关键词: 折叠引物, 多重PCR, 恶性疟原虫, 疟疾诊断

Abstract:

【Abstract】 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. ovale 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.

Key words: Folding primer, Multiplex PCR, Plasmodium falciparum, Malaria diagnosis