An internally controlled, one-step, real-time RT-PCR assay for norovirus detection and genogrouping.

ABSTRACT

BACKGROUND: Reverse transcription (RT)-PCR for norovirus detection is prone to false-negative results due to inhibitory substances in faeces. An internal control is needed to monitor extraction efficiency and to detect inhibition. OBJECTIVES: To further develop a one-step RT-PCR assay for norovirus detection/genogrouping by addition of MS2 bacteriophage as an internal control. STUDY DESIGN: Our norovirus RT-PCR assay was modified by addition of MS2 phage to the extraction tray and primers/probe for MS2 detection to the reaction mix. The effect of addition of MS2 phage and MS2 primers/probe on the sensitivity/specificity of the PCR assay was examined. RESULTS: The addition of MS2 as an internal control showed no loss of sensitivity or specificity for norovirus detection. CONCLUSIONS: A triplex, one-step, type-specific, real-time RT-PCR with MS2 internal control has been developed for use in routine laboratory diagnosis of norovirus infection.