Development And Validation Of Diarrhea Virus By Chemiluminescence DNA Microarray

Objective: Diarrhea is a common disease and one million children die from diarrhea-related diseases or complications every year around the world. Diarrhea Virus infection is a major cause of diarrheal diseases. The main diarrhea viruses are Sapovirus, Astrovirus,Enteric adenovirus,Rotavirus A, Rotavirus B, Norovirus GI and Norovirus G Ⅱ.There is no effective drugs against diarrhea viruses which are characteristic of short incubation period, infectious, easy outbreak, therefore, fast and accurate detection of the viruses in diarrhea treatment and prevention is particularly important.Current methods for diarrhea viruses, such as electron microscopy,immunological detection, PCR method, can not achieve high throughput detection.This paper aims to establish a diarrhea viruses gene chip chemiluminescence detection method for detecting seven kinds of diarrhea virus, which can simultaneously detect seven kinds of diarrhea virus, providing a new high-throughput assay for the detect of diarrhea virus.Method:First, survey research literature to determine target pathogen and target genes. Second, collect pathogen samples. Third, use Clustalx1.8.msf Alignment,Primer5.0 biology and other software design primers and probes, and synthesize screening-specific primers and probes. Fouth, optimize multiplex RT-PCR System. Fifth,prepare plasmid reference materials and in vitro transcribed RNA reference materials,then evaluate the specificity, sensitivity and reproducibility of the chip and detect clinical samples.Result:First, seven diarrhea viruses, namely, Rotavirus A, Rotavirus B, Norovirus GI,Norovirus GⅡ, Sapovirus, Astrovirus and Enteric adenovirus, were determined through researching literature. Second, seven pairs of specific primers and twelve ties of specific probes were selected. Third, seven kinds of diarrhea viruses plasmid reference materials and in vitro transcribed RNAs were completed. Forth, in order to ensure each target gene amplification efficiency in multiplex RT-PCR system, the system for the primer composition, the primer concentration ratio and other conditions of the multiplex RT-PCR system had been optimized. Eventually, multiplex RT-PCR system was divided into A, B groups and established diarrhea virus gene chip chemiluminescence detection method. Fifth, to verify the reliability of the method, in vitro transcribed RNA reference materials as templates were used under optimized RT-PCR system and other chip hybridization conditions to assess the chip’s performance. The sensitivity of RNA reference product of the chip was 3.0 × 103copies/μl. The microarray demonstrated upstanding specificity as well as sensitivity. In 100 clinical samples detections, the sensitivity rate and specificity rate were 95.2% and 92.1%, respectively,as well as the accuracy rate 95.1%.Conclusion: A detective method for diarrhea viruses is successfully established based on chemiluminescence DNA microarray. It can simultaneously detect seven kinds of diarrhea viruses. The performance evaluation results show that the chip’s specificity,sensitivity and reproducibility can reach the desired performance requirements. The method provides a new high-throughput method for the clinical diagnosis of viral diarrhea and epidemiological investigations.