LAMP技术检测弓形虫感染方法的建立

目的建立一种快速、简便的检测弓形虫感染的环介导等温扩增方法(LAMP)。方法根据弓形虫RH株SAG1基因序列合成2对特异性LAMP引物,优化扩增体系与参数。通过与常规PCR方法的比较,评估优化后的LAMP反应的特异性和灵敏性。利用建立的LAMP初步检测人工感染弓形虫4 d后白兔与大鼠的肌肉组织。结果 LAMP法检测弓形虫基因组DNA的灵敏度是传统PCR方法的100倍。LAMP法检测与其他常见寄生原虫、寄生虫无非特异性阳性结果。LAMP法检测人工接种动物的肌肉组织,阳性率达到100%,对照组为0,差异有统计学意义(P0.01)。结论 LAMP法检测弓形虫感染快速简便、敏感性高、设备要求低,具有较好的应用前景。

Establishment of loop-mediated isothermal amplification for the detection of Toxoplasma gondii

Objective To explore a quick, simple and convenient method of loop-mediated isothermal amplification（LAMP）for detection of Toxoplasma gondii in row meat product. Methods Two pairs of specific LAMP primers were deigned according to the sequences of Toxoplasma gondii RH strain surface antigen I（SAG1） gene, and then LAMP reaction system and condition were optimized. The sensitivity and specificity of optimized LAMP was evaluated by comparing with traditional PCR. Furthermore, LAMP was test by application on muscle tissue of artificial infected SD rat and New Zealand white rabbit.Results The sensitivity of detection of Toxoplasma gondii genome DNA by LAMP was 100-fold higher than traditional PCR.There was no false positive result observed by LAMP in other parasites and medical protozoan. Moreover, muscle tissue of artificial infected SD rat and New Zealand white rabbit were all positive, while the blank control was negative in the LAMP test. Conclusion The method is rapid, easy to operate, highly sensitive and low requirement for equipment, with preferable application prospects.