Research On Molecular Detection Technology Of Tospovirus

Tospovirus, belongs to the family Bunyaviridae, the viruses are transmited by thrips in a circulative and propagative manner, are considered to be noteworthy for causing direct economic losses of many crops and ornamental plants. Because of the representative species-TSWV(Tomato spottde wilt visus) that had the broad host range and caused significant economic, was listed as one of the 10 kinds of the world’s most harmful plant viruses. European and Mediteranean Plant Protection Organization, EPPO determined TSWV as a quarantine virus, and the thrips as A2 quarantine pests. The studies found that TSWV-infected plants are always attract more thrips feeding, so strengthening the detection of the tospoviruses is helpful for the prevention and control of thrips.This study established several detection methods of tospoviruses, the major elements are as follow:According to the comparison of N gene in the S segment of six the Tospovirus RNA, six pairs of specific primers were designed, and a multiplex polymerase chain reaction assay had been established. Amplifications resulted in a 776-bp PCR product for Tomato zonate spot virus,505-bp for Melon yellow spot virus,296-bp for Iris yellow spot virus, 221-bp for Impatiens necrotic spot virus,175-bp for Tomato spotted wilt virus, and a 110-bp for Tomato chlorotic spot virus. The multiplex PCR assay presented here could acquire clear target bands of each virus, and the primers possess good specificity and no non-specific amplification is produced during the detection of tospoviruses.According to the sequence of TSWV N gene, with the use of Express 3.0 to design the primers and probe, a real-time fluorescent PCR is established to detect TSWV and with the literature primers and probe to compare.With the plant blade infected by MYSV as the template, according to the published MYSV sequence of taiwan isolate in NCBI, the primers for RT-PCR are disigned. The 5’ sequence are acquired by the method of 5’RACE, and the other seven sequences that overlap each other and cover the whole segment are acquired by the nomal RT-PCR. After the clone sequencing of the sequences, we can get a sequence of MYSV S fragment of 3242 nt, the sequence homology of the two MYSV isolate is 94%.