Objective: To establish a fluorescent quantitative PCR (FQ-PCR) method for quantitative detection and species identification of Plasmodium sporozoites in Anopheles mosquitoes.
Methods: One pair of human Plasmodium genus-specific primers based on 18S rRNA genes were used and the reaction system and reaction condition of FQ-PCR were optimized by using the mixture of Plasmodium 18S rRNA gene recombinant plasmids and Anopheles DNA as a template. The specificity was verified by using four Plasmodium spp. 18S rRNA gene plasmid DNA as well as mosquito DNA and the Plasmodium species was identified according to the value of melting temperature (Tm). The standard curve was made by using P. vivax 18S rRNA gene recombinant plasmids which were serially diluted by negative Anopheles DNA as a template. The sensitivity was analysed by using plasmid DNA and laboratory infected sporozoite positive mosquito DNA, respectively. The different parts and different amounts of Anopheles DNA were added into the reaction system to investigate the influence of Anopheles DNA on the assessment.
Results: There was no specific amplification for mosquito DNA and human blood DNA. There was specific amplification for Plasmodium 18S RNA gene recombinant plasmids and the Tm(s) of P. malariae, P. falciparum, P. ovale and P. vivax were 71.0, 72.7, 73.9 degrees C and 75.9 degrees C, respectively, which were easy to be identified. The standard curve indicated a good linear relationship between the cycle threshold (Ct) and template concentration (r = -0.99). The sensitivity was 50 copies of plasmid DNA or one sporozoite positive mosquito DNA diluted by 32 times of mosquito DNA. Anopheles DNA could inhibite the FQ-PCR reaction. The Ct value of amplification showed a good reproducibility both within the same experiment and among different experiments.
Conclusion: The novel SYBR Green I based FQ-PCR method developed in this study shows a high sensitivity and specificity and it can be used for quantitative detection and species identification of sporozoites in mosquitoes.