Nucleotide sequences of Plasmodium vivax A-type rDNA ITS1, 5.8S, and ITS2.Elaboration of retrospective PCR diagnostics of malaria using stained thick blood smears

Stages life cycle of the malaria parasite differ in the rate of replication and the structural properties of functionally active A-, S-, and O-type ri bosomes. Regions of A-type rDNA including ITS1, 5.85, and ITS2 from two str ains of Plasmodium vivax with different incubation periods were amplified a nd sequenced. No substantial differences in the sequences of two strains we re revealed. Phylogenetic analysis of the obtained and homologous sequences of ITS I rDNA of A, S, and O types of P. vivax; A and S types of P. falcip arum; and Cryptosporidium parvum, Eimeria maxima, Toxoplasma gondii as outg roup, by the maximum parsimony method using PAUP 4.0 revealed that divergen ce of ITS I might have occurred after speciation and at different rates in individual lineages of the Plasmodium genus. Basing on the results of the a nalysis of orthologous sequences of I! I vivax and P. falciparum, we develo ped genus- and species-specific primers for PCR diagnostics of malaria, as well as a one-step effective method of DNA isolation from Giemsa-Romanovsky -stained thick blued smears. It was demonstrated that stained preparations could he a reliable source of plasmodial DNA, and the quality of preparatio ns and storage time (10-20 years) did not interfere with the results of PCR analysis.