Development of a One Step Reverse Transcription-Loop-Mediated Isothermal Amplification System for a Highly Sensitive Detection of Rabbit Hepatitis E Virus.

BACKGROUND: Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in areas with poor sanitation. Rabbit is one of important animal reservoirs of the virus. Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) system is a novel nucleic acid amplification assay, for which the specificity and sensitivity are much higher than that of conventional PCR. However, previously reported RT-LAMP system cannot detect rabbit HEV, which was identified recently and seems to be another potential source of human HEV infection.

METHODS: In this study, genotype 4 HEV strains and rabbit HEV strains were used to verify the applicability of the previously reported HEV RT-LAMP. A new specific one step RT-LAMP system was developed and evaluated to amplify rabbit HEV RNA. In order to test the sensitivity of the newly-developed assay, serial dilutions (from 5 x 103 to 5 x 10-1/µL) of the rabbit HEV RNA were used as template to be detected by RT-LAMP, real-time RTPCR, and nested RT-PCR. Specificity of the new assay was further evaluated using HAV, HBV, and HCV. With this new assay, 46 rabbit fecal samples were retrospectively investigated with real-time RT-PCR and nested RTPCR in parallel.

RESULTS: The detection limit of this newly-developed RT-LAMP assay reached as low as 10 copies/reaction and no cross-reactivity was observed with other hepatitis viruses including hepatitis A, B, and C virus, which indicated that this assay has much higher sensitivity and specificity than that of nested RT-PCR and real time RT-PCR. Furthermore, among 46 rabbit fecal samples, there were four positive samples determined by those three assays and one positive sample only detected by HEV RT-LAMP and real-time RT-PCR.

CONCLUSIONS: Using a combination of sensitivity, specificity, and evaluation of clinical samples, this study provides the first data on the usefulness of RT-LAMP in the diagnosis of rabbit HEV RNA.