高灵敏度等温扩增法可视化检测血液中的病毒核酸

Highly sensitive and visible detection of virus by loop-mediated isothermal amplification

目的探讨便携式、可灵敏、快速检测血液标本中病毒核酸的方法。方法以HBV病毒为例,针对其基因组保守序列设计特异性引物,采用环介导等温扩增法(LAMP)对待测HBV标本做核酸目标序列扩增,以高灵敏双链DNA嵌合荧光染料为指示剂,根据荧光信号指示扩增反应产物的有无;将本法与实时荧光PCR法对临床标本做HBV检测的对照分析。结果建立了血液病毒核酸的可视化检测方法,研制出扩增检测一体化仪器;灵敏度达到可在<1 h完成10 cp/管的HBV核酸扩增反应和产物检测。62例实时荧光PCR检测为HBV阳性的临床标本,本法亦都检测为阳性;而从42例实时荧光PCR检测为HBV阴性的临床标本中,本法检出了6例HBV阳性。结论所建立的方法和装置可实现高灵敏度单管快速检测血液病毒核酸,为特殊现场环境中的血液安全性筛查提供了新的手段。

Objective To develop a sensitive, rapid, and convenient method for detection of viral nucleic acid in blood samples, such as HBV. Method The primers were designed according to the conservative regions of HBV genome, and the loop-mediated isothermal amplification （LAMP） was carried out to amplify the target sequences of HBV. SYBR Green I was used as the indicator for the PCR products. The assay would be scored as positive if a fluorescent signal was detected. Resuits We have successfully developed a method for visible detection of blood virus and invented a portable device that combined the amphfication and detection in one platform. The sensitivity of our proposed method is 10 copies of HBV per tube. The amplification and detection process could be done within 1 hour. In the testing of 62 elinieal specimens, which have been determined as HBV positive by quantitative PCRIall specimens were positive and an accuracy of 100 % was achieved. We also detected 6 positive specimens from 42 clinical samples which have been determined as HBV negative by quantitative PCR. Conclusion The established method and device are highly sensitive and can realize single-tube detection of the blood virus, providing a powerful tool for point-of-care testing in resource-limited circumstances.