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J Virol Methods. 2014 Sep;206:84-8. doi: 10.1016/j.jviromet.2014.06.005. Epub 2014 Jun 9.

New method for the visual detection of human respiratory syncytial virus using reverse transcription loop-mediated amplification.

Author information

1
College of Life and Environmental Science, Shanghai Normal University, Shanghai, China; Pathogen Diagnostic Center, Institut Pasteur of Shanghai, Chinese Academy of Science, Shanghai, China.
2
Dujiangyan Medical Center, Dujiangyan City, Sichuan, China.
3
Pathogen Diagnostic Center, Institut Pasteur of Shanghai, Chinese Academy of Science, Shanghai, China; Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu, China.
4
Pathogen Diagnostic Center, Institut Pasteur of Shanghai, Chinese Academy of Science, Shanghai, China.
5
College of Life and Environmental Science, Shanghai Normal University, Shanghai, China.
6
Pathogen Diagnostic Center, Institut Pasteur of Shanghai, Chinese Academy of Science, Shanghai, China. Electronic address: lanke@sibs.ac.cn.

Abstract

Human respiratory syncytial virus (HRSV) is a seasonal respiratory pathogen that causes respiratory infection in children and the elderly. A new, reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid (within 1h), simultaneous detection of A and B group HRSV. Primers specific for groups A and B were designed to amplify the N and L genes of HRSV, respectively. A fluorescent dye, calcein, was used as an indicator for the endpoint visual detection and/or real-time amplification of HRSV RNA. The detection limit of the new method was 281.17 50% tissue culture infective doses (TCID50)/ml for HRSV A and 1.58 TCID50/ml for HRSV B. To evaluate the validity of this method, a comparison with RT-PCR was performed using 77 nasopharyngeal swabs as samples. Both RT-LAMP and RT-PCR detected HRSV in 38 HRSV samples, yielding a positive rate of 49%. Of the RT-LAMP positive samples, 36 (95%) were also positive by RT-PCR, while two were negative by RT-PCR. Among the 36 RT-LAMP and RT-PCR positive samples, 11 belonged to HRSV group A, while 25 belonged to group B. The results show that the new RT-LAMP is simple, rapid and well suited for HRSV diagnosis, especially in a limited-resource setting.

KEYWORDS:

Field test; Human respiratory syncytial virus; Molecular diagnosis; RT-LAMP; Visual detection

PMID:
24925133
DOI:
10.1016/j.jviromet.2014.06.005
[Indexed for MEDLINE]
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