Advancing broad bean true mosaic virus detection using conventional RT-PCR and real-time RT-PCR with novel primer set design.

ABSTRACT

Broad bean true mosaic virus (BBTMV) infects broad beans and peas, reducing yield. As BBTMV is transmitted through broad beans, many countries have implemented regulations to prevent the distribution of infected seeds. Currently, enzyme-linked immunosorbent assay (ELISA) is commonly used to detect BBTMV. While the PCR-based method is preferred for seed virus detection due to its sensitivity and speed. A BBTMV-specific PCR detection method has not yet been reported. A universal detection method currently exists that utilizes reverse transcription PCR (RT-PCR) for the Comovirus genus, to which BBTMV belongs. However, sequence analysis is required for species identification. To address this limitation, we developed and verified RT-PCR detection methods using newly designed BBTMV-specific primers. RT-PCR and real-time RT-PCR with these primers were approximately 5 × 105-106 times more sensitive than ELISA and 100-1000 times more sensitive than previously reported RT-PCR methods. Using RT-PCR and real-time RT-PCR employing these primers, we could detect BBTMV with same sensitivity when more than 3.0 × 105 copies were present per gram of broad bean seeds. Our newly developed detection methods can test for BBTMV with high sensitivity and speed.