Evaluation of loop-mediated isothermal amplification (LAMP) assays based on 5S rDNA-IGS2 regions for detecting Meloidogyne enterolobii
Evaluation of loop-mediated isothermal amplification (LAMP) assays based on 5S rDNA-IGS2 regions for detecting Meloidogyne enterolobii
复制标题
基于 5S rDNA-IGS2 区域的环介导等温扩增 (LAMP) 检测用于检测肠根结线虫的评估
DOI：
10.1111/j.1365-3059.2011.02562.x
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发表时间：
2012-08-01
期刊：
影响因子：
2.7
通讯作者：
Guo, Y. D.
中科院分区：
文献类型：
--
作者：
Niu, J. H.;Jian, H.;Guo, Y. D.
根据5S rDNA和IGS 2基因片段的序列，建立了一种环介导等温扩增（loop-mediated isothermal amplification，LAMP）方法，用于诊断根结线虫病。LAMP扩增在65 ℃等温条件下11.5 h内完成。其扩增子通过凝胶电泳、SacI酶分析、侧向流试纸（LFD）测定以及通过SYBR绿色I和钙黄绿素染色的目视检查来确认。结果表明，Me-LAMP能够特异性地检测M.来自不同地理来源的enterolobii种群，检测限约为10 fg M。与常规PCR相比，该方法的灵敏度提高了10100倍。此外，LAMP的适用性被证实后，其在检测害虫的根和土壤样品的成功表现在现场检测。Me-LAMP方法具有简便、灵敏、特异的特点，是一种很有前途的、实用的M.在害虫检疫和现场调查中应用。
A loop-mediated isothermal amplification (LAMP) assay for detection of Meloidogyne enterolobii (Me-LAMP) was developed based on the sequences of the 5S ribosomal DNA (5S rDNA) and intergenic spacer 2 (IGS2) segment. The LAMP amplification was achieved at 65 degrees C isothermal conditions within 11.5 h. Its amplicons were confirmed using gel electrophoresis, SacI enzyme analysis, lateral flow dipstick (LFD) assay, and visual inspection through SYBR Green I and calcein staining. The results demonstrated that the Me-LAMP was able to specifically detect M. enterolobii populations from different geographical origins, with a detection limit of about 10 fg M. enterolobii genomic DNA, which was 10100 times more sensitive than conventional PCR. In addition, the applicability of LAMP to field detection was confirmed following its successful performance in detecting the pest on root and soil samples. The Me-LAMP assay possessed the characteristics of simplicity, sensitivity and specificity, and is a promising and practical molecular tool for M. enterolobii diagnosis in pest quarantine and field surveys.