DDPCR Identification Of Influenza-like Clinical Samples And Sequence Analysis Of One H7N9 Virus

Influenza A(IVAs)is an acute respiratory infection disease caused by influenza A virus.The genes of the influenza virus are prone to reassortment,especially the influenza A virus has the strongest variability.Influenza A viruses not only cause severe respiratory symptoms,but can also cause pandemics worldwide,as they are widely known.In recent years,there have been outbreaks of the H7N9 subtype,which has brought certain threats to people's physical and mental health and social stability.In view of the increasing proportion of people suffering from influenza virus and the number of hospital visits,the isolation and detection of influenza viruses is partic?iarly important.The first part is the detection of influenza virus nucleic acid molec?ies using Real-Time PCR and Droplet Digital PCR(DDPCR)techniques.We extracted viral RNA from clinical respiratory tract infection samples and amplified it by one-step PCR.According to the standard curve and standard product amplification curve,the positive res?its were screened by comparing the Cq values of the samples and standard products.In view of the low positive rate of Real-Time PCR,we designed primer sequences of type A(A1 subtype,A3 subtype,A5 subtype and A7 subtype)and type B,and tested all samples with DDPCR.From the res?its,we can get the specific copy number of samples.Comparing the number of positive samples in Real-Time PCR and DDPCR,we found that the number of positives obtained by DDPCR was much higher than that of Real-Time PCR.Because of the time relationship and in order to further identify the influenza virus infection,we finally selected the strain with the highest copies to be inoc?iated into MDCK cells,and the cells developed lesions after subc?iture for several generations.It is concluded that the detection sensitivity of DDPCR is better.The second part is the isolation and identification of a H7N9 virus.The sample with TRIZOL added was taken from the CDC of Kunming City.First,the nucleic acid was extracted and then amplified by PCR reaction.The gene fragments of HA and NA were obtained by PCR with specific primers.After BLAST,it was found to have high similarity with another H7N9 virus.Then we performed PCR detection on another six fragments of this virus.Comparative analysis showed that their nucleotide homology in HA,NA,M,NS,NP,PA and PB1 genes was more than 99%,the homology of PB2 gene is 100%.Evolutionary tree analysis also showed that they came from the same evolutionary branch and belonged to the Yangtze River Delta pedigree.Based on the analysis of mutation of family loci and epidemiological investigation,we found that the two viruses originated from a pair of mother and daughter,and the mother had the possibility of being infected by her daughter.Finally,we identified the virus as H7N9 subtype.We detected R292K mutation in the NA gene of the virus,indicating oseltamivir resistance.Through this experiment,we isolated a strain of H7N9 virus and found that H7N9 infection has limited transmission possibility between people.