Abstract
Melon yellow spot virus (MYSV) is a new tospovirus which is spreading quickly in Kochi Prefecture, Japan. A PCR-microplate hybridization (PCR-MPH) method for the detection of MYSV from plants (cucumber) and thrips was developed. A primer set (NF3, 5'-TTAAACTTCAATGGACTTAGAYCTGG-3': NR3, 5'-TTAAACTTCAATGGACTTAGAYCTG-3') was designed based on degenerate sequences of the nucleocapsid protein gene on the S RNA of MYSV. A DNA fragment of approximately 900 by was amplified from MYSV-infected plant tissues and labeled with digoxigenin (DIG). The sensitivity of PCR-MPH was compared with those of ELISA and RT-PCR. The total RNA was purified from 0.02 g of MYSV-infected plant tissue, and diluted in 5-fold steps from 51 to 510. MYSV was detectable from a 510-fold dilution by PCR-MPH, which is 625 times more sensitive than ELISA or RT-PCR. Moreover, MYSV was also detected from individual thrips by PCR-MPH with a higher sensitivity than RT-PCR.
