title: Development and clinical validation of multiplex TaqMan® assays for rapid diagnosis of viral gastroenteritis.
abstract: There is a need to provide rapid, sensitive, and often high throughput detection of pathogens in diagnostic virology. Viral gastroenteritis is a serious health issue often leading to hospitalization in the young, the immunocompromised and the elderly. The common causes of viral gastroenteritis include rotavirus, norovirus (genogroups I and II), astrovirus, and group F adenoviruses (serotypes 40 and 41). This article describes the work-up of two internally controlled multiplex, probe-based PCR assays and reports on the clinical validation over a 3-year period, March 2007 to February 2010. Multiplex assays were developed using a combination of TaqMan™ and minor groove binder (MGB™) hydrolysis probes. The assays were validated using a panel of 137 specimens, previously positive via a nested gel-based assay. The assays had improved sensitivity for adenovirus, rotavirus, and norovirus (97.3% vs. 86.1%, 100% vs. 87.8%, and 95.1% vs. 79.5%, respectively) and also more specific for targets adenovirus, rotavirus, and norovirus (99% vs. 95.2%, 100% vs. 93.6%, and 97.9% vs. 92.3%, respectively). For the specimens tested, both assays had equal sensitivity and specificity for astrovirus (100%). Overall the probe-based assays detected 16 more positive specimens than the nested gel-based assay. Post-introduction to the routine diagnostic service, a total of 9,846 specimens were processed with multiplex 1 and 2 (7,053 pediatric, 2,793 adult) over the 3-year study period. This clinically validated, probe-based multiplex testing algorithm allows highly sensitive and timely diagnosis of the four most prominent causes of viral gastroenteritis.
authors: Regional Virus Laboratory, Royal Victoria Hospital, Belfast, Northern Ireland, United Kingdom. susan.feeney@belfasttrust.hscni.net
authors: S A
authors: SA
authors: Feeney
authors: V J
authors: VJ
authors: Armstrong
authors: S J
authors: SJ
authors: Mitchell
authors: L
authors: L
authors: Crawford
authors: C
authors: C
authors: McCaughey
authors: P V
authors: PV
authors: Coyle
mesh_terms: D000258:Adenovirus Infections, Human / Q000175:diagnosis*; D000328:Adult; D019350:Astroviridae Infections / Q000175:diagnosis*; D017250:Caliciviridae Infections / Q000175:diagnosis*; D002648:Child; D017931:DNA Primers; D005243:Feces / Q000821:virology; D005759:Gastroenteritis / Q000175:diagnosis* / Q000821:virology; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction; D029322:Norovirus; D012400:Rotavirus Infections / Q000175:diagnosis*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers