Rapid Detection of Cactus virus X in Pitaya by Efficient Reverse Transcription Loop-Mediated Isothermal Amplification

Cactus virus X (CVX) is a recently reported virus in pitaya that can significantly reduce crop yield and quality. According to the CVX genome sequence in GenBank (accession no. NC_002815), primers for reverse transcription loop-mediated isothermal amplification (RT-LAMP) and RT-PCR were designed to detect this virus. Our results indicated that the best LAMP reaction conditions were as follows: 1.2-1.6 μM of internal primers, 0.2-0.25 μM of external primers, 0.4-0.8 μM of loop primers, and incubation at 62°C/63°C for 30 min. Our data also showed that the LAMP primers specifically targeted CVX and resulted in the typical waterfall-like bands after gel electrophoresis and sigmoidal amplification curves. Ten-fold serial dilutions of CVX cDNA indicated that LAMP is much faster and at least 10-100 times more sensitive than RT-PCR in detecting this virus. In field identification using either RT-LAMP or RT-PCR, the majority of samples (86.7%) was positive for CVX. This is the first report of CVX diagnosis in China and of the use of the efficient RT-LAMP technology to identify CVX in the field. RT-LAMP meets the requirements for rapid diagnosis analysis needed for control and management of this emerging pathogen.