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Reverse transcription loop-mediated isothermal amplification of DNA for detection of Barley yellow dwarf viruses in
Kun Zhao1, Yan Liu, Xifeng Wang
1State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
Abstract:
A reverse transcription loop-mediated isothermal amplification of DNA (RT-LAMP) for detection of Barley yellow dwarf viruses (BYDVs) was developed. In this procedure, three sets of four primers matching a total of six sequences of the coat protein or read-through protein genes of BYDVs - one each for three species, namely BYDV-GAV, -GPV and -PAV were synthesized for developing a specific and sensitive RT-LAMP assay for total RNA extracts from field-infected wheat plants in such a way that a loop could be formed and elongated during DNA amplification. RT-LAMP assays for each of three species of BYDV/CYDVs in China exhibited high specificity and could detect viral sequences in total RNA extracts from infected oat tissues at dilutions of 1 x 10(-5). All field samples collected from different regions of China showed the same result using both RT-LAMP and RT-PCR. This relatively simple and sensitive technique showed excellent potential with field-collected samples and for use in grassroots agencies in developing countries with limited resources.
