[Establishment of a multiplex PCR system to detect plasmodium]

Abstract

Aim: To establish a simple, rapid and practical multiplex PCR system to detect Plasmodium vivax(P. v) and Plasmodium falciparum(P. f).

Methods: A common upper primer S1 and two species-specific lower primers of P. v and P. f, S2, S3 were designed according to the sequences of the small subunit ribosomal DNA (SSUrDNA) fragments of the two Plasmodium species. Using these three oligonucleotide primers, the two-temperature-point multiplex PCR system was established and applied to detect P. v and P. f in the stock blood samples of clinically confirmed patients.

Results: DNA fragments of about 705 and 575 base pairs were successfully amplified by multiplex PCR from the genomic DNA of P. v and P. f, but no fragments were obtained from that of P. knowlesi, P. cynomolgi and blood of healthy persons. By means of restriction endonuclease digestion, the amplified fragments were confirmed to be the SSUrDNA fragments of P. v and P. f as expected. This method was successfully used in detecting parasitemia 2-10 parasite/microliter whole blood. Of 104 samples tested by this system, 81 were coincident with microscopic examination. The multiplex PCR system also found 17 samples of mixed infection, which were not detected microscopically. Another 2 samples detected as P. v by microscopic examination were verified to be P. f infection by the multiplex PCR.

Conclusion: The ease of operation together with high sensitivity and specificity, particularly the sensitive detection for mixed infection in a single run of amplification suggests that the multiplex PCR system might be a useful tool for malaria diagnosis.