Optimization of a real-time RT-PCR assay reveals an increase of genogroup I norovirus in the clinical setting

• Published in: Journal of virological methods Vol. 175; no. 1; pp. 80 - 84
• Main Authors: Van Stelten, A., Kreman, T.M., Hall, N., DesJardin, L.E.
• Format: Journal Article
• Language: English
• Published: Kidlington Elsevier B.V 01.07.2011; Elsevier
• Subjects: Base Sequence; Biological and medical sciences; Caliciviridae Infections - diagnosis; Caliciviridae Infections - virology; Clinical specimens; detection limit; DNA Primers; Feces - virology; Fundamental and applied biological sciences. Psychology; Gastroenteritis; Gastroenteritis - diagnosis; Gastroenteritis - virology; Genogroup I; Humans; Limit of Detection; Microbiology; Norovirus; Norovirus - genetics; Norovirus - isolation & purification; patients; Real-Time Polymerase Chain Reaction - methods; Real-time RT-PCR; reverse transcriptase polymerase chain reaction; RNA, Viral - analysis; RNA, Viral - genetics; Techniques used in virology; Virology; Iowa; Real-time RT-PCR; Norovirus; Gastroenteritis; Genogroup I; Clinical specimens; Method; Real time; Optimization; Virus; Caliciviridae; Digestive diseases; Intestinal disease; Clinical isolate; Reverse transcription polymerase chain reaction; Gastric disease; Genogroup 1
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2011.04.022

► We optimized a real-time RT-PCR assay for improved detection of GI norovirus in patient specimens. ► Assay demonstrated an increase in sensitivity and amplification efficiency. ► Assay detected GI norovirus in clinical specimens that were initially negative. Although norovirus has been identified as the most common cause of gastroenteritis, the majority of cases have no etiologic agent identified. In this study, we describe the optimization of a real-time RT-PCR assay for the improved detection of genogroup I norovirus in patient specimens based upon sequence data from a collection of representative clinical norovirus sequences. The redesigned assay demonstrated a 64 fold increase in sensitivity, a 2 log decrease in the limit of detection, and an 18% increase in amplification efficiency, when compared to the standard assay. The optimized test also detected GI norovirus in clinical specimens that were initially negative by the standard assay. Use of the optimized assay increased the annual positivity of GI norovirus in Iowa from 1.2% to 4.5%, indicating the prevalence of GI norovirus may be higher than previously identified. Laboratory confirmation of the etiologic agent involved in gasteroenteritis cases is essential for better understanding of the prevalence and transmission of noroviruses.