene remains a used method for the rapid detection of norovirus (NV) in clinical laboratories. The incidence of and factors associated with false positives in this assay have not been previously evaluated.
Methods/principal findings: After an NV outbreak caused by the GII.4 Sydney strain in 2012, we reanalysed 250 stool samples positive for NV by RdRp gene detection. True positives were confirmed in 154 (61.6%) samples by successful amplification and sequencing confirmation of the viral protein 1 gene. Of the remaining 96 samples that underwent RT-PCR for the RdRp gene, 34 samples yielded PCR products of the expected length. However, the sequences of the amplicons belonged to the human genome, with 91-97% matched nucleotide sequences, indicating false positives. Multivariate analysis of the clinical features of the patients identified a positive stool culture for bacteria (adjusted odds ratio [aOR] 9.07, 95% adjusted confidence interval [aCI] 2.17-37.92, P = .003) and the use of parenteral antibiotics (aOR 5.55, 95% aCI 1.21-24.73, P = .027) as significant and independent factors associated with false positives.
Conclusion: Conventional RT-PCR targeting the RdRp gene of NV can lead to false positives in patients with bacterial enterocolitis by incidental amplification of DNA from a human source.