Simultaneous visual detection of multiple viral amplicons by dipstick assay
Simultaneous visual detection of multiple viral amplicons by dipstick assay
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DOI：
10.1128/jcm.43.8.4015-4021.2005
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发表时间：
2005-08-01
影响因子：
9.4
通讯作者：
Lee, H
中科院分区：
文献类型：
--
作者：
Dineva, MA;Candotti, D;Lee, H
开发了一种灵敏、简单且不依赖于仪器的方法，用于通过试纸条目视检测和鉴定多个核酸扩增子。该方法基于试纸条膜上的核酸杂交和允许目视检测的信号放大系统。以B型肝炎病毒（HBV）、丙型肝炎病毒（HCV）和人类免疫缺陷病毒1型（HIV-1）为模型分析物，证明目视试纸条检测结合多重逆转录（RT）-PCR扩增病毒核酸提供了一种特异、灵敏的检测方法。RT-PCR产物的检测试纸的效率类似于一个复杂的，昂贵的，和仪器依赖性的方法的基础上荧光寡核苷酸探针。试纸条结合多重RT-PCR对HBV DNA、HCV RNA和HIV-1 RNA的检测限分别为50、125和500 IU/ml。试纸条检测试剂盒以相似的效率检测来自HBV基因型A至F、HCV基因型1至6和HIV-1亚型A至H以及CRF 02循环重组形式HIV-1的扩增子。对295份临床样本和10份献血者血浆样本的19个样本池的分析表明，多重试纸条检测具有重现性、灵敏度和特异性。因此，多个扩增子的视觉试纸检测为目前用于核酸测试的复杂的仪器依赖性检测方法提供了有吸引力的替代方案。这种新的和敏感的核酸检测方法应增加在资源有限的环境中基因组筛查的可用性及其对近患者检测的适用性。
A sensitive, simple, and instrument-independent method for the visual detection and identification of multiple nucleic acid amplicons by dipstick has been developed. This method is based on nucleic acid hybridization on the dipstick membrane and a signal amplification system to allow visual detection. With hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1) as model analytes, it is demonstrated that the visual dipstick test combined with multiplex reverse transcription (RT)-PCR for the amplification of viral nucleic acid provides a specific and sensitive detection method. The RT-PCR products were detected by the dipstick with an efficiency similar to that of a complex, expensive, and instrument-dependent method based on fluorogenic oligonucleotide probes. The detection limits of the dipstick combined with multiplex RT-PCR were 50, 125, and 500 IU/ml for HBV DNA, HCV RNA, and HIV-1 RNA, respectively. The dipstick assay detected with similar efficiencies amplicons derived from strains of HBV genotypes A through F, HCV genotypes 1 to 6, and HIV-1 subtypes A through H as well as CRF02 circulating recombinant forms of HIV-1. Analysis of 295 clinical samples and 19 pools of 10 plasma specimens from blood donors revealed that multiplex dipstick detection was reproducible, sensitive, and specific. The visual dipstick detection of multiple amplicons thus provides an attractive alternative to complex, instrument-dependent detection methods currently in use for nucleic acid testing. This new and sensitive method for nucleic acid detection should increase the availability of genomic screening in resource-limited settings and its applicability to near-patient testing.