高灵敏可视化核酸试纸条法快速检测HBV DNA

目的：本研究旨在建立一种基于试纸条的快速、灵敏及可视化检测乙型肝炎病毒核酸的方法。方法：利用聚合酶链反应扩增乙肝病毒的保守区,其中上、下游引物的5＇端分别修饰异硫氰酸荧光素和生物素。核酸试纸条上的胶体金以及检测线处分别标记有链霉亲和素以及抗荧光素抗体。将扩增产物与展开液混合后点样,10 min后即可用肉眼判读结果。在优化了展开液成分、上样体积以及上样浓度之后,对该方法的灵敏度进行了评价。最后收集15例阴性样本及33例HBsAg阳性样本,按血清标志物结果进行分类后使用核酸试纸条进行检测,并与实时荧光PCR的结果进行了比较。结果：试纸条检测乙肝病毒核酸的灵敏度为250copies/mL。在临床样本的测定中,该方法与实时荧光定量PCR的特异性均为100%。且两种方法检测不同血清标志物类型的阳性检出率无差异。结论：核酸试纸条技术能够用于乙肝病毒核酸的可视化检测,与实时荧光PCR相比检测速度快,具有较好的灵敏度和特异性,适合流行病学调查以及在基层医院体检使用。

Development of a sensitive,visual nucleic acid dipstick assay for rapid detection of hepatitis B virus DNA

Objective：We aimed to develop a nucleic acid dipstick assay（NADA） for rapid and visual detection of Hepatitis B virus（HBV） with a high specificity and sensitivity.Methods：PCR amplification of HBV conserved sequences was carried out by a pair of primers labeled with FITC and biotin at the 5＇ end respectively.The gold nanoparticles and test zones in the dipstick were modified with anti-fluorescein and strepavidin respectively.PCR products mixed with developing buffer were applied to the sample-loading area of the dipstick and the detection result can be observed 10 min afterward with naked eyes.As the buffer migrates along the strip by capillary action,the PCR products were captured at the test zones by immobilized anti-fluorescein and detected by strepavidin-funtcionalized gold nanoparticles,thus generating characteristic red lines.The excess nanoparticles were captured by immobilized biotinylated albumin at the control zone of the strip and then form another red line.We have optimized the developing buffer as well as the volume and concentration for sample-loading.The sensitivity of the optimized NADA has also been evaluated.To demonstrate the utility of NADA,a total of 48 clinical samples containing 15 negative control and 33 HBsAg positive cases were detected.The results from NADA were further com-pared with that from qPCR.Results：The sensitivity of NADA for visual detection of HBV was 250 copies/mL.In the clinical tests,the specificity of DADA and qPCR was 100%,respectively.There were no differences for the detection rates of discriminating the different serum-marker patterns by NADA and qPCR.Conclusions：Our newly developed NADA could be applied for visual detection of HBV.Compared with qPCR,NADA have the advantages of the rapidity,higher sensitivity and specificity,holding a great promise for wide ap-plications in epidemiological surveys and routine physical examinations in hospitals.