Detection of respiratory syncytial virus genome by subgroups-A, B specific reverse transcription loop-mediated isothermal amplification (RT-LAMP)

✍ Scribed by Masanobu Ushio; Ikuko Yui; Naoko Yoshida; Motoko Fujino; Toshihiro Yonekawa; Yoshinori Ota; Tsugunori Notomi; Tetsuo Nakayama

• Publisher: John Wiley and Sons
• Year: 2005
• Tongue: English
• Weight: 276 KB
• Volume: 77
• Category: Article
• ISSN: 0146-6615
• DOI: 10.1002/jmv.20424

✦ Synopsis

Abstract

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 RT‐PCR, 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, Bgl 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. J. Med. Virol. 77:121–127, 2005. © 2005 Wiley‐Liss, Inc.