多重RT-RPA技术检测常见儿童腹泻病原体的方法研究OA

Objective To establish a reverse transcription recombinase polymerase amplification(RT-RPA)that can simultaneously detect multiple common pathogens causing diarrhea in children.Methods Conservative sequences of rotavirus,adenovirus,and norovirus genomes were selected to design recombinase polymerase amplification(RPA)primer and probe,and a multiplex RT-RPA detection system was established.The sensitivity,specificity,and repeatability of the established multiplex RT-RPA reaction system were validated,and small clinical sample validation was conducted on the established system.Results The multiplex RT-RPA established in this study has a detection sensitivity of 101copies/μl,102copies/μl and 102copies/μl for rotavirus,adenovirus,and norovirus,respectively,and there was no cross-reaction with other common viruses.Different concentrations of samples were tested 10 times,and the standard deviation of Ct values was small,with coefficients of variation less than 5%.There is no significant difference in the detection rate of common pediatric diarrhea pathogens in clinical samples between PCR method and the colloidal gold method.Conclusion The multiplex RT-RPA established in this study has good sensitivity,specificity,and reproducibility,and can provide new ideas for the detection of pathogens in childhood diarrhea.