OBJECTIVE: To develop a new method of genotyping circumsporozoite protein (CSP) gene for identification of field isolates of Plasmodium vivax. METHODS: Improved Chelex-100 ion-exchange method was used to extract DNA from blood filter paper samples, nested PCR and allele-specific PCR techniques, agarose gel electrophoresis analysis and dot/southern blotting-probe hybridization were employed for amplification, resolution and identification of the diagnostic fragments. RESULTS: Using the nest-allele-specific PCR assay reported here, small amounts of DNA extracted from a piece of blood filter paper sample were amplified which produced three different size ranges of diagnostic bands: 650-770 bp PV-species-specific band, 170-230 bp diagnostic band for temperate zone family and 588 bp band for PV type-2. The sizes and patterns of the bands produced by the reference strains were consistent with those of designed target sequences. Of 59 examined isolates from 6 provinces of China, 42 temperate zone family strains, 15 tropical zone family strains and two PV type-2 strains were identified. CONCLUSION: 1, Three genotype strains of P. vivax mentioned above could be identified by this method with only two rounds of PCR and without probe hybridization. 2, The preliminary results showed that PV type-1 including temperate zone family strains and tropical zone family strains as well as PV type-2 strains are present in China. In addition, another CSP genotype with both sequence characteristics of temperate zone and tropical zone family might also be present in China.
OBJECTIVE: To develop a new method of genotyping circumsporozoite protein (CSP) gene for identification of field isolates of Plasmodium vivax. METHODS: Improved Chelex-100 ion-exchange method was used to extract DNA from blood filter paper samples, nested PCR and allele-specific PCR techniques, agarose gel electrophoresis analysis and dot/southern blotting-probe hybridization were employed for amplification, resolution and identification of the diagnostic fragments. RESULTS: Using the nest-allele-specific PCR assay reported here, small amounts of DNA extracted from a piece of blood filter paper sample were amplified which produced three different size ranges of diagnostic bands: 650-770 bp PV-species-specific band, 170-230 bp diagnostic band for temperate zone family and 588 bp band for PV type-2. The sizes and patterns of the bands produced by the reference strains were consistent with those of designed target sequences. Of 59 examined isolates from 6 provinces of China, 42 temperate zone family strains, 15 tropical zone family strains and two PV type-2 strains were identified. CONCLUSION: 1, Three genotype strains of P. vivax mentioned above could be identified by this method with only two rounds of PCR and without probe hybridization. 2, The preliminary results showed that PV type-1 including temperate zone family strains and tropical zone family strains as well as PV type-2 strains are present in China. In addition, another CSP genotype with both sequence characteristics of temperate zone and tropical zone family might also be present in China.