Accurate diagnosis of viral diarrhea can avoid the abuse of antibiotics after misdiagnosis as bacterial diarrhea, and carry out targeted treatment. At the same time, the clinical symptoms of these viruses are similar in the early stage of infection, but there are great differences in the clinical severity in the later stage. Rotavirus A can cause severe clinical symptoms such as dehydration and electrolyte turbulence, while Enteroadenovirus can cause mild to moderate dehydration in most children. Norovirus and sapovirus can cause significantly less clinical symptoms in children than rotavirus A [14–16]. Our results also revealed that the median age of children infected with each diarrhea virus was very close (1–2 years), except for astrovirus. Therefore, the accurate identification of viral pathogens early in the infection can individualize the treatment and management of children to avoid the occurrence of serious clinical symptoms. In order to detect common viruses in children diarrhea with high throughput, multiplex real-time RT-PCR was developed for the detection of adenovirus, astrovirus, rotavirus and sapovirus from stool samples in previous study . However, the detection capacity of this method is not large enough.
This paper describes the development and validation of a real-time RT-PCR plus melting profile analysis (RRCMC) assay, which will allow rapid and simultaneous detection of rotavirus A, B, C, norovirus GI and GII, adenovirus, astrovirus and sapovirus in stool samples. The cost of RRCMC assay is about 10 dollars each sample, and it takes about 3–5 hours to finish the testing process. Because of the conservative characteristics of primers and probes and the control of melting curve, RRCMC assay has high specificity. There is no internal cross-reaction between 8 diarrhea viruses. The specificity of the assay was also confirmed by testing a panel of other commonly intestinal pathogens and no-template controls and no false positive results were encountered. Our study showed that the detection limit of the RRCMC method was range from 1 × 102 to 1 × 105 copies/ml for each diarrhea associated virus. Among them, the sensitivity of rotavirus detection is the highest (1 × 102 copies/ml) and the sensitivity of norovirus (GI and GII) detection is the lowest (1 × 105 copies/ml).
In 160 patients with acute diarrhea, a total of 71 patients were tested positive by RRCMC. In our study, rotavirus A, C, norovirus GI and II, adenovirus, astrovirus and sapovirus were detected by RRCMC, and norovirus, adenovirus and rotovirus were the most common viruses caused acute diarrhea in children in study period. Rotavirus B was not detected in this study. In further studies, we will enroll more clinical samples from children with acute diarrhea.