Detection of respiratory syncytial virus genome by subgroups-A, B specific reverse transcription loop-mediated isothermal amplification (RT-LAMP).
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.
Ushio
Masanobu
M
Department of Pediatrics, Tokyo Medical College, Shinjuku-ku, Tokyo, Japan.
Yui
Ikuko
I
Yoshida
Naoko
N
Fujino
Motoko
M
Yonekawa
Toshihiro
T
Ota
Yoshinori
Y
Notomi
Tsugunori
T
Nakayama
Tetsuo
T
D016679:Genome, Viral; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D018357:Respiratory Syncytial Virus Infections / Q000175:diagnosis* / Q000821:virology*; D012136:Respiratory Syncytial Viruses / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D012367:RNA, Viral