Abstract: To optimize the application of the nested PCR method for the detection of malaria according to the working practice， so as to improve the efficiency of malaria detection. Methods Premixing solution of PCR， internal primers for further amplification and new designed primers that aimed at two Plasmodium ovale subspecies were employed to optimize the reaction system， reaction condition and specific primers of P. ovale on basis of routine nested PCR. Then the specificity and the sensitivity of the optimized method were analyzed. The positive blood samples and examination samples of malaria were detected by the routine nested PCR and the optimized method simultaneously， and the detection results were compared and analyzed. Results The optimized method showed good specificity， and its sensitivity could reach the pg to fg level. The two methods were used to detect the same positive malarial blood samples simultaneously， the results indicated that the PCR products of the two methods had no significant difference， but the non?specific amplification reduced obviously and the detection rates of P. ovale subspecies improved， as well as the total specificity also increased through the use of the optimized method. The actual detection results of 111 cases of malarial blood samples showed that the sensitivity and specificity of the routine nested PCR were 94.57％ and 86.96％， respectively， and those of the optimized method were both 93.48％， and there was no statistically significant difference between the two methods in the sensitivity （P > 0.05）， but there was a statistically significant difference between the two methods in the specificity （P < 0.05）. Conclusion The optimized PCR can improve the specificity without reducing the sensitivity on the basis of the routine nested PCR， it also can save the cost and increase the efficiency of malaria detection as less experiment links.

Key words: Malaria； Nested PCR； Plasmodium ovale； Optimizing application； Sensitivity； Specificity

摘要： 目的 结合疟疾检测工作实际，对疟疾基因检测的巢式PCR方法进行优化应用，以提高疟疾检测效率。方法 通过采用PCR预混液、内引物一步扩增、重新设计针对卵形疟多个亚种的新引物，在疟疾巢式PCR的基础对反应体系、反应条件和卵形疟原虫特异性引物进行优化。对优化后的方法进行特异性和敏感性分析，并用巢式PCR和优化后的方法同时对疟疾阳性血样和疟疾送检血样进行检测，对检测结果进行比较分析。结果 优化后的方法特异性较好，敏感性可达pg至fg级。两种方法同时检测疟疾阳性血样的结果表明，优化后的方法PCR扩增产物的产量无明显变化，非特异性扩增明显减少，卵形疟不同亚种的检出率提高，总体特异性提高。对111份疟疾送检血样的实测结果表明，巢式PCR的敏感性和虫种特异性分别为94.57％和86.96％，优化后方法的敏感性和虫种特异性均为93.48％，两种方法敏感性差异无统计学意义（P > 0.05），但优化后的方法特异性明显提高（P < 0.05）。结论 优化后的PCR检测在保持常规巢式PCR方法敏感性的基础上提高了特异性，同时因其实验环节减少而节省了检测成本、提高了检测效率。

关键词: 疟疾；巢式PCR；卵形疟原虫；优化应用；敏感性；特异性