Loop-mediated Isothermal Amplification Assay for Rapid Diagnosis of Aphelenchoides besseyi
Loop-mediated Isothermal Amplification Assay for Rapid Diagnosis of Aphelenchoides besseyi
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DOI：
10.16819/j.1001-7216.2017.7001
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发表时间：
2017-07-01
影响因子：
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通讯作者：
Xie Hui
中科院分区：
文献类型：
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作者：
Bai Zongshi;Qin Meng;Xie Hui
【目的】本研究为贝塞阿芬氏蜱的快速检测和早期诊断提供一种新的方法。[方法]以线虫18S核糖体RNA基因为基础，设计贝塞依虫环介导等温扩增（LAMP）特异性引物，包括2个外部引物、2个内部引物和2个环引物。DNA样品用LAMP引物等温扩增。扩增产物经电泳和荧光染料检测。如果在电泳结果中有清晰的DNA阶梯，或者在荧光染料结果中显示绿色荧光，则证明样品中存在贝赛伊拟虫。[结果]结果表明，LAMP引物可检测鉴定贝赛依螨的单卵、幼虫、雌虫和雄虫，并可直接从多种线虫种样品和植物组织样品中检测贝赛依螨，LAMP检测的灵敏度为单个线虫DNA的1/1000。【结论】该诊断方法具有准确、灵敏、稳定、结果直观等优点，且操作简便，实用性强。
[Objective] This study was performed to provide a new method for the rapid detection and early diagnosis of Aphelenchoides besseyi. [Method] The loop-mediated isothermal amplification (LAMP) specific primers of A. besseyi were designed based on the nematode 18S ribosomal RNA gene, including two external primer, two inner primer and two loop primer. The DNA sample was amplified by LAMP primers using isothermal amplification. The amplified products were detected by electrophoresis and fluorescent dye. It proved that there was A. besseyi in the test sample if there was clear DNA ladder in the electrophoresis results or it showed green fluorescence in the fluorescent dye results. [Result] The results showed that LAMP primers could detect and identify single egg, juvenile, female and male of A. besseyi, and could also directly detect A. besseyi from multiple nematode species samples and plant tissue samples, the sensitivity of LAMP assay was 1/1000 of single nematode DNA. [Conclusion] This diagnosis method has the advantages of accuracy, sensitivity, stability and intuitive results, and is also simple and more practical.