Detection of equine rotavirus by reverse transcription loop-mediated isothermal amplification (RT-LAMP).
Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was applied to detection of equine rotavirus. Because equine rotavirus of the single P genotype, P[12], is predominant in the equine population worldwide, an RT-LAMP primer set was designed to target the genotype P[12] sequence and thus detect equine rotavirus. The detection limit of the RT-LAMP assay was 10(3) copies of viral RNA, whereas that of semi-nested RT-PCR for genotype P[12] was 10(5) copies. The RT-LAMP assay specifically amplified genotype P[12] but did not amplify the other P genotype strains. The RT-LAMP assay did not amplify any pathogens related to equine intestinal disorder other than rotavirus. Using 96 diarrheal stools, the RT-LAMP assay detected equine rotavirus in 58 samples, whereas semi-nested RT-PCR only detected equine rotavirus in 25 samples. The RT-LAMP assay did not detect equine rotavirus with fecal samples collected from nine healthy foals. These results indicate that the RT-LAMP assay is specific for equine rotavirus and more sensitive than semi-nested RT-PCR. Because it is easy to manipulate without the need for a thermal cycler or gel electrophoresis, the RT-LAMP assay should be applicable to diagnosis of equine rotavirus infections in diagnostic laboratories.
Nemoto
Manabu
M
Epizootic Research Center, Equine Research Institute, Japan Racing Association, Shiba, Shimotsuke, Tochigi, Japan. nemoto_manabu@epizoo.equinst.go.jp
Imagawa
Hiroshi
H
Tsujimura
Koji
K
Yamanaka
Takashi
T
Kondo
Takashi
T
Matsumura
Tomio
T
D000818:Animals; D001424:Bacterial Infections / Q000175:diagnosis / Q000662:veterinary; D001483:Base Sequence; D002213:Capsid / Q000276:immunology; D036022:Capsid Proteins / Q000276:immunology; D017931:DNA Primers; D003967:Diarrhea / Q000662:veterinary / Q000821:virology; D005784:Gene Amplification; D016679:Genome, Viral; D006734:Horse Diseases / Q000175:diagnosis / Q000821:virology*; D006736:Horses; D007410:Intestinal Diseases / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D048348:Reverse Transcription; D012401:Rotavirus / Q000235:genetics* / Q000302:isolation & purification; D012400:Rotavirus Infections / Q000175:diagnosis / Q000662:veterinary*
D036022:Capsid Proteins; D017931:DNA Primers