3型腺病毒免提取核酸重组酶介导的等温扩增实时荧光检测方法的建立及应用

Development and evaluation of real-time fluorescence recombinase aided amplification assay without extracting nucleic acid for detection of adenovirus type 3

目的 建立3型腺病毒(human adenovirus 3,HAdV-3)免提取核酸的重组酶介导的等温扩增(recmbinase acid amplification,RAA)实时荧光检测方法。方法 根据HAdV-3基因保守序列分别设计1对引物和1条探针,通过对免提核酸的条件摸索和优化,建立1种免提取核酸,重组酶介导的等温扩增实时荧光检测方法;通过对培养的毒株进行系列稀释,免提取核酸分析该方法的灵敏度;通过检测其他呼吸道病毒的原始样本,评价该方法的特异性;并检测HAdV-3临床样本,与传统的提核酸实时荧光定量PCR方法进行比较。结果 本研究建立的免提取核酸实时荧光RAA方法对10倍系列稀释的HAdV-3毒株进行检测,和实时荧光定量PCR方法相比,灵敏度相当,对应的最低浓度稀释样本的Ct值为36.87,对常见其他型别的呼吸道病毒检测无交叉反应。两种方法对56例HAdV-3阳性临床样本同时检测,结果完全一致。结论 本文首次报道了免提取核酸的实时荧光RAA检测HAdV-3的方法,该方法具有较高的灵敏度和特异性,适合应用于临床实验室及基层单位快速检测HAdV-3。

Objective To establish a real-time fluorescence recombinase acid amplification(RAA)method for the detection of adenovirus type 3(HAdV-3)without extraction nucleic acid.Methods According to the conserved sequence of adenovirus type 3 gene,a pair of primers and a probe were designed,and a real-time fluorescence RAA without extracting nucleic acid was established and optimizing the condition of DNA-free extraction.The sensitivity of the method was analyzed by a series of dilution and the specificity of the method was evaluated by detecting the original samples of other respiratory viruses.The clinical samples of HAdV-3 were detected and compared with the traditional real-time fluorescence quantitative PCR method for nucleic acid extraction.Results The sensitivity of the real-time fluorescence RAA method was as high as that of qPCR in the detection of 10 series diluted HAdV-3 strains.The highest corresponding CT value of qPCR was 36.87.The sensitivity of the real-time fluorescence RAA method was similar to that of the real-time fluorescence quantitative PCR method.There was no cross-reaction to other common types of respiratory viruses.The two method were used to detect 56 clinical samples at the same time,and the result were completely consistent.Conclusions We provide the first report of the real-time fluorescent RAA assays for the detection of HAdV-3 without extracting nucleic acid and it has high sensitivity and specificity.Is suitable for rapid detection of HAdV-3 in clinical laboratories and on-site unite.