Research And Development Of Genetic Diagnostic Method Of Malaria Based On Fluorescent Quantitative Polymerase-Chain-Reaction(FQ-PCR)

Malaria remains one of the leading causes of morbidity and mortality in the tropical and subtropical regions. 300-500 million people are infected and nearly 3 million are killed by this disease in the world each year. Therefore, finding better ways to prevent and treat malaria is a high priority for malaria research. Fast and accurate diagnosis at early stage, which is one of the key measures for malaria control, remains to be improved urgently. At the same time, convenient and accurate quantitative detection method, which will be very useful for evaluation of new antimalaria drugs or vaccine, is very necessary to be developed. Although the PCR assay and some relative quantitative assays have been widely used for their convenience and high-sensitivity, the relatively high false positive results and deficiency in accurate quantitation have restricted its application. A real-time quantitative PCR method---TaqMan assay (also named as Fluorescent Quantitative Method, FQ-PCR), which was developed in 1995, is being attached a lot of importance because it can overcome the aforesaid, shortcomings. In this project, we aimed to establish FQ-PCR methods to detect two common malaria parasites--- Plasrnodi urn falciparuni and P/a smodiurn vi~抋x respectively. According to the nucleotide sequence of small subunit rRNA (SSUrRNA) of human malaria parasite, we designed specific primers and 5 probes for each (the probe is overlap the sequence between the primers), and labeled fluorescent material to the probes. The PCR products were then cloned into T-vector separately and the recombinant plasmids were constructed. The recombined plasmids, used as standard quantitative control, were diluted into 10% iOn,. 10% iOn,. 10% 102,. 10?copies / ~?1 and run the PCR program in PE 7700 instrument to evaluate the PCR amplifying efficiency under different conditions. Thus, the amplifying conditions were optimized and the standard quantitative curves were gained. Finally, referred to the standard curves, blood samples were tested by the newly developed method. Comparing the results by FQ-PCR with those by common PCR and microscopy, the efficacy of this new method was estimated. The results showed that the cloned genes of Pf and P v in recombined plasmids were accordant with the target genes in their size and nucleic acid sequences. As to the FQ-PCR amplifying, under the condition that Mg2~ at the concentration of 5.Ommol/L, annealing and extending temperature at 55 0C, the kinetics curves showed the highest efficiency and best relationship between the 搕hreshold cycle(CT)?and the initiate DNA concentration, with the coefficient above 0.990. Both the threshold values of detection for Pf and Pv are 100 Copies per microliter. An appropriate DNA extraction method was selected before blood samples were detected. Positive quantitative control and negative control were also set up as well each time when the samples?DNA templates were amplified. And the samples?initiate concentrations were acquired by comparing their CT value with the CT-concentration curves from the standard controls. According to the results of 127 blood samples from 6 malaria area detected by three methods respectively , the FQ-PCR has showed the highest positive rate. The detective rate of P.f po~tiv...