环介导同温DNA扩增技术快速检测弓形虫DNA的初步研究

Preliminary study on detecting Toxoplasma gondii DNA rapidly by loop-mediated isothermal amplification

目的建立一种快速的弓形虫活动性感染检测方法。方法根据刚地弓形虫特异性基因B1的基因序列设计一套用于环介导同温DNA扩增(loop-mediated isothermal amplification,LAMP)的特异性引物,用DNA聚合酶Bst在65℃水浴箱中对含有弓形虫基因组DNA的样本进行LAMP扩增,用荧光染料SYBRⅡ染色及琼脂糖凝胶电泳,溴化乙啶染色观察结果。同时进行常规PCR对照试验。结果琼脂糖凝胶电泳,溴化乙啶染色发现LAMP扩增产物为大小不等的DNA片段,在扩增产物中加入荧光素SYBRⅡ顔色由紫红色变成绿色荧光,而阴性对照管仍保持紫红色。环介导同温DNA扩增的敏感性为103个弓形虫/ml,高于常规PCR。结论LAMP扩增检测弓形虫DNA的方法已经建立,为发展弓形虫病快速诊断方法奠定了良好的基础。

Objective To construct a rapid method for detecting the activity infection of Toxoplasma gondii.Methods A set of specific primers used to amplify the genomic DNA of T.gondii by loop-mediated isothermal amplification（LAMP） was designed according to the B1 gene sequence of T.gondii.The sample containing Toxoplasma gondii was amplified by LAMP at 65 ℃ in a water bath,and the amplification products were detected by staining it with the fluorescence dye of SYBRⅡ.Meanwhile,the regular PCR was done as a control,and the PCR product was detected by agarose gel electrophoresis and staining by ethidium bromide.Results The amplification products of LAMP was a mixture composed of different size DNA fragments on agarose gel staining by EB,and the color of amplification products changed from purple to green fluorescence after it was mixed with SYBRⅡ,and the color of control tube remained purple.The sensitivity of LAMP is 103 T.gondii per milliliter,which was higher than regular PCR.Conclusion The LAMP method for detecting T.gondii DNA has been established,and provided a good foundation for developing rapid diagnostic method of toxoplasmosis.