Development of a Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Phytopythium vexans
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abstract
Brown root rot caused byPhytopythium vexansis a new destructive root disease on many plants such as Gingko, Citrus, kiwifruit, and ramie. The establishment of loop-mediated isothermal amplification (LAMP) technology for detectingP. vexanscan help monitor and control brown root rot quickly, efficiently, and accurately. LAMP technology is known for its simplicity, sensitivity, and speed; and it does not require any specialized equipment – a water bath or a thermoblock is sufficient for isothermal amplifications. LAMP products can be visualized by using hydroxy naphthol blue (HNB) dye or agarose gel electrophoresis. In this study, by searching and comparing the internal transcribed spacer (ITS) sequences ofP. vexansand the related species in oomycete generaPythium, Phytopythium, andPhytophthora, we designed specific primers targeting the ITS gene region ofP. vexans. Using HNB dye, we established a LAMP technique for rapid detection ofP. vexansby visible color change. In addition, we optimized the protocol to enhance both sensitivity and specificity forP. vexansdetection. Under the optimized condition, our protocol based on LAMP technology could detect as low as 24 copies of theP. vexansgenomic DNA, which is ∼100 times more sensitive than conventional PCR. This method can successfully detectP. vexansusing cell suspensions fromP. vexans– infected ramie root tissues.
