Simultaneous detection of four causal agents of tobacco bushy top disease by a multiplex one-step RT-PCR.
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, 792bp for TVDVaRNA, 598bp for Sat-TBTV and 357bp 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 naturally with one to four target viruses, transmission vector of aphids and field samples collected from Yunnan, Hunan, and Guizhou province, 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.
Key Laboratory of Agricultural Biodiversity for Pest Management of China Education Ministry, Yunnan Agricultural University, Kunming 650201, China; College of Agriculture and Biotechnology, Yunnan Agricultural University, Kunming 650201, China.
Fang
F
Liu
Modern Education Technology Center, Yunnan Agricultural University, Kunming 650201, China.
Guanlin
G
Tan
Key Laboratory of Agricultural Biodiversity for Pest Management of China Education Ministry, Yunnan Agricultural University, Kunming 650201, China.
Xiaojing
X
Li
Key Laboratory of Agricultural Biodiversity for Pest Management of China Education Ministry, Yunnan Agricultural University, Kunming 650201, China.
Hairu
H
Chen
USDA-ARS, National Germplasm Resources Laboratory, Beltsville, MD 20705, USA. Electronic address: Ruhui.Li@ars.usda.gov.
Ruhui
R
Li
Key Laboratory of Agricultural Biodiversity for Pest Management of China Education Ministry, Yunnan Agricultural University, Kunming 650201, China. Electronic address: fanli@ynau.edu.cn.
Fan
F
Li
D017931:DNA Primers / Q000235:genetics; D052679:Luteoviridae / Q000235:genetics / Q000302:isolation & purification*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D010935:Plant Diseases / Q000821:virology*; D010942:Plant Viruses / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000032:analysis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D013045:Species Specificity; D014026:Nicotiana / Q000821:virology*; D019183:Tombusviridae / Q000235:genetics / Q000302:isolation & purification*
D017931:DNA Primers; D012367:RNA, Viral
Multiplex RT-PCR; Sat-TBTV; TBTV; TVDV; TVDVaRNA; Tobacco bushy top disease