Detection of Papaya leaf distortion mosaic virus by reverse-transcription loop-mediated isothermal amplification

被引:25
作者
Shen, Wentao [1 ,2 ]
Tuo, Decai [1 ,2 ,3 ]
Yan, Pu [1 ,2 ]
Li, Xiaoying [1 ,2 ]
Zhou, Peng [1 ,2 ]
机构
[1] Chinese Acad Trop Agr Sci, Inst Trop Biosci & Biotechnol, Minist Agr, Key Lab Biol & Genet Resources Trop Crops, Haikou 571101, Peoples R China
[2] Chinese Acad Trop Agr Sci, Anal & Testing Ctr, Haikou 571101, Peoples R China
[3] Hainan Univ, Coll Agr, Haikou 570228, Hainan, Peoples R China
基金
中国国家自然科学基金;
关键词
Papaya leaf distortion mosaic virus; PLDMV detection; Reverse-transcription loop-mediated isothermal amplification; RT-PCR; RINGSPOT-VIRUS; RAPID DETECTION; NUCLEOTIDE-SEQUENCE; TRANSGENIC PAPAYA; PROTEIN GENE; DNA; VARIABILITY; GENERATION; CHINA; LAMP;
D O I
10.1016/j.jviromet.2013.09.011
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Papaya leaf distortion mosaic virus (PLDMV) can infect transgenic papaya resistant to a related pathogen, Papaya ringspot virus (PRSV), posing a substantial threat to papaya production in China. Current detection methods, however, are unable to be used for rapid detection in the field. Here, a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of PLDMV, using a set of four RT-LAMP primers designed based on the conserved sequence of PLDMV CP. The RT-LAMP method detected specifically PLDMV and was highly sensitive, with a detection limit of 1.32 x 10(-6) mu g of total RNA per reaction. Indeed, the reaction was 10 times more sensitive than one-step RT-PCR, while also requiring significantly less time and equipment. The effectiveness of RT-LAMP and one-step RT-PCR in detecting the virus were compared using 90 field samples of non-transgenic papaya and 90 field samples of commercialized PRSV-resistant transgenic papaya from Hainan Island. None of the non-transgenic papaya tested positive for PLDMV using either method. In contrast, 19 of the commercialized PRSV-resistant transgenic papaya samples tested positive by RT-LAMP assay, and 6 of those tested negative by RT-PCR. Therefore, the PLDMV-specific RT-LAMP is a simple, rapid, sensitive, and cost-effective tool in the field diagnosis and control of PLDMV. (C) 2013 Elsevier B.V. All rights reserved.
引用
收藏
页码:174 / 179
页数:6
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