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Research and Development of Genetic Diagnostic Method of Malaria Based on Fluorescent Quantitative Polymerase-Chain-Reaction(FQ-PCR)

Author: JiangXiaoLing
Tutor: LiMing
School: First Military Medical University
Course: Immunology
Keywords: Malaria P. vivax Plasmodium falciparum PCR Quantitative PCR Quantitative PCR
CLC: R446.9
Type: Master's thesis
Year: 2000
Downloads: 343
Quote: 2
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Abstract

Malaria is widely prevalent in tropical, subtropical and even temperate edge of an important insect-borne diseases pose a great risk to human health, the prevention and treatment of attention by the international medical community. Fast and effective malaria diagnostic methods, has become an urgent need of the malaria problem; development of simple, rapid quantitative detection of Plasmodium, more accurate quantitative detection of Plasmodium, not only in the degree of infection with malaria research Plasmodium, the relationship as well as the monitoring of antimalarial treatment to deepen malaria research, but also to provide a more objective and credible quantitative data for vaccine development and evaluation of new drugs and malaria. Over the past decade the rapid development of the polymerase chain reaction (PCR) technology and its based on quantitative techniques, its fast, simple and high sensitivity in the diagnosis of malaria has been widely appreciated, but there is product pollution prone to cause false-positive PCR amplification of the late \1995 a probe real-time quantitative monitoring is characterized by quantitative PCR method can effectively overcome the above disadvantages, and caused a high degree of attention of the international medical community, to replace traditional PCR pathogen detection trend. The purpose of this study is to establish the two most common malaria parasite - Plasmodium falciparum and P. vivax quantitative PCR method and its evaluation for the development of rapid and accurate quantitative detection of Plasmodium quantitative PCR Kit lay the foundation. Conservative sequence the two Plasmodium SSUrRNA gene, were designed for P. falciparum and P. vivax-specific primers and probes (probes are located between the upstream and downstream primers), and fluorescently labeled probe. The PCR product was cloned falciparum malaria were constructed between R malaria SSUrRNA target fragment of recombinant plasmid, positive standard control used for quantitative PCR, quantitative PCR amplification conditions optimization and the establishment of the standard curve. Finally, as a basis for sample testing to evaluate the microscopic comparison of the results with conventional PCR, quantitative PCR method for Plasmodium falciparum and Plasmodium vivax. The results showed that the cloned P. falciparum and P. vivax recombinant plasmid by restriction enzyme digestion and DNA sequencing of the cloned gene, indicating the size of the fragments of the cloned genes and gene sequence of the target gene is exactly the same. Quantitative PCR' dissertation">Quantitative PCR conditions worry for Mg 'best concentration of knives. 5 mmol / L, annealing extension optimum temperature of 55 ° C. Under these conditions, the malaria parasite, Plasmodium falciparum and Plasmodium vivax positive quantitative gradient template for amplification to obtain the PCR amplification kinetics curved line graph analysis showed that the type of the initial concentration of template. plateau phase after the reaction, the amount of product phase differential is small, and if the detection point as a product generated a large number of the initial number of cycles of a cycle threshold UT value), then found that the initial template concentration values ??and the CT value was good linear relationship before 12 the relationship between the number of 0.998 (P < 0 20 001, Plasmodium falciparum) and 0.996 ... tight corner .001, between R malaria), both Plasmodium detection threshold mean 100 copy / ul sample fluorescence quantitative detection are set the positive quantitative criteria template control and negative control of the CT value of each sample detection resulting curve CT template concentration curve compared, that is, to draw qualitative and quantitative results. qualitative results of the blood samples of patients 127 cases of malaria, Plasmodium falciparum microscopy, conventional PCR, quantitative PCR positive rate of detection 33.l0 39.4o, 40.2O: Q H Plasmodium microscopy, conventional Pm, fluorescence quantitative bite R positive rates were 36%, busy .5 and busy factory. Statistical analysis showed that between any two of three methods consistent with h.001), there is a difference between quantitative PCR and microscopic ... tight corner .01 There was no significant difference between the conventional PCR method towel Sichuan .05). quantitative test results of microscopic examination of blood samples showed that the linear correlation between the quantitative detection of copy number and the microscopic infection, correlation coefficients another 0.961 (p (0.001, malignant malaria) and 0.95 (a tight corner .001, vivax malaria \\ In addition, The fluorescence quantitative MR also showed high sensitivity and specificity of the sensitivity of detection of Plasmodium falciparum IXIO '% (protozoa infection rate per 100 red 3 cells, protozoa), between R malaria could occur 1.26XIO '% (the protozoan infection rate), this concentration of blood DNA template using quantitative PCR amplification typical' S-shaped curve, and microscopic in the the protozoa rate of 4x10-'% has been difficult to come to a positive result, the test results were negative and non-endemic areas of m copies of normal human blood samples. above results show that quantitative PCR has high sensitivity and specificity, and to some extent better than microscopic examination and conventional PCR method. Studies have shown that we have established at home and abroad for the first time falciparum and P. vivax quantitative PCR assay, detection of sample blood samples of wood reflects the accuracy of this method and superiority. laid the foundation for the development of the malaria parasite fluorescence quantitative PCR detection kit also provides a feasible way for more in-depth malaria research.

Research and Development of Genetic Diagnostic Method of Malaria Based on Fluorescent Quantitative Polymerase-Chain-Reaction(FQ-PCR)

Abstract

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