Identification Of Type 3 Porcine-derived Reovirus By Small RNA Deep Sequencing And Establishment And Application Of Its TaqMan QPCR Assay

There appeared frequent occurrence of new or reemerging infect ious diseases around the world,and the increased probability of new viruses have posed serious challenges to traditional virus det ect ion methods,and required novel and improved det ection methods.Tradit ional virus detection relies on knowledge of virus information,and not all viru ses can be isolat ed and cultured.Relying only on tradit ional virus det ection methods to identify unknown or emerging viruses,will face the disadvantages of long t imes and large manpower and material resources.Small RNA deep sequen cing is a second-generat ion sequencing technology,wh ich can identify unknown or n ovel viru ses without knowledge of the virus,and facilitate further development of virus-specific diagnostic methods.Mammalian orthoreovirus（MRV）is widely dist ributed in nature,and MRV infect ion is common in humans and animals,causing respiratory and intest inal symptoms and myocarditis.MRV strains isolat ed from animals are closely related t o those in human,and pose significant threat to public safety.In order t o provide technical support for ep idemiological investigation,pathogenic diagnosis and prevention and control of MRV-3 infection.In this study,a tot al of 0.73G Reads with Q30 above 95.46 were obtained by small RNA deep sequencing of unknown viru s samples;after removing the host s RNA,758 viral unique were obtained from the samples after assembly and redundancy;234 viral contigs were obtained by comparing the viral contig sequen ces t o nucleic acid and protein library databases.Finally,19 viral sequences were detected,11 of them were MRV（in which has 1MRV-1,3 MRV-2,6 MRV-3,and 1 MRV-4）,and the remaining 8 were alpha papillomavirus,cyprinid herpesvirus,cytomegalovirus,lentivirus,ovine herpesviru s,blueton gue virus,parainfluenza virus and classical swine fever virus,respectively.Since the S1 gene of MRV determines its serotype,the primers and TaqMan probes were designed by selecting the con served region of 985～1130 bp of MRV-3 S1 gene.After const ructing the posit ive plasmid stan dards,we established a TaqMan RT-qPCR assay for MRV-3 st rain.Th e results showed that the Ct value and the concentration of the positive plasmid st andard were linear in the range of 1.50×10 ⁷～1.50×10⁰ copies/μL with the correlat ion coefficient（R ²）of 0.994.The optimum primer concentration and TaqMan probe concentration were 0.4μmol/L and 0.2μmol/L,respectively.The sensitivity of the TaqMan RT-qPCR established in this study was better than that of convention al PCR,and the minimum detection concentration reached 1.50×10^-1 copies/μL;The reprodu cibility experiments demonstrated that the method is reproducible and stable;the assay established in this study was proved to be superior t o the conventional PCR and tradit ional PCR recommended by the national standard T/CALAS 50-2017 and can be used for clinical test to check the clinical samples.In summary,small RNA deep sequencing technology can rapidly identify viruses in clinical samples and establish a TaqMan RT-qPCR assay for MRV-3 strains with high specificity,sensitivity and stability,providing a technical means to detect MRV-3 strains.