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Position: Home > Articles > Development of a multiplex reverse transcription-PCR assay for simultaneous detection of garlic viruses Journal of Integrative Agriculture 2015,14 (5)

Development of a multiplex reverse transcription-PCR assay for simultaneous detection of garlic viruses

作者：

Hu Xin-xi;Lei Yan;Wang Pei;Tang Lin-fei;He Chang-zheng;Song Yong;Xiong Xing-yao;Nie Xian-zhou

单位：

Agr & Agri Food Canada, Potato Res Ctr, Fredericton, NB E3B 4Z7, Canada;Hunan Agr Univ, Coll Hort & Landscape, Hunan Prov Engn Res Ctr Potatoes, Hunan Prov Key Lab Crop Germplasm Innovat & Utili, Changsha 410128, Hunan, Peoples R China

关键词：

garlic;garlic viruses;RT-PCR;detection;multiplex PCR

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 nnRT-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.

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Development of a multiplex reverse transcription-PCR assay for simultaneous detection of garlic viruses

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