Development and Evaluation of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Tylenchulus semipenetrans Using DNA Extracted from Soil

• Published in: The plant pathology journal Vol. 33; no. 2; pp. 184 - 192
• Main Authors: Song, Zhi-Qiang, Cheng, Ju-E, Cheng, Fei-Xue, Zhang, De-Yong, Liu, Yong
• Format: Journal Article
• Language: English
• Published: Korea (South) 한국식물병리학회 01.04.2017; Korean Society of Plant Pathology; Hanrimwon Publishing Company
• Subjects: citrus slow decline disease; DNA extraction; loop-mediated isothermal amplification; nematode detection; Tylenchulus semipenetrans; 농학; citrus slow decline disease; DNA extraction; nematode detection; loop-mediated isothermal amplification; Tylenchulus semipenetrans
• ISSN: 1598-2254; 2093-9280
• DOI: 10.5423/PPJ.OA.10.2016.0224

is an important and widespread plant-parasitic nematode of citrus worldwide and can cause citrus slow decline disease leading to significant reduction in tree growth and yield. Rapid and accurate detection of in soil is important for the disease forecasting and management. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed to detect using DNA extracted from soil. A set of five primers was designed from the internal transcribed spacer region (ITS1) of rDNA, and was highly specific to . The LAMP reaction was performed at 63°C for 60 min. The LAMP product was visualized directly in one reaction tube by adding SYBR Green I. The detection limit of the LAMP assay was 10 J2/0.5 g of soil, which was 10 times more sensitive than conventional PCR (10 J2/0.5 g of soil). Examination of 24 field soil samples revealed that the LAMP assay was applicable to a range of soils infested naturally with , and the total assay time was less than 2.5 h. These results indicated that the developed LAMP assay is a simple, rapid, sensitive, specific and accurate technique for detection of in field soil, and contributes to the effective management of citrus slow decline disease.