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Title: Simultaneous detection of four causal agents of tobacco bushy top disease by a multiplex one-step RT-PCR

item LIU, FANG - Yunnan Agricultural University
item TAN, GUANLIN - Yunnan Agricultural University
item LI, XIAOJING - Yunnan Agricultural University
item CHEN, HAIRU - Yunnan Agricultural University
item Li, Ruhui
item LI, FAN - Yunnan Agricultural University

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/2/2014
Publication Date: 5/14/2014
Citation: Liu, F., Tan, G., Li, X., Chen, H., Li, R., Li, F. 2014. Simultaneous detection of four causal agents of tobacco bushy top disease by a multiplex one-step RT-PCR. Journal of Virological Methods. 205:236-248.

Interpretive Summary: Tobacco bushy top disease (TBTD) is disease complex caused by mixed infection of four virus/viral RNA derived pathogens. The disease was observed in western Yunnan, China in 1993 and has since become widespread in many tobacco growing areas of the province. The viruses also infect other solanaceous crops. The symptoms include small leaves, irregular necrotic lesions on leaves, yellowing or chlorosis, internode shortening and stunting. The disease agents are transmitted easily by green peach aphids in the field. Once the disease is introduced, it spreads quickly and causes irreversible crop losses. A simple, sensitive, and cost-effective method was developed to detect the four pathogens in one tube; it detects and amplifies the pathogen RNA genetic material. The assay was reliable using plant samples from both greenhouses and tobacco fields. The assay should be very useful as a routine TBTD diagnostic tool, including the ability to monitor aphid populations to support disease management decisions such as insecticide applications.

Technical Abstract: Tobacco bushy top disease is a complex disease caused by mixed infection of Tobacco bushy top virus (TBTV), Tobacco vein distorting virus (TVDV), satellite RNA of TBTV (Sat-TBTV) and Tobacco vein distorting virus associate RNA (TVDVaRNA). A one-tube multiplex reverse transcription-PCR (RT-PCR) assay was developed for simultaneous detection of the four causal agents of the disease. Four pairs of specific primers based on the conserved regions of each of the four disease agents were used in the one-tube RT-PCR. The RT-PCR products consisted of fragments of 1049 base pairs (bp) for TBTV, 792 bp for TVDVaRNA, 598 bp for Sat-TBTV and 357 bp for TVDV, and their origins were confirmed by sequencing. Primer concentrations and cycling condition were optimized for the multiplex RT-PCR. The detection limit of the assay was up to 10-4 dilution. The assay was evaluated using tobacco plants infected with one to four target viruses, transmission vector of aphids and field samples collected from Yunnan province, southwestern China. The results show that the multiplex RT-PCR is reliable and sensitive as a simple, rapid and cost-effective method to detect these pathogens in tobacco and aphid. This assay will be useful for virus surveys when large numbers of samples are tested.