Development and Evaluation of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Tylenchulus semipenetrans Using DNA Extracted from Soil.
Development and Evaluation of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Tylenchulus semipenetrans Using DNA Extracted from Soil.
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DOI：
10.5423/ppj.oa.10.2016.0224
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发表时间：
2017-04
期刊：
影响因子：
--
通讯作者：
Liu Y
中科院分区：
文献类型：
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作者：
Song ZQ;Cheng JE;Cheng FX;Zhang DY;Liu Y
半反茎线虫（Tylenchulus semiacetrans）是一种重要的柑橘植物寄生线虫，可引起柑橘慢衰病，严重影响柑橘的生长和产量。快速、准确地检测T.土壤中半胱氨酸的含量对病害的预测和防治具有重要意义。本研究建立了环介导等温扩增（LAMP）检测T。使用从土壤中提取的DNA进行半穿透。根据rDNA的ITS1区设计了一组5对引物，对T.半反式LAMP反应在63 ℃下进行60 min。通过加入SYBR绿色I，在一个反应管中直接观察LAMP产物。LAMP检测的检测限为10 − 2 J2/0.5 g土壤，比常规PCR（10 − 1 J2/0.5 g土壤）灵敏10倍。对24个田间土壤样品的检测表明，LAMP检测方法适用于自然感染T.半反式，总测定时间小于2.5 h。这些结果表明，建立的LAMP检测方法是一种简单、快速、灵敏、特异和准确的T.半反式半
Tylenchulus semipenetrans 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 T. semipenetrans in soil is important for the disease forecasting and management. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed to detect T. semipenetrans 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 T. semipenetrans. 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 10−2 J2/0.5 g of soil, which was 10 times more sensitive than conventional PCR (10−1 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 T. semipenetrans, 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 T. semipenetrans in field soil, and contributes to the effective management of citrus slow decline disease.