Study On Gene Detection Techniques For Malaria Vector Investigation

Malaria is the arthropod-borne infectious disease which is seriously endangered to the human's health. Malaria, AIDS (Acquired Immune Deficiency Syndrome) and TB (Tuberculosis) are recommended to be three urgent public health problems in the world by the world health organization (WHO). Vector control is the principal component in malaria integrated control measures and the effect of vector control measures relies on the accurate vector investigation data to a great degree. The malaria vector's species, density, blood preference and their competence of transmissing malaria are important contents in vector investigation and the scientific evidence to implement integrated control measures. With the development of molecular biology, the technique for anopheles mosquito classification in species population and infra-species has been developed. However, the determination of sporozoite rate and the detection of human blood index now mainly depend on the microscopic examination and the serological methods. These methods are not suitable for the large-scale malaria vector investigation because of their complex operation and limited sensitivity and specificity.In view of this, this study establishes the gene-based methods for detecting human blood index and determining the sporozoite rate of anopheles mosquito standing on the requirements of vector investigation in malaria field control. The study includes two parts:Part I: Study on gene detection technique for detecting the human blood index of Anopheles mosquitoObjective: To develop a gene-based technique to detect human blood index of anopheles mosquito.Methods: A pair of specific primer was designed according to the sequence of human rDNA. The blood DNA from different sources, DNA extracted from mosquito without bloodsucking and DNA from the mosquitoes after their bloodsucking for different time(such as 1 h, 6 h, 12 h, 18 h, 24 h, 27 h, 30 h, 33 h, 36 h, 40 h, 44 h, 48 h)were detected to verify the specificity and to determine the sensitivity of the new gene detection method.Results:i. The specific PCR product(519 bp)was amplified from the DNA extracted from human blood using the new gene detection method. No specific PCR product was found either from the blood of other animals or from the mosquitoes without bloodsucking.ii. The specific bands were produced from all the mosquitoes within bloodsucking for 24 h. After bloodsucking for 27 h, 30 h, 33 h and 36 h, only 4, 4, 2, 1 mosquito could produce specific bands in the total of 5 tested mosquitoes, respectively. No specific PCR product was amplified after feeding for 40 h.iii. Logistic regression analysis indicated there was a negative correlation between the bloodsucking time and the quantity of positive mosquitoes detected by PCR after bloodsucking for 24-40 h(P<0.01).Conclusion: The new gene detection technique developed in this study can identify human blood in Anopheles sinensis within bloodsucking within 24h accurately, and this method can replace the traditional immunological method and can be used to detect human blood index of anopheles mosquito in vector investigation.Part II: Study on gene detection technique for detecting the plasmodium sporozoite in mosquitoI Establishment of SYBR Green I real-time PCR method for detecting and identifying plasmodium sporozoite in mosquitoObjective: To develop a SYBR Green I real-time PCR method for detecting and identifying plasmodium sporozoite in mosquito.Methods: Based on the sequence of Plasmodium 18S rDNA, the primer set was designed based on the genus-specific region around the species-specific region to detect the sporozoite in mosquito. The real-time PCR reaction was optimized using the plasmodium vivax 18S rDNA recombinant plasmid (pGEM-V) which was constructed in our laboratory. The mixture of serial dilution pGEM-V and negative anopheles mosquito DNA was used as the PCR template to make the standard curve and study the sensitivity of the new method. The four plasmodium 18S rDNA recombinant plasmid(pGEM-V,pGEM-F,pGEM-O,pGEM-M) and three species of anopheles mosquito(Anopheles sinensis, Anopheles anthropophagus, Anopheles stephensi) reared in our laboratory were used to study the specificity. According to the different sequence of Plasmodium spp.18S rDNA region, the melting curve analysis was conducted following real-time PCR to identify species of four Plasmodium spp.by the different melting temperature(Tm).Results:i. The optimum reaction system of the real-time PCR: In a total volume of 20μl,containing 1xPower SYBR Green PCR Master Mix, primer 0.2μM and 1μl template DNAii. The optimum reaction condition of the real-time PCR: After pre-denaturing at 95℃for 10min,under the condition of denaturing at 95℃for 15s,annealing/extension at 60℃for 1min,amplifying for 45 cycles. The instrument gives the melting curve analysis automatically after the PCR.iii. The sensitivity of the real-time PCR: The standard curve indicated a good linear relationship between the cycle threshold (Ct) and template concentration(r= -0.99).The minimum detection level is 50 copies plasmid DNA in 1μl mosquito DNA.iv. The specificity of the real-time PCR: The effect of amplification for four plasmodium 18S rDNA recombinant plasmid is good and there is no specific amplification for mosquito DNA and human blood DNA. The Tm of Plasmodium malariae, falciparum, ovale and vivax are 71.0℃, 72.7℃, 73.9℃, 75.9℃, respectively. The four plasmodium species can be identified by the analysis of the Tm.v. The reproducibility of the experimental results: The amplification curve and the Tm showed good reproducibility both in the same and different assay.Conclusion: The SYBR Green I real-time PCR technology developed in this study shows the high sensitivity and specificity and it can be used for quantitative detection and species identification of sporozoite in mosquito. II Study on detection of plasmodium vivax sporozoite using real-time PCRObjective: To verify the feasibility of the SYBR Green I real-time PCR technology in detecting plasmodium vivax sporozoite in Anopheles sinensis.Methods: Collect the blood samples of the vivax malaria patients and feed the Anopheles sinensis using the laboratory in vitro artificial mosquito feeding system. The DNA was extracted from the cephalothoraxes of mosquitoes 14-16 days post-infection and was detected using the real-time PCR.Results:i. The new developed real-time PCR can amplify the DNA of the sporozoite in Anopheles sinensis specifically and the Tm is the same as the plasmodium vivax 18S rDNA recombinant plasmid. The sequence of the product was conformed to be the plasmodium 18S rDNA specific by the sequence alignment. There is no specific amplification from uninfected Anopheles sinensis.ii. Using the new real-time PCR, the traditional dissection technique and the nested-PCR to detect the eight mosquitoes 15days post-infected, five ones were positive, three ones were negative. The positive rate is higher than that of the nested-PCR and similar with the result of the microscopic examination.iii. The DNA of infected mosquito can be detected when it was diluted 32 times by negative mosquito DNA. This suggests that the method can be used in pool sample detection for its high sensitivity.Conclusion: The SYBR Green I real-time PCR technique developed in this study can be used for detecting sporozoite in mosquito vector with the high sensitivity and specificity. The operation of the technique is simple and fast and it holds the promise to be popularized and applied in the field vector investigation after further optimization.