不同核酸提取方法在恶性疟原虫实时荧光定量PCR检测中的效果  

Effects of different nucleic acid extraction methods in real⁃time fluorescence quantitative PCR detection of Plasmodium falciparum

作　　者：董小蓉[1] 孙凌聪[1] 周晓蓉[1] DONG Xiaorong;SUN Lincong;ZHOU Xiaorong(Hubei Provincial Center for Diseases Control and Prevention,Wuhan,Hubei 430079,China)

机构地区：[1]湖北省疾病预防控制中心,湖北武汉430079

出　　处：《热带医学杂志》2024年第4期490-493,508,共5页Journal of Tropical Medicine

基　　金：湖北省自然科学基金(2020CFB130)。

摘　　要：目的比较不同核酸提取方法在恶性疟原虫荧光定量PCR检测中的扩增效果,为实验室的不同需求选择不同方法和试剂盒提供依据。方法以健康人群抗凝血对恶性疟血样标本按照1∶10、1∶100、1∶1000、1∶10000、1∶100000、1∶1000000进行倍比稀释,采用磁珠法、滤纸血离心柱法和检测试剂盒自带试剂抽提法对每个稀释度样本进行核酸提取和检测,每个方法每份样本分成3份平行开展。结果磁珠法提取的核酸样本稀释度到10-6时,3份平行样本检测均为阴性;滤纸血离心柱法提取的核酸样本稀释度到10-4时,检测均为阴性;检测试剂盒自带抽提试剂提取的核酸样本除了原血样有1份检测为阳性外,其余所有样本检测均为阴性。磁珠法提取的核酸,原血样、1∶10、1∶100、1∶1000稀释度下其检测CT均值分别为16.18、20.48、22.76、24.51,明显小于采用滤纸血离心柱法的CT值22.50、26.51、30.58、32.63,差异均有统计学意义(t=-16.041、-6.133、-8.737、-5.580,P均<0.05)。磁珠法除了1∶10稀释度的CV值小于滤纸血离心柱法,其他浓度的样本CV值均大于滤纸血离心柱法。根据绝对定量标准曲线计算的初始拷贝数,磁珠法提取的各稀释度DNA初始拷贝数大于滤纸血离心柱法。结论3种核酸提取方法中,磁珠法和滤纸血离心柱法效果较好,磁珠法适合大规模样品筛查及突发疟疾疫情的高效检测,滤纸血离心柱法适合长期保存样本的检测研究。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∶1000,1∶10000,1∶100000,1∶1000000,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∶1000 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

关 键 词：核酸 恶性疟原虫 磁珠法 离心柱法 实时荧光定量PCR 

分 类 号：R382.31[医药卫生—医学寄生虫学]