Detection of respiratory syncytial virus genome by subgroups-A, B specific reverse transcription loop-mediated isothermal amplification (RT-LAMP)
Detection of respiratory syncytial virus genome by subgroups-A, B specific reverse transcription loop-mediated isothermal amplification (RT-LAMP)
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DOI：
10.1002/jmv.20424
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发表时间：
2005-09-01
影响因子：
12.7
通讯作者：
Nakayama, T
中科院分区：
文献类型：
--
作者：
Ushio, M;Yui, I;Nakayama, T
呼吸道合胞病毒（RSV）感染的年度季节性爆发发生在每年冬天。大多数患者通过鼻咽分泌物（RSV）蛋白的快速检测试剂盒进行临床诊断，但是已经报道了这种快速检测试剂盒的特异性和灵敏度的一些问题。为了证实这些问题，一个敏感的，特异的，简单的，快速的基于分子的诊断方法，预计将被引入，我们已经开发了一种方法来检测RSV基因组的亚组A和B独立的逆转录环介导的等温扩增（RT-LAMP）。在等温条件下，在提取RNA后60 min内，我们通过RT-LAMP检测到样品中约0.1 TCID 50的RSV亚组的基因组RNA。通过实时浊度计监测特异性DNA扩增，并计算RNA的量。RSV基因组检测在47 50例中的RT-LAMP，42巢式RTPCR，而病毒分离阳性的29和酶联免疫测定（EIA）的34。RT-LAMP A法检出RSV A亚群25例，RT-LAMP B法检出RSV B亚群23例，其中1例为A、B亚群双重感染。用特异性限制性内切酶BgI II消化确认。结果表明，RT-LAMP作为一种有用的诊断工具，用于检测RSV的潜在的临床可行性与巢式RT-PCR相似的高灵敏度。(c)2005 Wiley-Liss，Inc.
Annual seasonal outbreaks of respiratory syncytial virus (RSV) infection occur every winter. Most patients are diagnosed clinically by a rapid detection kit for RSV protein(s) from nasopharyngeal secretion (NPS), but some problems have been reported on the specificity and sensitivity of such rapid detection kits. To ratify these issues, a sensitive, specific, simple, and rapid molecular based diagnostic method is expected to be introduced and we have developed a method to detect the RSV genome of subgroups A and B independently by reverse transcription loop-mediated isothermal amplification (RT-LAMP). We detected the genomic RNA corresponding approximately to 0.1 TCID 50 in the sample by RT-LAMP for both RSV subgroups under isothermal condition within 60 min after extraction of RNA. Specific DNA amplification was monitored by a real-time turbidimeter and the quantity of RNA was calculated. The RSV genome was detected in 47 of 50 NPS by RT-LAMP, and in 42 by nested RTPCR, whereas virus isolation was positive for 29 and enzyme-linked immunoassay (EIA) for 34. RSV subgroup A was detected in 25 by RSV RT-LAMP A, RSV subgroup B in 23 by RSV RT-LAMP B, and dual infection with RSV subgroups A and B was identified in one case. They were confirmed with digestion with a specific restriction enzyme, BgI II. The results showed the potential clinical feasibility of RT-LAMP as a useful diagnostic tool for the detection of RSV with high sensitivity similar to nested RT-PCR. (c) 2005 Wiley-Liss, Inc.