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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(P<0.01)。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。
Objective To establish a rapid and simple loop-mediated isothermal amplification assay (LAMP) for the detection of Toxoplasma gondii infection. Methods Two pairs of specific LAMP primers were synthesized according to the sequence of SAG1 gene of Toxoplasma gondii RH strain, and the amplification system and parameters were optimized. The specificity and sensitivity of the optimized LAMP response were evaluated by comparison with conventional PCR methods. The LAMP was used to detect the muscle tissue of white rabbits and rats after artificial infection with Toxoplasma gondii for 4 days. Results The sensitivity of LAMP method to detect Toxoplasma gondii genomic DNA was 100 times that of the traditional PCR method. LAMP method with other common parasitic protozoan parasites no non-specific positive results. LAMP method was used to detect the muscle tissue of artificial inoculated animals. The positive rate was 100% in control group and 0, the difference was statistically significant (P <0.01). Conclusion The detection of Toxoplasma gondii by LAMP method is rapid and simple, sensitive and requires low equipment. It has a good prospect of application.