Application Of Loop-mediated Isothermal Amplification Assay Of Giant Panda Rotavirus And G/P Serotype Identification

Giant Panda Rotavirus（GPRV）mainly affects giant panda cubs and causes intractable diarrhea and even death.Such circumstances pose a major threat to breeding,health and reintroduction of reared giant pandas.Therefore,it is of paramount importance to strengthen the monitoring of GPRV infection and to develop simple,rapid and high-sensitive laboratory diagnostic methods for giant pandas.Through the identification of GPRV serotype,the epidemic trend and genetic evolution law are analyzed to provide basic data for the selection and development of vaccine.1.Loop-Mediated Isothermal Amplification（LAMP）assay for detection of GPRVLoop-Mediated Isothermal Amplification（LAMP）is a fast and sensitive molecular biological diagnostic method for virus detection.It is cheap and easy to operate,through which the results can be observed directly by naked eyes without leading-edge instruments.However,it has not been applied for detection of GPRV.Thereby,in this study,three pairs of primers were designed according to VP4 gene of CH-1 strain of GPRV,and the visual LAMP assay for GPRV was established to optimize its application and detect its specificity and sensitivity.The visual LAMP assay was used to detect the GPRV of 91 stool samples of giant panda in 2017-2019,and the goodness of fit was verified by fluorescence quantitative polymerase chain reaction（PCR）.The results showed that the visual observation of LAMP detection could be achieved by adding 1×visible light dye in the amplification system.The color of positive amplification samples changed from purple to blue,while the color of negative amplification samples did not change.Under the condition of Mg²⁺concentration at 8.0 m M,betaine concentration at 0.6 M,and Bst DNA polymerase concentration at 12 U,the best amplification effect can be obtained after reaction at 62℃for 40 minutes.This assay was highly specific without no cross-reaction to some common giant panda infectious viruses such as canine distemper virus and parvovirus.Its sensitivity was 100 times higher than that of conventional PCR,and the lowest detection limit was 50 fg RNA.Among the enrolled 91 stool samples of giant pandas from 2017 to 2019,39 samples were positive for GPRV,with a total positive rate of 42.86%.The coincident rate for positive was 97.50%and for negative was 98.08%by fluorescent quantitative PCR.2.G/P serotype identification and phylogenetic analysis for GPRVIn this study,GPRV-positive stool samples of giant pandas were screened by LAMP assay.Nested PCR technique was utilized to identify the G/P serotype of these positive samples,and p MD19T-VP7 and p MD19T-VP4 recombinant plasmids were constructed and sequenced.The sequencing results were compared with gene sequences of rotavirus VP4and VP7 reported in BLAST and Gen Bank by homology alignment.The phylogenetic tree was constructed using maximum likelihood method to analyze the average evolution rate and selection pressure.The results noted that the epidemic serotype of GPRV in 2017-2019was G2P[8],and G1,G4 and P[11]serotypes were occasionally detected.The new G/P serotypes were over 98%homologous to VP7 and VP4 genes of GPRV CH-1 strain.It was speculated that the new serotypes were resulted from the evolution of CH-1 strain.The average evolutionary rates of VP7 and VP4 genes were 1.93×10^-4 and 1.81×10^-4substitutions/site/year,respectively.In addition,VP7 had 1 positive selected site,while VP4had 6 positive selected sites and 6 negative selected sites respectively.The analysis of positive selected sites is important on the recognition of immune escape,resistance mutation and disease prevention and treatment.VP4 also endured a certain degree of negative selected pressure during adaptive evolution,which ensured the stability of RNA virus.The visual LAMP assay established in this study has the advantages of low cost,fast response,high specificity and sensitivity compared with the conventional PCR.The fluorescent quantitative PCR verified its reliability and assured it as an effective tool to detect GPRV in the early clinical stage,thus achieving efficient monitoring of the disease.G/P serotype identification through stool samples of giant pandas discovered the epidemic serotype of giant pandas was G2P[8],and phylogenetic analyses speculated it was caused by the evolution of GPRV strain CH-1.Taken together,it is of great value to master the molecular epidemic trend of GPRV and analyze its genetic evolution for the development and evaluation of vaccines.