Comparative evaluation of RT-PCR, nucleic acid sequence-based amplification (NASBA) and real-time RT-PCR for detection of noroviruses in faecal material.
Using different primer and probe sets, RT-PCR, NASBA and TaqMan RT-PCR molecular methods were compared to detect NoV GII in 13 clinical stool samples. The RT-PCR results observed by gel electrophoresis (Ando, Kageyama and Anderson amplification and probe systems), dot blot hybridization (Ando and Kageyama) and real-time TaqMan assay (Ando and Kageyama) were shown to be consistent and reproducible for the detection of NoV GII. Whereas, the NASBA assay using Ando primers showed some reproducibility discrepancies. Detection limits of the NoV GII/Kageyama system were found to be equal or significantly higher than the Ando system. Real-time TaqMan RT-PCR assay showed similar detection limits to that of NASBA with the Kageyama amplification and detection system, while it was 1log less sensitive than the Ando system. In a clinical context, RT-PCR, NASBA and real-time TaqMan RT-PCR methods using undiluted samples were all suitable for the detection of NoV GII, however the NASBA assay provided less consistent signals. The NoV GII Kageyama real-time TaqMan RT-PCR assay was reliable with a high analytical sensitivity and has shown the capability of detecting one genomic equivalent copy.
Houde
Alain
A
Agriculture and Agri-Food Canada, Food Research and Development Centre, 3600 Casavant Blvd. West, Saint-Hyacinthe, Que., Canada J2S 8E3. houdea@agr.gc.ca
Leblanc
Danielle
D
Poitras
Elyse
E
Ward
Pierre
P
Brassard
Julie
J
Simard
Carole
C
Trottier
Yvon-Louis
YL
D001483:Base Sequence; D005243:Feces / Q000821:virology*; D006801:Humans; D008969:Molecular Sequence Data; D029322:Norovirus / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity