The validation and utility of a quantitative one-step multiplex RT real-time PCR targeting Rotavirus A and Norovirus

Trần Thị Ngọc Dung ,Nguyen Thanh Vinh,James I Campbell,Pham Van Minh,Voong Vinh Phat,Ha Manh Tuan,Duong Thi Hue Kien,Nguyen Tran Chinh,Tran Vu Thieu Nga,Huynh Le Anh Huy,Stephen Baker,Pham Thanh Duy,Cao Thu Thuy,Tang Chi Thuong,Ha Vinh,Hoang Le Phuc,Pham Thi Ngoc Tuyet,Nguyen Thi Thu Hau,Nguyễn Văn Minh Hoàng ,Jeremy Farrar ,Cameron P Simmons ,My V T Phan

doi:10.1016/j.jviromet.2012.09.021

Abstract

Rotavirus (RoV) and Norovirus (NoV) are the main causes of viral gastroenteritis. Currently, there is no validated multiplex real-time PCR that can detect and quantify RoV and NoV simultaneously. The aim of the study was to develop, validate, and internally control a multiplex one-step RT real-time PCR to detect and quantify RoV and NoV in stool samples. PCR sensitivity was assessed by comparing amplification against the current gold standard, enzyme immunoassay (EIA), on stool samples from 94 individuals with diarrhea and 94 individuals without diarrhea. PCR detected 10% more RoV positive samples than EIA in stools samples from patients with diarrhea. PCR detected 23% more NoV genogroup II positive samples from individuals with diarrhea and 9% more from individuals without diarrhea than EIA, respectively. Genotyping of the PCR positive/EIA negative samples suggested the higher rate of PCR positivity, in comparison to EIA, was due to increased sensitivity, rather than nonspecific hybridization. Quantitation demonstrated that the viral loads of RoV and NoV in the stools of diarrheal patients were an order of magnitude greater than in individuals without diarrhea. This internally controlled real-time PCR method is robust, exhibits a high degree of reproducibility, and may have a greater utility and sensitivity than commercial EIA kits.

Highlights

Diarrhea remains a major cause of childhood morbidity and mortality globally (Thapar et al, 2004; Wardlaw et al, 2010), with the vast majority of the 2 billion annual infections and 2.5 million resulting deaths occurring in low and middle-income countries
This study was conducted according to the principles expressed in the Declaration of Helsinki, and was approved by the ethical review boards of the Hospital for Tropical Diseases (HTD), Children’s Hospital 1 (CH1) and Children’s Hospital 2 (CH2) in Ho Chi Minh City in Viet Nam, and the Oxford Tropical Research Ethics Committee (OxTREC) in the United Kingdom
Stool samples were obtained from a group of symptomatic children and a group of asymptomatic children

Summary

Introduction

Diarrhea remains a major cause of childhood morbidity and mortality globally (Thapar et al, 2004; Wardlaw et al, 2010), with the vast majority of the 2 billion annual infections and 2.5 million resulting deaths occurring in low and middle-income countries. Rotavirus A (RoV) and genogroup I and II Norovirus (NoVI and II) are predominant causes of viral gastroenteritis worldwide, and are responsible for over 40% of all cases of diarrhea in developing countries T.T.N. Dung et al / Journal of Virological Methods 187 (2013) 138–143 Viral target RoV. Non-structural protein 3 (NSP3)/87 bp Primer/probe name NVP3-FDeg NVP3-R1 NVP3-Probe NoVII.

Objectives

Methods

Results

Conclusion