Establishment And Application Of A Loop-mediated Isothermal Amplification Method For Detection Of Toxoplasma Gondii

Objective: To establish a high sensitivity, specificity, simple, rapid and low-cost method to detect Toxoplasma gondii DNA .Methods: The reaction system of detection of Toxoplasma gondii DNA was set up by loop-mediated isothermal amplification (LAMP) technology, and the reaction system was optimized, the specificity and sensitivity were evaluated. On the basis, a simple method to observe the results and a quantitative protocol with SYBR Green I were set up. Finally 57 peripheral blood samples from pregnant women were tested by our LAMP system .Results: By optimizing the established LAMP system to detect Toxoplasma gondii DNA , We found that the most important influencing fact was Mg2+ ,which was more important than betaine and temperature .Only Toxoplasma gondii DNA could be amplified ,but pork tapeworm, schistosoma were failed. Enzyme digestion further proved the specificity of amplification. The detection limit reached 10 copies per reaction. Cycle threshold and concentration of template had a good linear relationship in the stand curve made by recombinant plasmid .The correlation coefficient was 0.929,and the amplification efficiency was 0.961. 2 samples were positive when detected by our LAMP system from 57 pregnant women ,and the contents were both about 103copies/μl by quantitative detection.Conclusion: The results indicated that LAMP system, which we established to detect Toxoplasma gondii DNA, not only had good specificity, sensitivity, but simple, rapid and low-cost, might play a role in clinical detection.