Development Of A Multiplex Real-time Fluorescence Quantitative PCR For Human Diarrhea-associated Viruses

Objectives To prepare recombinant plasmids and corresponding DNA or RNA standards as reference materials for laboratory evaluation for 17 kinds of diarrhea-associated viruses,establish a multiplex Real-time PCR platform for the simultaneous detection of17 diarrhea-associated viruses in one sample,and achieve rapid detection of viral pathogens in diarrhea outbreaks.Methods 1.Biology software Primer Express3.0 was applied to design and evaluate primers and probes of 17 viruses.2.Based on gene cloning technology,the target viral gene fragment was amplified by PCR and connected to T-easy Vector,and transformed into bacteria host cells.3.DNA or RNA standards were prepared by PCR or in vitro transcription.4.Single/multiplex detection methods for 17 viruses were developed by optimizing the reaction system and reaction conditions,and the sensitivity(lowest detection limit),specificity(cross-reaction),and stability of these methods(repeat within and between groups)were evaluated further.5.Fecal samples of children under 5 years old with diarrhea were collected in a hospital in Chongqing in 2020 from September to November for for the evaluation of these methods.Results 1.The recombinant plasmids and standard products of 17 viruses as reference materials for laboratory evaluation were developed successfully.2.The lowest detection limit of single-plex Real-time PCR of 17 viruses can reach as low as 10~0?10~3copies/?L.There is no obvious cross-reaction between these methods,and the intra-and inter-batch coefficients of variation of each method were less than 3%and 4%respectively.3.6 combinations of multiplex Real-time PCR for simultaneous detection of 17 viruses were established.The lowest detection limit of them was observed to 10~0?10~3copies/?L,and they demonstrated even neglect significant difference compared with the single reaction.There is no obvious cross-reaction between intra-and inter-groups.Both of the coefficients of variation within and between batches were less than 5%.4.79 cases were tested positive in 100 samples from children with diarrhea from Chongqing in 2020 by the multiplex Real-time PCR method,including 5 for GI norovirus,52 for GII norovirus,and 6 for enteric adenovirus,14 for astrovirus,6 for sapovirus,5 for Group A rotavirus,1 for GIV norovirus,and of which 7 for mixed infection.Conclusions.The recombinant plasmids and corresponding DNA or RNA standards as reference materials for laboratory evaluation for 17 kinds of diarrhea-associated viruses were prepared successfully.The established single-plex and multiplex Real-time PCR detection methods for 17 diarrhea-related viruses in this study showed high sensitivity,strong specificity and good stability,which indicates these methods might be applied in laboratories.The present study provides technical alternatives for the rapid detection of multiplex diarrhea-related pathogens,and also lay foundation for new method with fewer combinations for more pathogens.Figure22;Table36;Reference 185...