Pediatria e Terapêutica
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Development of Two Multiplex Real-Time PCR Assays for the Rapid Detection of RNA and DNA Viruses Associated with Gastroenteritis in Pediatric Patients

Dongmei Chen,Yu Sun,Linqing Zhao*,Yuan Qian,Runan Zhu,Liying Liu,Liping Jia,Huijin Dong,Jie Deng,Li Deng

Background: Viral gastroenteritis is one of the most common causes of morbidity and mortality in infants and young children in China. Objectives: To develop a rapid and sensitive real time PCR method capable of detecting several viruses associated with gastroenteritis in pediatric patients simultaneously. Methods: Two multiplex real-time PCR assays, the first targeting the RNA viruses: rotavirus, norovirus and parachovirus and the second the DNA viruses: human adenovirus and human bocavirus 2, were designed and assessed for their specificity and sensitivity. The multiplex assays were evaluated using clinical samples and compared to conventional RT-PCR/PCR. Results The multiplex assays developed in the study were successful in detecting the five target viruses. No cross-reactions with a panel of other human viruses were presented. The assays were sensitive enough to detect as little as one copy of in vitro transcribed target RNA or plasmid DNA in a single reaction. Compared to conventional RT-PCR or PCR, the multiplex assays showed acceptable sensitivity from 86.2% to 95.7% together with high specificity (97.2% to 99.5%) in detecting rotavirus, norovirus and adenovirus.   Conclusion: The development of these two multiplex assays should result in significant improvement in the screening of viral pathogens associated with pediatric gastroenteritis in China.