Application of Pyrosequencing Technology in Confirmation of Swine Influenza A H1N1 Virus

Sun Tao1,Zhang Taixiang2,Xu Biao1,Liang Chengzhu1,Ling Zongshuai1,Zhu Laihua1,Yue Zhiqin1(1.Shandong Exit-Entry Inspection and Quarantine Bureau,Qingdao 266002;2.Weifang Exit-Entry Inspection and Quarantine Bureau,Weifang 261041;3.Jinan Exit-Entry Inspection and Quarantine Bureau,Jinan 250014)

swine influenza A virus H1N1;squence analysis;Pyrosequecing

Objective To establish a new molecular method for simultaneoulsly rapid detection and confirmation of subtype H1N1 swine influence virus by using sequence analysis combined with pyrosequencing technology.Methods According to the published sequences in GenBank,two amplification primers and one sequencing primer were designed from conserved regions of the H1HA and N1NA genes.Total SIV RNA was extracted from chorioallantois fluid infected with H1N1 swine influence virus,and RT-PCR were developed for conserved regions of HA and NA gene respectively.The RT-PCR products were pysrosequenced by pyrosequencing.The specificity and the reproductivity were tested by using the amplification primer andthe sequencing primer.The method was used to detect the 221 clinical samples and compared to the viruse isolation and real-time PCR.Results Two nucleotide segments which characterized by H1N1 were abtained by sequence analysis.And then the two segments were confirmed as H1N1 swine influence virus by pyrosequeing.The reproducibility result showed 100% in agreement and no cross-reaction was discovered with other swine disease vaccines.The method was used to detect the 221 clinical samples and the results were 96.8% in agreement with those of virus isolation in embryonated eggs.Compared to the Taqman fluorescent quantitation method,they showed 90.3% agreement.Conclusion Pyrosequencing technology based on sequences analysis can be used to confirm the subtype H1N1 swine influenza A virus.