New Methods Of Molecular Diagnosis Of Malaria Plasmodium And Polymorphism Of Plasmodium Falciparum Histidine-rich Protein 2 Gene

Malaria is a kind of infections caused by plasmodium parasitized in human beings, and together with AIDS and tuberculosis ranked as the world’s three major public health problems by the World Health Organization (WHO). The cure and elimination of malaria is widespread concerned around the world. Clinical diagnosis mainly depends on the microscopic examination and immunology rapid diagnostic test (RDT) method. However, microscopic examination in the diagnosis is low sensitive and difficulty and heavily dependent on experienced microscopists. Immunological method is strongly influenced by environmental conditions and malaria parasite polymorphism, and the misdiagnosis rate is higher when parasite density is low. Certain gene’s (pfhrp2) absence would also affect diagnostic sensitivity. In molecular diagnosis method, the PCR technology has the advantages of high sensitivity, and strong specificity etc., which can make accurate test and diagnosis for the different types of malaria parasite. But due to the expensive cost, high demand of template preparation and complicated operation of this method, the application of this technology was limited in mass malaria epidemic investigations. Aim at the present problems existing in the malaria parasite test and diagnosis, this research developed new malaria diagnosis method and improved the test means. We analyzed the plasmodium falciparum hrp2 gene polymorphism of specific areas, in order to provide theory basis for the use of immunology rapid test methods in diagnosis of malaria.1) This research firstly set up a malaria parasite test method (FP-PCR) by directly used malaria parasite filter paper blood samples of saponin normal temperature processed. This method reduced the time of single sample preparation to 10-12 minutes from the required 90-100 minutes by using Qiagen kit, which greatly improved the timeliness of test. The preparation cost was also reduced to one over forty of the original, effectively reduced the test cost. The test limit of this method reaches to 0.2 parasite/μL. The test sensitivity is 50 times higher than the current clinical microscopy method. At the same time, the template preparation does not need heating procedure, avoiding samples cross contamination.2) On the basis of simplifying the tedious operation of template preparation and reducing the cost of PCR reaction, further optimized the second step reaction system of PCR set, the original four tubes reaction to identify different plasmodium species was simplified and four different malaria parasite species can be identified in single tube reaction system. And a multiplex system of the malaria parasite PCR diagnostic technology (Multi-FP-PCR) was established. The method has the advantages of lower reactions, reducing reagent consumption, simplifing the configuration and operation, and at the same time, reducing more than half of the test time of the PCR reaction. The optimized specific primers improved clinical practicality of the test.3) This research also explored the polymorphism of plasmodium falciparum hrp2 gene, and found 4 cases missing parasite strains. Further studies will focus on how plasmodium falciparum hrp2 gene would affect the sensitivity of RDT.