Abstract: Objective To establish a sensitive, simple to use and low noise nested/multiplex PCR for simultaneously detection of Plasmodium falciparum （P.f） and Plasmodium vivax （P.v）. Methods The tag primer amplification technique, software Primer Premier 5.0, NCBI-BLAST web resources and the matrix test were used to optimize the nested / multiplex PCR for detection of P.f and P.v with filter paper blood samples taken from malaria patients diagnosed by microscopy, and the results of the optimized nested/multiplex PCR and microscopy were evaluated. Results The sensitivity of the optimized PCR, determined by the examination of imitative filter paper blood samples, was about 1-2 parasites / μl for P.f and 5-10 parasites / μl for P.v. Primer-dimer and other PCR noise were removed. When 71 field filter paper blood samples taken from microscopically diagnosed patients (24 P.f, 47 P.v) were examined, the concordance between the optimized PCR and microscopy was 87.5% for P.f and 100% for P.v. Conclusion The nested/multiplex PCR optimized by tag primer amplification technique is simple, with low noise and being able to detect P.f and P.v simultaneously. It is more sensitive in detecting cases with low parasitaemia and more accurate in identifying Plasmodium species than microscopy.

Key words: Plasmodium falciparum, Plasmodium vivax, PCR, Tag primer, Malaria diagnosis