Novel light-upon-extension real-time PCR assays for detection and quantification of genogroup I and II noroviruses in clinical specimens.
Norovirus is now recognized as the leading cause of nonbacterial acute gastroenteritis in adults, causing numerous outbreaks worldwide. We have developed two novel light-upon-extension (LUX) real-time PCR assays for detection and quantification of norovirus genogroups I and II. The LUX system uses a fluorophore attached to one primer having a self-quenching hairpin structure, making it cost-effective and specific. The assays were evaluated against clinical stool specimens (n = 103) from Sweden and Nicaragua and compared to established methods. The norovirus assay detected more positive stool specimens (47/103) than conventional PCR (39/103) and corresponded to a TaqMan real-time PCR, with the exception of one specimen. Furthermore, the assays correctly identified all (n = 11) coded control specimens in a reference panel containing various genogroups and genotypes. Both LUX real-time PCR assays had a wide dynamic range, detecting from < or = 10(1) to 10(7) genes per reaction, resulting in a theoretical lower limit of < or = approximately 20,000 viruses per gram of stool. No cross-reactivity was noticed with specimens containing other enteric viruses, and by using melting curve analysis we could differentiate between norovirus genogroups I and II.
Nordgren
Johan
J
Department of Clinical and Experimental Medicine, Linköping University, 58185 Linköping, Sweden.
Bucardo
Filemón
F
Dienus
Olaf
O
Svensson
Lennart
L
Lindgren
Per-Eric
PE
D000328:Adult; D002648:Child; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D005243:Feces / Q000821:virology; D005453:Fluorescence; D005759:Gastroenteritis / Q000821:virology; D006801:Humans; D009527:Nicaragua; D029322:Norovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013548:Sweden
D017931:DNA Primers