کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
6133297 1593463 2015 7 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Advanced loop-mediated isothermal amplification method for sensitive and specific detection of Tomato chlorosis virus using a uracil DNA glycosylase to control carry-over contamination
موضوعات مرتبط
علوم زیستی و بیوفناوری ایمنی شناسی و میکروب شناسی ویروس شناسی
پیش نمایش صفحه اول مقاله
Advanced loop-mediated isothermal amplification method for sensitive and specific detection of Tomato chlorosis virus using a uracil DNA glycosylase to control carry-over contamination
چکیده انگلیسی
In 2013, Tomato chlorosis virus (ToCV) was identified in symptomatic tomato plants in Korea. In the present study, a loop-mediated isothermal amplification (LAMP) method was developed using four specific primers designed against ORF6 in ToCV RNA2 to detect ToCV rapidly and with high sensitivity. The optimized reaction involved incubation of a reaction mixture containing 2 U Bst DNA polymerase and 4 mM MgSO4 for 1 h at 60-62 °C. Although specific and rapid detection of ToCV by LAMP was confirmed, false-positive reactions caused by carry-over contamination sometimes occurred because of the high sensitivity of LAMP compared with other detection methods. To prevent false-positive reactions, dUTP was substituted for dTTP and uracil-DNA glycosylase (UDG) was added to the LAMP reaction. First, the LAMP reaction was conducted successfully with substitution of dUTP for dTTP. Before the next reaction, LAMP products with incorporated dUTP were cleaved selectively by UDG without any effect on thymine-containing DNA (template DNA). This modified LAMP method complemented with UDG treatment to prevent carry-over contamination offers a potentially powerful method for detecting plant viruses.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Virological Methods - Volume 213, 1 March 2015, Pages 68-74
نویسندگان
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