疟疾巢式PCR检测方法的优化应用

发布时间：2018-02-22 04:46

本文关键词： 疟疾巢式PCR 卵形疟原虫优化应用敏感性特异性　出处：《中国血吸虫病防治杂志》2017年04期 　论文类型：期刊论文

【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(P0.05),但优化后的方法特异性明显提高(P0.05)。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。
[Abstract]:Objective to optimize the application of nested PCR method for malaria gene detection in order to improve the efficiency of malaria detection. A new primer for multiple subspecies of Plasmodium ovum was redesigned to optimize the reaction system, reaction conditions and specific primers of Plasmodium ovum on the basis of malaria nested PCR. The specificity and sensitivity of the optimized method were analyzed. Nested PCR and optimized method were used to detect both malaria positive blood samples and malaria blood samples, and the results were compared and analyzed. The sensitivity of the two methods was from PG to FG. The results of the two methods showed that the yield of the PCR amplification products of the optimized method had no significant change, the non-specific amplification was significantly reduced, and the detection rate of different subspecies of oval malaria was increased. The overall specificity was improved. The measured results of 111 malaria blood samples showed that, The sensitivity and species specificity of nested PCR were 94.57% and 86.96, respectively. The sensitivity and species specificity of the optimized method were 93.48. There was no significant difference in sensitivity between the two methods (P 0.05), but the specificity of the optimized method was significantly improved. The optimized PCR detection improved the specificity on the basis of keeping the sensitivity of the conventional nested PCR method. At the same time, the cost of detection is saved and the efficiency of detection is improved because of the reduction of experimental links.
【作者单位】：上海市疾病预防控制中心;
【分类号】：R531.3

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