环介导等温扩增技术检测蓝氏贾第鞭毛虫

Detection of Giardia lamblia by loop-mediated isothermal amplification

目的 建立应用环介导等温扩增技术（loop-mediated isothermal amplification,LAMP）检测蓝氏贾第鞭毛虫的方法. 方法 体外培养蓝氏贾第鞭毛虫滋养体,提取DNA.根据GenBank显示的贾第虫序列及环介导等温扩增技术的原理,设计4条贾第虫特异引物,利用LAMP检测蓝氏贾第鞭毛虫DNA,以隐孢子虫卵囊DNA、疟原虫DNA为对照,并将不含病原体DNA的纯水作为阴性对照.LAMP产物经SYBR green I显色后观察结果,绿色为阳性,棕色为阴性;对LAMP产物进行琼脂糖凝胶电泳分析,观察其特征条带的情况. 结果 蓝氏贾第鞭毛虫DNA检测管经显色后呈绿色,隐孢子虫卵囊DNA、疟原虫DNA及水阴性对照管呈棕色.含有蓝氏贾第鞭毛虫DNA的LAMP产物经电泳后呈LAMP特征性梯状条带,安氏隐孢子虫DNA、恶性疟原虫DNA及阴性对照水无扩增产物. 结论 成功建立了检测蓝氏贾第鞭毛虫的LAMP方法.

Objective To develop a method for detecting the DNA of Giardia lamblia by loop-mediated isothermal amplification （LAMP）. Methods The DNA was extracted from cultured in vitro G. lamblia trophozoites. According to the DNA sequence of G. lamblia in GenBank and the principal of LAMP, 4 specific primers were designed for LAMP assay detection on G. lamblia, the DNA of Cryptosporidium andersoni and Plasmodium falciparum were also detected as control, and pure water without DNA as negative control. The LAMP products were stained by SYBR green I and analyzed by agarose gel electrophoresis. Results After LAMP reaction, the positive signal from G. lamblia DNA turned green while the other two kinds DNA of pathogens and negative control kept brown. Moreover,electrophoresis analysis showed the characterized ladder of the bands for LAMP product of G. lamblia. By contrast,no bands observed for the LAMP products of andersoni, Plasmodium fakiparum and water. Conclusion A simple method for detecting G. lamblia by LAMP assay was established.