Effects of different nucleic acid extraction methods in real-time fluorescence quantitative PCR detection of Plasmodium falciparum
Objective To compare the amplification effects of different nucleic acid extraction methods in fluorescence quantitative PCR detection of Plasmodium falciparum,and provide a basis for selecting different methods and kits for different laboratory needs.Methods The anticoagulated blood of healthy people was used to dilute the falciparum malaria blood samples according to the ratio of 1∶10,1∶100,1∶1 000,1∶10 000,1∶100 000,1∶1 000 000,using the magnetic bead method and the filter paper blood spin column method.The reagent extraction method provided with the detection kit was used to extract and detect the nucleic acid of each dilution sample.Each method was repeated three times for each sample to be carried out in parallel.Results When the dilution of the nucleic acid sample extracted by the magnetic bead method reached 10-6,the three parallel samples were all negative.When the dilution of the nucleic acid samples extracted by the filter paper blood spin column method reached 10-4,the test was negative.Except for one original blood sample,the nucleic acid samples extracted by the extraction reagents included in the test kit tested positive,and all other samples tested negative.For nucleic acids extracted by the magnetic bead method,the average CT values of the original blood sample,1∶10,1∶100,and 1∶1 000 dilutions were 16.18,20.48,22.76,and 24.51 respectively,which were significantly lower than the CT values of using the filter paper blood spin column method(22.50,26.51,30.58,32.63);the differences were all statistically significant(t=-16.041,-6.133,-8.737,-5.580;all P＜0.05).According to the initial copy number calculated by the absolute quantification standard curve,the initial copy number of each dilution of DNA extracted by the magnetic bead method was greater than that of the filter paper blood spin column method.Conclusions Among the three nucleic acid extraction methods,magnetic bead method and dried blood spot spin column method were more effective than the method provided by the kit.Magnetic bead method is suitable for large-scale sample screening and efficient detection of malaria outbreaks while dried blood spot spin column method is suitable for long-term preservation of samples.
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万方数据
