Development of a multiplex reverse transcription-PCR assay for simultaneous detection of garlic viruses
HU Xin-xi, LEI Yan, WANG Pei, TANG Lin-fei, HE Chang-zheng, SONG Yong, XIONG Xing-yao, NIE Xian-zhou
1、Hunan Provincial Key Laboratory of Crop Germplasm Innovation and Utilization, Hunan Provincial Engineering Research Center
for Potatoes, College of Horticulture and Landscape, Hunan Agricultural University, Changsha 410128, P.R.China
2、Potato Research Centre, Agriculture and Agri-Food Canada, Fredericton, E3B 4Z7, New Brunswick, Canada
摘要 A preliminary screening for garlic viruses in garlic plants in Hunan, China, using existing monoplex (simplex) reverse transcription- polymerase chain reaction (RT-PCR) procedures detected four viruses/virus groups. These viruses/virus groups were Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), Shallot latent virus (SLV) and allexiviruses (e.g., garlic viruses A, B, C, D, E, X). Sequence analysis of the projected allexivirus amplicons revealed the allexivirus in the infected garlic plants was Garlic virus D (GarV-D), which shared 92–97% sequence identities with various isolates from the world. A multiplex RT-PCR (mRT-PCR) was therefore developed to simultaneously detect and differentiate the four viruses/virus groups. To achieve this, four primer pairs targeting allexiviruses, OYDV, LYSV and SLV were designed. The anticipated amplicon sizes are 183 bp (allexiviruses), 265 bp (OYDV), 404 bp (LYSV) and 592 bp (SLV), respectively. All primer pairs produced virus-specific fragments in both simplex and multiplex formats, thus confirming the efficacy of the newly developed mRT-PCR for detection of these viruses. The mRT-PCR further was evaluated by applying it to garlic plant samples collected in two geographic locations in Hunan. Allexiviruses, OYDV, LYSV and SLV were detected in 50.9, 40.3, 28.3 and 58.5% of leaf samples, respectively; and mixed infections with two or more viruses accounted for 54% of the garlic samples. The results obtained by mRT-PCR were confirmed by simplex RT-PCR assays. In conclusion, this newly developed mRT-PCR provides a rapid, sensitive and reliable method for the detection and identification of major garlic viruses.
Abstract A preliminary screening for garlic viruses in garlic plants in Hunan, China, using existing monoplex (simplex) reverse transcription- polymerase chain reaction (RT-PCR) procedures detected four viruses/virus groups. These viruses/virus groups were Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), Shallot latent virus (SLV) and allexiviruses (e.g., garlic viruses A, B, C, D, E, X). Sequence analysis of the projected allexivirus amplicons revealed the allexivirus in the infected garlic plants was Garlic virus D (GarV-D), which shared 92–97% sequence identities with various isolates from the world. A multiplex RT-PCR (mRT-PCR) was therefore developed to simultaneously detect and differentiate the four viruses/virus groups. To achieve this, four primer pairs targeting allexiviruses, OYDV, LYSV and SLV were designed. The anticipated amplicon sizes are 183 bp (allexiviruses), 265 bp (OYDV), 404 bp (LYSV) and 592 bp (SLV), respectively. All primer pairs produced virus-specific fragments in both simplex and multiplex formats, thus confirming the efficacy of the newly developed mRT-PCR for detection of these viruses. The mRT-PCR further was evaluated by applying it to garlic plant samples collected in two geographic locations in Hunan. Allexiviruses, OYDV, LYSV and SLV were detected in 50.9, 40.3, 28.3 and 58.5% of leaf samples, respectively; and mixed infections with two or more viruses accounted for 54% of the garlic samples. The results obtained by mRT-PCR were confirmed by simplex RT-PCR assays. In conclusion, this newly developed mRT-PCR provides a rapid, sensitive and reliable method for the detection and identification of major garlic viruses.
This research was supported by the Non-Profit Industry Financial Program of Ministry of Agriculture of China (20130328) to Prof. Liu Yong and Prof. Dai Liangying, and the Natural Science Foundation of Hunan Province, China to Prof. Hu Xinxi (11JJ2018).
HU Xin-xi, LEI Yan, WANG Pei, TANG Lin-fei, HE Chang-zheng, SONG Yong, XIONG Xing-yao, NIE Xian-zhou.
2015.
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