title: Rapid detection of equine coronavirus by 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 40 min. 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 10(1.8)copies/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.
authors: Epizootic Research Center, Equine Research Institute, Japan Racing Association, 1400-4 Shiba, Shimotsuke, Tochigi 329-0412, Japan. Electronic address: nemoto_manabu@epizoo.equinst.go.jp.
authors: Manabu
authors: M
authors: Nemoto
authors: Tokachi Draft Horse Clinic, Nishi 13, Minami 9-1, Obihiro, Hokkaido 080-0023, Japan.
authors: Yoshinori
authors: Y
authors: Morita
authors: Epizootic Research Center, Equine Research Institute, Japan Racing Association, 1400-4 Shiba, Shimotsuke, Tochigi 329-0412, Japan.
authors: Hidekazu
authors: H
authors: Niwa
authors: Epizootic Research Center, Equine Research Institute, Japan Racing Association, 1400-4 Shiba, Shimotsuke, Tochigi 329-0412, Japan.
authors: Hiroshi
authors: H
authors: Bannai
authors: Epizootic Research Center, Equine Research Institute, Japan Racing Association, 1400-4 Shiba, Shimotsuke, Tochigi 329-0412, Japan.
authors: Koji
authors: K
authors: Tsujimura
authors: Epizootic Research Center, Equine Research Institute, Japan Racing Association, 1400-4 Shiba, Shimotsuke, Tochigi 329-0412, Japan.
authors: Takashi
authors: T
authors: Yamanaka
authors: Epizootic Research Center, Equine Research Institute, Japan Racing Association, 1400-4 Shiba, Shimotsuke, Tochigi 329-0412, Japan.
authors: Takashi
authors: T
authors: Kondo
mesh_terms: D000818:Animals; D017934:Coronavirus / Q000302:isolation & purification*; D018352:Coronavirus Infections / Q000175:diagnosis* / Q000821:virology*; D003429:Cross Reactions; D005243:Feces / Q000821:virology; D006734:Horse Diseases / Q000175:diagnosis* / Q000821:virology; D006736:Horses; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors
keywords: Diagnosis; Equine coronavirus; RT-PCR; Real-time RT-PCR; Reverse transcription loop-mediated isothermal amplification