A simplified crude sap-based detection of cucumber mosaic virus in chilli germplasm through reverse transcription recombinase polymerase amplification assay (RT-RPA)
摘要
Chilli cultivation is under constant threat by many pests and diseases, and about 35 viruses are known to infect chilli globally. Among the viruses, the cucumber mosaic virus (CMV) is the most significant constraint to chilli production worldwide. Detection of CMV in infected plants, reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) methods are employed frequently. These conventional methods have been time consuming, laborious and expensive for detection of samples in low resource laboratories. In order to provide alternatives for the conventional methods currently various isothermal amplification methods have been employed. The present study exhibits the optimisation of the reverse transcription recombinase polymerase amplification (RT-RPA) assay by eliminating the steps of ribonucleic acid extraction, cDNA conversion, and the use of a thermal cycler. The optimized RT-RPA assay successfully detected CMV at concentrations as low as 10–10 (100 fg) and 10–9 dilutions of both RNA and crude sap templates, demonstrating high sensitivity comparable to the routine RT-PCR assay. Specificity tests confirmed that the RT-RPA assay did not exhibit cross-reactivity with other common chilli-infecting viruses, such as chilli leaf curl virus (ChiLCV), chilli veinal mottle virus (ChiVMV), capsicum chlorosis virus (CaCV), and groundnut bud necrosis virus (GBNV). Its ability to function efficiently with crude sap as a template further enhances its field applicability, making it a promising tool for early virus detection in low-resource laboratories and onsite diagnostics. The integration of such isothermal amplification techniques could revolutionize plant virus detection and management strategies, ultimately aiding in the mitigation of crop losses and ensuring food security.