Experience from the development of a diagnostic single tube real-time PCR for human caliciviruses, Norovirus genogroups I and II.
Detection of caliciviruses requires high mutation tolerance and throughput. The development of a rational simple, single tube reverse transcription-real-time quantitative PCR (QPCR) technique for human noroviruses (NV) is reported here. A dual-probe, triple-primer system (NM system) was used for simultaneous detection and preliminary differentiation of NV genogroups in fecal samples. The design was based on a comprehensive analysis of all 1140 NV sequences available in GenBank. A touch-down amplification protocol improved the frequency of detection. The final QPCR was evaluated with 71 fecal samples from outbreak and sporadic cases in Sweden (1997-2004), all calicivirus-positive by electron microscopy. Up to 56 (79 %) were positive. The method is more rational than NV detection methods described previously, and should be a developmental basis for large-scale routine methods for detection of NV.
Mohamed
Nahla
N
Section of Clinical Virology, Department of Medical Sciences, Academic Hospital, Uppsala University, Sweden.
Belák
Sándor
S
Hedlund
Kjell-Olof
KO
Blomberg
Jonas
J
D000328:Adult; D017250:Caliciviridae Infections / Q000175:diagnosis* / Q000821:virology; D002648:Child; D017931:DNA Primers; D005243:Feces / Q000821:virology; D005759:Gastroenteritis / Q000821:virology; D006801:Humans; D008854:Microscopy, Electron; D029322:Norovirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D012367:RNA, Viral