Development of an RT-LAMP Assay for Rapid Detection of hippeastrum chlorotic ringspot virus (HCRV)

In order to quickly detect the hippeastrum chlorotic ringspot virus(HCRV), five sets of primers were designed and screened according to the conserved nucleotide sequence of the nucleocapsid (N) protein gene of HCRV, and the RT-LAMP system reaction temperature, time, betaine concentration, dNTP concentration, Mg²⁺ concentration, and concentration ratio of internal and external primers were optimized by single variable method. A detection system of HCRV reverse transcription loop mediated isothermal amplification (RT-LAMP) was established. The results showed that the optimal primer group was P1, the optimal reaction temperature was 64 ℃, and the optimal final concentrations of Betaine, dNTPs, and Mg²⁺ were 0.8, 0.2, 6 mmol/L. The optimal concentration ratio of internal and external primers is 5∶1, and the optimal reaction time is 50 minutes. The test results showed that after staining with SYBR Green I, the optimized RT-LAMP can be directly evaluated by naked eyes, with high specificity and sensitivity, 100 times higher than that of conventional RT-PCR. This study provides a convenient, efficient, and reliable method for the detection of HCRV.