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


OBJECTIVE
To further develop a one-step RT-PCR assay for norovirus detection/genogrouping by addition of MS2 bacteriophage as an internal control.


METHODS
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.
Rolfe
K J
KJ
Health Protection Agency, East of England Laboratory, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QW, United Kingdom. kathryn.rolfe@addenbrookes.nhs.uk
Parmar
S
S
Mururi
D
D
Wreghitt
T G
TG
Jalal
H
H
Zhang
H
H
Curran
M D
MD
D017250:Caliciviridae Infections / Q000821:virology*; D017931:DNA Primers; D005243:Feces / Q000821:virology; D005759:Gastroenteritis / Q000821:virology*; D005838:Genotype; D006801:Humans; D017909:Levivirus / Q000235:genetics / Q000302:isolation & purification; D029322:Norovirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification / Q000592:standards*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity
D017931:DNA Primers; D012367:RNA, Viral