OBJECTIVE: To develop a real-time PCR method for human Plasmodium spp. qantitative detection and species identificaton. METHODS: According to the sequence of Plasmodium 18S rRNA, the primer set was designed based on the genus-specific region around the species-specfic region. The PCR products were amplified and cloned into pGEM-T vector to produce standard plasmids of real-time PCR, and melting curve analysis was conducted following real-time PCR for Plasmodium species indentification. RESULTS: By using the primer set, specific PCR products were produced from all of 4 human malaria parasites. The correlation of real-time PCR standard curve was good enough (r = -1.00) for quantitation. According to the melting curve analysis, the melting temperatures (Tm) of Plasmodium malariae, falciparum, ovale and vivax were significantly different, being 71.3, 72.8, 74.6 degrees C and 75.8 degrees C, respectively. CONCLUSION: The SYBR Green I based real-time PCR method developed in this study can be used for human Plasmodium spp. quantitative detection and species identificaton.
OBJECTIVE: To develop a real-time PCR method for humanPlasmodium spp. qantitative detection and species identificaton. METHODS: According to the sequence of Plasmodium 18S rRNA, the primer set was designed based on the genus-specific region around the species-specfic region. The PCR products were amplified and cloned into pGEM-T vector to produce standard plasmids of real-time PCR, and melting curve analysis was conducted following real-time PCR for Plasmodium species indentification. RESULTS: By using the primer set, specific PCR products were produced from all of 4 humanmalaria parasites. The correlation of real-time PCR standard curve was good enough (r = -1.00) for quantitation. According to the melting curve analysis, the melting temperatures (Tm) of Plasmodium malariae, falciparum, ovale and vivax were significantly different, being 71.3, 72.8, 74.6 degrees C and 75.8 degrees C, respectively. CONCLUSION: The SYBR Green I based real-time PCR method developed in this study can be used for humanPlasmodium spp. quantitative detection and species identificaton.