柑橘黄龙病可视化LAMP检测技术的建立及应用

Establishment and Application of Visual Loop-mediated Amplification Assay on Citrus Huanglongbing

建立柑橘黄龙病(HLB)可视化环介导等温扩增(LAMP)检测方法,为HLB的快速检测提供有力的技术支持。根据LAMP技术原理,针对柑橘黄龙病菌16S rDNA靶序列的6个位点设计4条特异性引物,通过对反应体系和反应条件的优化,并在反应前的体系中加入1∶10的钙黄绿素(calcein)和氯化锰(MnCl2)混合液作为荧光显色剂,建立可视化LAMP检测技术,同时对该技术的特异性、灵敏度和样品检测进行了测试。该检测方法具有很高的特异性,反应体系中除了HLB具有特异性的扩增,产生黄绿色荧光扩增产物,其他供试菌株均无特异扩增。对柑橘黄龙病菌总DNA的检测限为1.51×10-3 ng/μL,而对含有目的基因的CG-pMD18-T质粒DNA的检测限为2.12×10-6 ng/μL,与定量PCR灵敏度相当,而比常规PCR灵敏度高1 000倍。利用可视化LAMP技术和定量PCR对来自漳州、南平、福州等地的样品进行检测,结果 2种技术的检出率均为76.7%,符合率达到100%。

A novel method namely visual loop-mediated isothermal amplification （LAMP） was established. It would provide strong support for Huanglongbing （HLB） quick detection. The method is based on LAMP reaction. The conserved region, 16S ribosomal DNA in the genomic DNA of Candidatus Liberibacter asiaticus （Las） was amplified by a set of four specific primers that recognize six distinct sequences of target. Calcein and MnCl2 （1：10） reagent were added into the mix before reaction and used in LAMP product judgment with naked eyes. A visual LAMP method was established by the optimized reaction systems and condition in this study, and the specificity and sensitivity was test in the field. This visual LAMP method was tremendously high specificity. Only Las in the reaction system was specifically amplified with greenyellow fluorescence products, the other samples was not amplified, and without any products. The lowest detection concentrations of Las DNA and Las plasmid DNA in the method were 1.51×10^-3 ng/uL and 2.12×10^-6ng/uL, respectively. The sensitivity of visual LAMP was equivalency with real-time PCR and 1 000 times more than conventional PCR. Samples from Zhangzhou, Nanping and Fuzhou City were detected by the visual LAMP and real-time PCR, it showed in the results that the detection rate and coincidence rate of this two method were the same. They were 76.7% and 100%, respectively. The visual LAMP assay method with fluorescent coloration was successfully established, it can be applied into detect the Las in the field.