Establishment And Evaluation Of Whole Genome Amplification Methods For Domestic Enterovirus Epidemic Strains

BACKGROUND and AIM:Recently,Hand,foot and mouth disease(HFMD)has caused millions of infections in China and Southeast Asian countries,leading to serious public health problems.The pathogens of HFMD that are highly prevalent in China are enterovirus EV-71 and Coxsackie virus CV-A16.In-depth study of the genomic characteristics and evolutionary rules of enterovirus will play a vital role in the prevalence and prevention of the disease of the foot and mouth.Although the current clinical diagnosis of enterovirus is one of the diagnostic indicators for detecting viral nucleic acid,it only confirms the local characteristic fragments of the virus,and cannot obtain the whole genome sequence,and cannot support the in-depth study of enterovirus.Comparative analysis must first find primers that can amplify our Chinese enterovirus epidemic strains.In this study,we investigated the genome-wide sequence of Chinese enterovirus epidemic strains in recent years,and established a universal amplification method that can obtain the enterovirus sequence in China,laying a foundation for the epidemiological analysis and pathogenic mechanism of HFMD in the future..METHODS:The whole genome sequence of enterovirus epidemic strains reported in China in the past five years was collected as a reference sequence,and the reference sequences were compared and analyzed.The universal primers were designed in the conserved region to the primers of the enterovirus strains preserved in the laboratory.Screen and verify.The enterovirus coding region and the 3'UTR region were amplified using optimized 3'RACE,long-distance PCR,and the 5'UTR region of the enterovirus was amplified using degenerate primers to obtain the whole genome sequence of the enterovirus.The amplified products were subjected to deep sequencing using Ion Torrent PGM to verify and evaluate the amplification method.Further,a standard curve of enterovirus copy number was established by qRT-PCR,and a series of gradient virus concentration original virus liquid samples were constructed,and the detection limit of the amplification method was explored,and the sensitivity of the detection method was evaluated.On this basis,a mixture of two enteroviruses was simulated by a series of molar ratios to simulate mixed enterovirus infection,and the specific amplification method was established by our universal amplification method to verify the specificity of the amplification method.We then validated the amplified method using a validated recombinant strain of enterovirus.RESULTS:In this study,the general amplification method of domestic enterovirus epidemic strains was established by optimizing the primer sequence and reaction system conditions.The whole genome sequence of enterovirus was obtained by the second-generation sequencing experiment,which reached more than one thousand times of sequencing depth.Further,16 strains of EV-71 or CV-A16 enterovirus were used for verification,and the whole genome sequence of enterovirus with high sequencing depth was obtained.Both amplification methods have the same specificity and accuracy in a single enterovirus infection compared to specific primer segmentation amplification.In the evaluation of the lower limit of detection,when the viral load of the enterovirus is as low as 10~4,the amplification method can still obtain the whole genome sequence information.The mixed virus infection was simulated by a series of ratios.The amplification method was able to obtain the whole genome sequence of two enteroviruses simultaneously in a 1:10 ratio,and the proportion of mixed virus infections was basically estimated by sequencing the reads.A universal amplification method for domestic enterovirus epidemic strains Amplification of recombinant enteroviruses can completely reveal the recombination of enteroviruses.CONCLUSION:This study established a universal genome-wide amplification method for enterovirus epidemic strains in recent years.It shows high sensitivity in the extraction and amplification of enteroviruses in virus culture medium,and can reflect the real situation of mixed virus infection and recombinant virus.The amplification method will provide an efficient,accurate and convenient solution for the complete sequence amplification and virulence research of enteroviruses in the future.