Development and Validation of a Commercial TaqMan-Based RT-qPCR Kit for Rotavirus and Norovirus Detection in the Brazilian Acute Diarrhea Surveillance Network

Abstract

Acute gastroenteritis (AGE) is a major cause of illness and death in children under five, especially in low- and middle-income countries, and rotavirus A (RVA) and norovirus are the leading viral agents. The present study aimed to describe the development of a commercial multiplex TaqMan-based RT-qPCR assay to detect those viruses to enhance surveillance and public health responses in Brazil. The assay validation involved optimizing primers and probes for multiplex RT-qPCR, assessing analytical sensitivity, and confirming specificity. A multicenter pilot study across Brazil’s AGE surveillance network assessed the assay’s performance. The IBMP NAT assay demonstrated high specificity and sensitivity for detecting RVA and norovirus GI and GII. No cross-reactivity was observed. LoD95 values were low: 18.6 (GI), 71.2 (GII), and 12.3 (RVA) copies/reaction. In 379 clinical samples, diagnostic sensitivity and specificity exceeded 96% for all targets. The assay showed strong reproducibility across operators and instruments. Stability tests confirmed consistent performance under freeze–thaw, transport, and storage conditions. Compared to in-house RT-qPCR, the IBMP NAT test yielded lower Ct values, indicating improved detection of low viral loads. The IBMP NAT Kit significantly advances molecular diagnostics, enabling rapid, sensitive, and reliable detection of RVA and norovirus in fecal specimens. It strengthens public health surveillance and supports timely responses to AGE outbreaks, helping reduce disease burden in vulnerable populations.