Lin Borong, Wang Honghong, Zhuo Kan, Liao Jinling
Laboratory of Plant Nematology, South China Agricultural University, Guangzhou 510642, China; and Guangdong Province Key Laboratory of Microbial Signals and Disease Control, South China Agricultural University, Guangzhou 510642, China.
Laboratory of Plant Nematology and Guangdong Province Key Laboratory of Microbial Signals and Disease Control, South China Agricultural University; and Guangdong Vocational College of Ecological Engineering, Guangzhou 510520, China.
Plant Dis. 2016 May;100(5):877-883. doi: 10.1094/PDIS-07-15-0801-RE. Epub 2016 Feb 29.
Tylenchulus semipenetrans is an economically important plant-parasitic nematode occurring in all citrus-producing regions of the world and causing a disease called "slow decline". For the rapid detection of this nematode, a loop-mediated isothermal amplification (LAMP) assay was developed, based on the ribosomal DNA internal transcribed spacer sequence. The optimal condition for the LAMP assay was 65°C for 50 min. The LAMP products were confirmed using conventional polymerase chain reaction (PCR) and restriction analysis with the BamHI enzyme, and by adding SYBR Green I to the LAMP products for visual inspection. The LAMP assay was highly specific for the detection of T. semipenetrans populations from different geographical origins. It was also sensitive, detecting a tenth of the DNA from an individual specimen of T. semipenetrans, which was 10 times more sensitive than conventional PCR. The LAMP protocol was applied to natural citrus rhizosphere soil samples from several orchards in China and the results were fast, sensitive, robust, and accurate. This study is the first to provide a diagnostic tool for T. semipenetrans using DNA extracted directly from citrus rhizosphere soils. This LAMP assay could be used as a practical molecular tool to identify T. semipenetrans and diagnose slow decline disease, even in remote locations.
半穿刺线虫是一种具有经济重要性的植物寄生线虫，存在于世界所有柑橘产区，会引发一种名为“缓慢衰退”的病害。为了快速检测这种线虫，基于核糖体DNA内转录间隔区序列开发了一种环介导等温扩增（LAMP）检测方法。LAMP检测的最佳条件是65℃反应50分钟。使用常规聚合酶链反应（PCR）和BamHI酶切分析对LAMP产物进行确认，并通过向LAMP产物中添加SYBR Green I进行目视检查。LAMP检测对来自不同地理区域的半穿刺线虫种群具有高度特异性。它也很灵敏，能检测到半穿刺线虫单个样本十分之一的DNA，比传统PCR灵敏10倍。LAMP方法应用于中国几个果园的天然柑橘根际土壤样本，结果快速、灵敏、可靠且准确。本研究首次提供了一种利用直接从柑橘根际土壤中提取的DNA对半穿刺线虫进行诊断的工具。这种LAMP检测方法可作为一种实用的分子工具，用于鉴定半穿刺线虫和诊断缓慢衰退病害，即使在偏远地区也适用。
