Development of RT-LAMP for rapid visual detection of

Abstract:

[Objective] A rapid, simple and sensitive visual reverse transcription loop-mediated isothermal amplification method was established to detect group A rotavirus. [Methods] The method employed a set of four specially designed primers that recognize six distinct sequences of the VP6 gene for amplification of nucleic acid under isothermal conditions at 64 °C for one hour. The amplification process of RT-LAMP was monitored by the addition of Calcein dye prior to amplification. The specificity and sensitivity of the RT-LAMP assay was assessed and the assay was further evaluated with 90 clinical specimens of diarrhea patients with RT-LAMP and RT-PCR detection. [Results] The results showed that the RT-LAMP was able to achieve a sensitivity of 103 copies/μl with a high specificity. The detection limit of RT-LAMP was 100-fold higher than that of RT-PCR. Positive rate of RT-LAMP in analysis of clinical specimens was approximately the same as that of RT-PCR as well. [Conclusion] The RT-LAMP assay has been proven to be a rapid, sensitive, specific and visual method for detection of the group A rotavirus, and that the RT-LAMP assay is potentially useful for the field detection and rapid detection on spot.