基于数字微流控技术的上呼吸道易感病毒多靶标快速检测方法的建立

Establishment of multi-target rapid detection method of upper respiratory susceptible viruses based on digital microfluidic technology

目的建立一种基于数字微流控RT-qPCR芯片技术的上呼吸道易感病毒多靶标快速检测方法。方法根据新型冠状病毒(2019-nCOV)、甲型流感病毒(FluA)、乙型流感病毒(FluB)、冠状病毒(SARS-CoV)、中东呼吸综合征冠状病毒(MERS-CoV)的保守区域,设计特异性引物和检测探针,通过将引物和探针预存到数字微流控芯片中,建立检测上述5种上呼吸道病毒的数字微流控RT-qPCR芯片检测技术;评价预存了引物的数字微流控RT-qPCR芯片的检测下限和特异性;使用20例临床样本以PCR仪检测结果作为对照组评价数字微流控RT-qPCR芯片的检测性能。结果建立的2019-nCOV、FluA、FluB、SARS-CoV、MERS-CoV的数字微流控RT-qPCR芯片检测下限为12拷贝/反应,而未使用数字微流控的RT-qPCR法检测下限为15拷贝/反应,两个方法的检测下限基本一致。对于临床样本提取的核酸样品,数字微流控RT-qPCR芯片检测结果均是阴性无非特异性扩增。同时使用RT-qPCR法和数字微流控RT-qPCR芯片法对20例临床样本分别进行5个项目的临床对比测试,共100个测试,结果显示为数字微流控RT-qPCR芯片灵敏度达94%,特异性为100%。使用SPSS对两个方法进行一致性分析,结果显示为两个方法具有高度的一致性(Kappa=0.962,P<0.05)。结论基于数字微流控RT-qPCR芯片技术建立了上呼吸道易感病毒多靶标快速检测方法,为临床早期鉴别呼吸道病原体提供了新的检测手段。

Objective Provide a digital microfluidic RT-qPCR chip for rapid detection of several upper respiratory diseases.MethodsSeveral specific primer-probe sets were designed according to the conserved sequences of 2019 novel coronavirus(2019-n COV),influenza A virus(Flu A),influenza B virus(Flu B),severe acute respiratory syndrome coronavirus(SARS-Co V),Middle East respiratory syndrome coronavirus(MERS-Co V),and then packaged into a digital microfluidic chipwhich allowed simultaneous detection of five upper respiratory tract pathogens with the help of reverse transcriptionquantitative PCR(RT-q PCR)technology.In the meanwhile,the detection limit,specificity and sensitivity of this digitalmicrofluidic chip were evaluated base on the clinical specimens,plasmids and unrelated pathogens.ResultsTheestablished digital microfluidic RT-q PCR chip for 2019-n COV,Flu A,Flu B,SARS-Co V,MERS-Co V had a detection limitof 12 copies/reaction,while the detection limit of the RT-q PCR method without digital microfluidics was 15 copy/reaction;the detection limit of the two methods was basically the same.For nucleic acid samples extracted from clinical samples,thedetection results of digital microfluidic RT-q PCR chips were all negative without non-specific amplification.At the same time,the RT-qPCR method and the digital microfluidic RT-qPCR chip method were used to carry out clinical comparativetests of 5 items in 20 clinical samples,total 100 tests.The results showed that the sensitivity of the digital microfluidic RT-q PCR chip reached 94%,the specificity was 100%.SPSS was used to analyze the consistency of the two methods,and theresults showed that the two methods had a high degree of consistency(Kappa=0.962,P<0.05).ConclusionBased ondigital microfluidic RT-q PCR chip technology,a multi-target rapid detection method of upper respiratory tract susceptiblevirus was established,which could provide a new detection method for early clinical identification of respiratory pathogens.