七重呼吸道病毒液相芯片核酸检测技术的初步建立

Preliminary Establishment of Nucleic Acid Detection Technology of Heptathyrovirus by Liquid Chip

由呼吸道病毒引起的疾病严重威胁着人类的生命健康,为了建立一种快速高通量的呼吸道病毒核酸检测方法,本研究将多重PCR技术同液相芯片技术结合起来,针对呼吸道合胞病毒A型和B型、乙型流感病毒Victoria系和Yamagata系、甲型流感病毒H1型和H3型以及新冠病毒等常见的七种呼吸道病毒,初步建立了七重呼吸道病毒液相芯片核酸检测技术,评价了方法的特异性、敏感性和重复性,并使用来自安徽省疾控的25份临床急性期样本核酸对方法进行验证。结果显示,建立起的基于液相芯片多重核酸检测方法可特异性识别七种目标呼吸道病毒的靶基因序列,与包括副流感病毒在内的9种非目标呼吸道病毒无交叉反应。对七种病毒核酸进行十倍稀释液相检测,其中H3、BV、RSVB可以检出102拷贝/μL,BY、RSVA和SARS-CoV-2可以检出103拷贝/μL,对H1的检测限为104拷贝/μL。25份样本核酸检测结果与实际相符。结果表明,本研究建立的七重呼吸道病毒液相芯片核酸检测技术具有特异性强、敏感性高、稳定性好等特点,可用于临床样本的快速检测,为呼吸道类传染病的液相芯片诊断奠定了实验室基础。

Diseases caused by respiratory viruses are a serious threat to human life and health.In order to establish a rapid and high-throughput nucleic acid detection method for respiratory viruses,this study combined the multiplex PCR technology with the liquid chip technology.For seven common respiratory viruses,including respiratory syncytial viruses A and B,influenza B viruses Victoria and Yamagata,influenza A viruses Hl and H3,and novel coronavirus,a liquid chip nucleic acid detection technology for Heptavirus was initially established.The specificity,sensitivity and repeatability of the method were evaluated.Nucleic acids from 25 acute phase-clinical samples from Anhui Province Center for Disease Control were used to verify the method.The results showed that the multiplex nucleic acid detection method based on liquid chip can specifically identify the target gene sequences each of the seven respiratory viruses,and showed no cross-reaction with nine non-target respiratory viruses including parainfluenza virus.Sensitivity test showed that H3,BV and RSVB could detect 10°copies/μL,BY,RSVA and SARS-CoV-2 could detect 10°copies/μL,and the detection limit for Hl was 10*copies/μL.The nucleic acid test results of 25 samples were consistent with the real situation.The results showed that the HPLC nucleic acid detection technology of heptathyronucleoviruses established in this study has the characteristics of strong specificity,high sensitivity and good repeatability,which can be used for rapid detection of clinical samples,and laid a laboratory foundation for the liquid chip diagnosis of respiratory viral infectious diseases.