Analytical performance of norovirus real-time RT-PCR detection protocols in Canadian laboratories.
BACKGROUND
Noroviruses (NoVs) are the leading cause of infectious gastroenteritis worldwide. Real-time reverse transcription PCR (real-time RT-PCR) is the preferred method of NoV detection for the majority of testing laboratories. Although the accepted target region for molecular detection assays is the conserved ORF1/ORF2 junction, multiple variations have been published with differences in primers, probes, reagents, multiplexing, etc.


OBJECTIVE
We assessed the detection limit for GII.4 NoV real-time RT-PCR assays as well as the ability to detect the non-GII.4 NoV genotypes in each participating laboratory.


METHODS
A panel of 25 RNA samples was circulated to 18 testing laboratories for comparison of their real-time RT-PCR procedures for NoV detection.


RESULTS
Multiple protocols with slight differences in reagents or conditions successfully detected 10 genome equivalents or fewer of NoV per reaction. Multiplex procedures were significantly associated (p=0.04) with false negative results, particularly for a GI.2 strain. Sensitive detection was associated with false positive results (p=0.03).


CONCLUSIONS
Overall, the data indicate that comparable results are produced under slightly different assay conditions.
Bureau of Microbial Hazards, Health Canada, Ottawa, ON, Canada.
Kirsten
K
Mattison
Elsie
E
Grudeski
Brian
B
Auk
Julie
J
Brassard
Hugues
H
Charest
Kerry
K
Dust
Jonathan
J
Gubbay
Todd F
TF
Hatchette
Alain
A
Houde
Julie
J
Jean
Tineke
T
Jones
Bonita E
BE
Lee
Hiroshi
H
Mamiya
Ryan
R
McDonald
Oksana
O
Mykytczuk
Xiaoli
X
Pang
Astrid
A
Petrich
Daniel
D
Plante
Gordon
G
Ritchie
Julie
J
Wong
Tim F
TF
Booth
D001483:Base Sequence; D017250:Caliciviridae Infections / Q000175:diagnosis* / Q000821:virology; D002170:Canada; D005243:Feces / Q000821:virology; D005759:Gastroenteritis / Q000175:diagnosis* / Q000821:virology; D005838:Genotype; D006801:Humans; D057230:Limit of Detection; D029322:Norovirus / Q000235:genetics / Q000302:isolation & purification*; D016366:Open Reading Frames; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D012367:RNA, Viral