Detection of Equine Rotavirus by Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP)

概要

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Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was applied to the 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 103 copies of viral RNA, whereas that of semi-nested RT-PCR for genotype P[12] was 105 copies. The RT-LAMP assay specifically amplified genotype P[12] but did not amplify the other P genotype strains. And the RT-LAMP assay did not amplify all 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 the diagnosis of equine rotavirus infections in diagnostic laboratories.