Development of a Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Phytopythium vexans

Abstract

Brown root rot caused by Phytopythium vexans is 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 detecting P. vexans can 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 of P. vexans and the related species in oomycete genera Pythium, Phytopythium, and Phytophthora, we designed specific primers targeting the ITS gene region of P. vexans. Using HNB dye, we established a LAMP technique for rapid detection of P. vexans by visible color change. In addition, we optimized the protocol to enhance both sensitivity and specificity for P. vexans detection. Under the optimized condition, our protocol based on LAMP technology could detect as low as 24 copies of the P. vexans genomic DNA, which is ∼100 times more sensitive than conventional PCR. This method can successfully detect P. vexans using cell suspensions from P. vexans – infected ramie root tissues.

Development of a Polymerase Spiral Reaction-Based Isothermal Assay for Rapid Identification of Thrips palmi

“…HNB is an indicator of metal ions, reported as a quantitative chemical indicator for Mg 2+ ions ( Ito and Ueno, 1970 ). It has been successfully employed by several researchers in LAMP and RPA assays ( Priti et al, 2021 ; Wang et al, 2021 ). The change in reaction color from violet (negative) to sky blue (positive) in PSR was induced by the chelation of Mg 2+ ions by dNTPs ( Gelfand, 1989 ).…”

Development of a Loop-Mediated Isothermal Amplification Assay for Rapid and sensitive Detection of Hemileia vastatrix in Coffee

“…Compared with PCR-based methods, the LAMP assay sometimes has the great advantages of high amplification efficiency, and high degree of sensitivity and specificity (Khan et al 2017). Therefore, LAMP technology has been widely developed to detect various plant and animal pathogens, including oomycete, fungi, bacteria, viruses, nematodes (Ortega et al 2018;Wang et al 2021;Shu et al 2022;Bertacca et al 2022). However, a LAMP detection method for H. vastatrix has not been reported.…”