| Objective:(1)To explore the Toxoplasma gondii tachyzoites culture in vitro, cryopreservation and purification methods which will provide rich and purified tachyzoites for the following researches (2)To compare five kinds of tachyzoites DNA extraction methods and select the relatively simple and efficient DNA extraction method for the following experiments (3)To find a high sensitivity, specificity, simple and rapid method to detect DNA of Toxoplasma gondii,the loop-mediated isothermal amplification (LAMP) reaction system were established and optimized.Methods:(1)The tachyzoites were inoculated in HeLa cells and continually observed the procession of tachyzoites invaded in HeLa cells.(2)7% dimethyl sulfoxide and 15% of ethylene glycol were respectively added to tachyzoites,which were observed of the two kinds of anti-freeze frozen protective effect for tachyzoites at -76℃within 6 months. (3)Comparison of the tachyzoites cultured in vivo and vitro purification effect by different kinds of filter membrane (diameter 3,5,8μm ).(4) To extract tachyzoites DNA in 10-fold serial dilutions at a concentration down to one tachyzoite by DNA extraction kit, schizolysis, boiling,phenol-chloroform extraction and salt fractionation of DNA extraction,then detected DNA quantity and purity by using eppendorf biophotometer and agarose gel electrophoresis (5) Based on Loop-mediated isothermal amplification have been established to detect Toxoplasma gondii DNA, and the reaction system was optimized. the specificity, sensitivity of this method was assessed by comparing with traditional PCR.Results:(1)Toxoplasma gondii tachyzoites growth well in Hela cells. Hela cells were completely destroyed and the tachyzoites increased peak during 96 ~ 144 hours after tachyzoites were inoculated in Hela cells. It was the best time to collect the tachyzoites for experiments. (2)Observing 7% dimethyl sulfoxide and 15% of ethylene glycol cryopreservation protective effect for tachyzoites at -76℃, The result suggested that at the end of the first month there was no significant difference between the two antifreezing agent. At the end of the third and the sixth month,7% dimethyl sulfoxide protective effect of cryopreservation was better than 15% of ethylene glycol. With time passing the survival rate of tachyzoites was downing, but at the end of the six month both the tachyzoites had successfully infected mice which frozen with the former two antifreezing agent. (3) When the tachyzoites cultured in vivo were purified , the white blood cell clearance rates, which filtered by 3 kinds of filter membrane(diameter 3,5,8μm ),were no significant difference(P >0.05).The red blood cell clearance rate was reducing with increasing of the membrane pore's diameter and the red blood cell clearance rate among the three different pore filter membrane has significant differences(P<0.05); tachyzoites recovery rate increased with the filter membrane pore size enlargement, and there were significant differences among the three different filter membranes(P<0.05). In vitro,the Hela cell clearence rate ,which purified by 3 kinds of filter membrane(diameter 3,5,8μm ),has no significant difference(P >0.05).Tachyzoites recovery rate increased with the filter membrane pore size enlargement and there were significant differences among the three different filter membranes(P<0.05). (4) The result showed that the DNA purity extraceted by phenol-chlorform extraction,boiling, salt fractionation, schizolysis method, DNA extraction kit in turn decreased. The electrophoresis strip of genome DNA which extract by the phenol-chlorform, schizolysis , salt fractionation.with 1×106 tachyzoite was present.Using DNA extraction kit,schizolysis method, boiling method,phenol-chloroform extraction method and salt fractionation method which could detect the limit tachyzoit in turn were 1×104 ,no strip, 1×102,1×101,1×103 by PCR. (5) Through the optimization of a LAMP amplification reaction system of Toxoplasma gondii DNA, It suggested that 1.2 mM dNTPs, 6mM MgSO4, 8U Bst DNA polymerase for 25μl reaction system were better,The optimization reaction temperature was 63℃and the reaction time could be selected 30 ~ 60min .It was failed to find the ring primers could speed up the reaction velocity. Betaine was little effect on the reaction. Specific assessment indicates that the only test positive for Toxoplasma gondii. LAMP sensitivity is 10 times higher than conventional PCR and could detect single-digit number of tachyzoites. When detected the tissue samples of mice infected by Toxoplasma gondii,the result of the two methods was non-discriminatory. In addition to agarose gel electrophoresis detection of reaction results, It also could detect by turbidimetric instrument, centrifugal sedimentation and UV lamp after adding fluorescent dye.Conclusion:Hela cells can be used to culture Toxoplasma gondii tachyzoites in vitro.The tachyzoites added antifreeze could be stored in refrigerator for more than half a year at -76℃.According following experiments to choose membranes of different pore sizes used in purifying Toxoplasma gondii tachyzoites.Tachyzoites DNA extracted by classic phenol-chloroform extraction had high purity, but the operation is relatively more complicated.Direct boiling method is convenient and can meet common requirements for DNA extraction.This study established the LAMP detection of Toxoplasma gondii genomic DNA,which is specificity, sensitivity, simple, fast and low cost.By continuous improvement,it is expected to be a new method applied to diagnosis of Toxoplasma gondii in Clinical Medicine.
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