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Rapid detection of equine coronavirus by reverse transcription loop-mediated isothermal amplification

Author:
Nemoto, Manabu, Morita, Yoshinori, Niwa, Hidekazu, Bannai, Hiroshi, Tsujimura, Koji, Yamanaka, Takashi, Kondo, Takashi
Source:
Journal of virological methods 2015 v.215-216 pp. 13-16
ISSN:
0166-0934
Subject:
Coronavirinae, RNA, equipment, feces, horses, monitoring, pathogens, quantitative polymerase chain reaction, rapid methods, reverse transcriptase polymerase chain reaction, reverse transcription loop-mediated isothermal amplification
Abstract:
A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid detection of equine coronavirus (ECoV). This assay was conducted at 60°C for 40min. Specificity of the RT-LAMP assay was confirmed using several equine intestinal and respiratory pathogens in addition to ECoV. The novel assay failed to cross-react with the other pathogens tested, suggesting it is highly specific for ECoV. Using artificially synthesized ECoV RNA, the 50% detection limit of the RT-LAMP assay was 10¹.⁸copies/reaction. This is a 50-fold greater sensitivity than conventional reverse transcription polymerase chain reaction (RT-PCR) assays, but a 4-fold lower sensitivity than quantitative RT-PCR (qRT-PCR) assays. Eighty-two fecal samples collected during ECoV outbreaks were analyzed. ECoV was detected in 59 samples using the RT-LAMP assay, and in 30 and 65 samples using RT-PCR or qRT-PCR assays, respectively. Although the RT-LAMP assay is less sensitive than qRT-PCR techniques, it can be performed without the need for expensive equipment. Thus, the RT-LAMP assay might be suitable for large-scale surveillance and diagnosis of ECoV infection in laboratories with limited resources.
Agid:
6026666