单管单轮多重PCR检测4种疟原虫混合血样

Single-tube Single-run Multiplex PCR Detects Mixed Samples with Four Species of Plasmodium

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。

Objective To establish a single-tube single-run multiplex PCR technique that can detect single or mixed samples with four species of Plasmodium. Methods Folding primers were designed based on the fast nested PCR. The reaction component concentrations were optimized and the primers were selected based on the annealing temperature. The established single-tube single-run folding-primer multiplex PCR （FP-PCR） was tested for its sensitivity and specificity to detect single-species and mixed samples with P. vivax, P. falciparum, P. ovale （including P. ova/e wallikeri） and/or P. malariae. Results In all the seven experimental repeats, FP-PCR successfully detected single- species infection for all the four species, with the detection limit reaching or close to 1 parasite/μl blood. For mixed infections with 2-4 species at different densities with the highest being 100 times of the lowest, FP-PCR identified all the species in each combination in 57 out of 84 tests. Further, in 10 dried blood samples on filter paper from healthy subjects, no FP-PCR amplification was found, except weak formation of dimers. Conclusion FP-PCR is a simple and sensitive method for detecting both single-species and mixed infections with human Plasmodium, and can be applied for malaria diagnosis, screening and monitoring.