单管单轮多重PCR检测4种疟原虫混合血样

发布时间：2018-05-26 08:00

本文选题：折叠引物 + 多重PCR　；参考：《中国寄生虫学与寄生虫病杂志》2015年03期

【摘要】：目的建立一种可同时检测4种疟原虫单一及混合感染的单管单轮多重PCR技术。方法在快速巢式PCR的基础上,设计折叠引物,优化PCR反应试剂的主要组分浓度及各条引物的退火温度,建立折叠引物多重PCR法(FP-PCR)。利用优化后的FP-PCR法,对间日疟原虫(Plasmodium vivax)、恶性疟原虫(P.falciparum)、卵形疟原虫(P.ovale,包括wallikeri亚种)和三日疟原虫(P.malariae)的单一及混合疟原虫DNA模板进行检测,分析该方法的敏感性和特异性。结果在7次重复实验中,4种疟原虫单一感染检出的最低浓度均接近1个/μl血。此外,在检测2~4种原虫以高、低密度相差100倍的模拟混合感染的滤纸血样中,FP-PCR法正确检出每个组合的所有虫种的成功率为68%(57/84)。在10份健康人滤纸血样中,FP-PCR法除产生少量二聚体外无任何扩增条带。结论 FP-PCR法仅需单管单轮扩增即可同时检测4种人疟原虫单一或混合感染,为疟疾的监测和筛查提供了简便、可靠的诊断方法。
[Abstract]:Objective to establish a single tube multiplex PCR technique for simultaneous detection of single and mixed infection of four Plasmodium species. Methods on the basis of fast nested PCR, the folding primers were designed, the main component concentration of PCR reaction reagent and annealing temperature of each primer were optimized, and the multiplex PCR method of folding primers was established. The single and mixed DNA templates of Plasmodium vivaxum, P. falciparumum, P.ovaleincluding wallikeri subspecies of Plasmodium vivaxi, P. falciparum, P.ovale and P. malariae were detected by optimized FP-PCR method. The sensitivity and specificity of the method were analyzed. Results in the 7 repeated experiments, the lowest concentration detected by single infection of four Plasmodium species was close to 1 / 渭 l blood. In addition, FP-PCR was used to detect four protozoa species with a high and low density difference of 100 times to simulate the mixed infection in filter paper blood samples. The success rate of detecting all species of each combination by FP-PCR was 68 / 57 / 84. There were no amplified bands except a small amount of dimer produced by FP-PCR in 10 healthy human filter paper blood samples. Conclusion the method of FP-PCR can be used to detect the single or mixed infection of four Plasmodium falciparum at the same time, which provides a simple and reliable diagnostic method for malaria surveillance and screening.
【作者单位】：贵州省疾病预防控制中心;
【基金】：贵州省卫生计生委科学技术基金(No.gzwjkj2014-1-001)~~
【分类号】：R531.3;R440

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