自疟原虫滤纸血样提取DNA的溶血保护法研究  

A hemolytic protection method for extracting DNA from Plasmodium dried blood spots

作　　者：胡家欢 黄天谊[2] 黄雨婷[2] Hu Jiahuan;Huang Tianyi;Huang Yuting(Department of Toxicology,Guizhou Center for Disease Control and Prevention,Guiyang 550004,China;Department of P3 Testing,Guizhou Center for Disease Control and Prevention,Guiyang 550004,China)

机构地区：[1]贵州省疾病预防控制中心毒理科,贵阳550004 [2]贵州省疾病预防控制中心P3实验科,贵阳550004

出　　处：《中华地方病学杂志》2025年第1期61-65,共5页Chinese Journal of Endemiology

摘　　要：目的研究和评价溶血保护法自疟原虫滤纸血样(DBS)提取DNA的效果。方法采用离心柱法、树脂法和溶血保护法分别提取统一标准制备保存的同一批DBS(1.0、10.0、100.0、1000.0、10000.0个虫/μl血)的DNA,将1.0个虫/μl血DBS提取DNA稀释10倍,制备0.1个虫/μl血的模板DNA;以提取DNA作为模板进行巢式PCR检测(≥1.0个虫/μl血DBS的模板DNA重复检测3次;0.1个虫/μl血的模板DNA重复检测30次),比较检出率和检出限(LOD);采用超微量紫外分光光度计测定3种方法自DBS(10.0、100.0、1000.0个虫/μl血)提取DNA的浓度和纯度,重复测定3次,计算DNA回收量。同时分析不同方法的适用性和所需成本等。结果3种方法自≥1.0个虫/μl血DBS提取的模板DNA的巢式PCR检出率均为3/3;0.1个虫/μl血的模板DNA,巢式PCR检出率分别为0(0/30)、73.33%(22/30)、100%(30/30),三者比较差异有统计学意义(χ^(2)=65.95,P<0.001);LOD分别为1.0、0.1、0.1个虫/μl血。3种方法提取DNA浓度、纯度、回收量比较,差异均有统计学意义(H=23.25、17.50、23.25,均P<0.001)。离心柱法步骤数为9步,提取时间为2.5 h,所需成本为33.32元/个;树脂法步骤数为7步,提取时间为20.0 h,所需成本为7.11元/个;溶血保护法,步骤数为4步,提取时间为1.0 h,所需成本为1.96元/个。结论溶血保护法是一种高效、灵敏、快速、经济的自疟原虫DBS提取DNA方法。Objective To study and evaluate the efficacy of hemolytic protection method for extracting DNA from Plasmodium dried blood spots(DBS).Methods Centrifugal column method,resin method,and hemolytic protection method were used,respectively,to extract DNA from the same batch of DBS(1.0,10.0,100.0,1000.0,10000.0 parasites/μl blood)that were prepared and preserved according to unified standards.Dilute DNA extracted from 1.0 parasites/μl blood DBS by 10 times to prepare 0.1 parasites/μl blood template DNA.Nested PCR was used to detect templates prepared by the three different methods(with template DNA of≥1.0 parasites/μl blood DBS repeated 3 times).The limit of detection(LOD)and detection rate were compared by repeating the detection of 0.1 parasites/μl blood template DNA for 30 times.The ultra-micro ultraviolet spectrophotometer was used to measure the concentration and purity of DNA extracted from DBS(10.0,100.0,and 1000.0 parasites/μl blood),three times each,and the DNA recovery amount was calculated.Meanwhile,the applicability and cost of different methods were analyzed.Results The nested PCR detection rate of the template DNA of≥1.0 parasite/μl blood DBS extracted by the three methods was all 3/3.For the 0.1 parasite/μl blood template DNA,the nested PCR detection rates were 0(0/30),73.33%(22/30),and 100%(30/30),respectively,and there was a significant difference(χ^(2)=65.95,P<0.001).The LOD was 1.0,0.1,0.1 parasite/μl blood,respectively.When comparing the DNA concentration,purity,and recovery amount extracted by the 3 methods,all showed significant differences(H=23.25,17.50,23.25,P<0.001).The centrifugal column method has 9 steps,an extraction time of 2.5 h,and the cost for each sample was 33.32 yuan.The resin method has 7 steps,an extraction time of 20.0 h,and the cost for each sample was 7.11 yuan.The hemolytic protection method has 4 steps,an extraction time of 1.0 h,and the cost for each sample was 1.96 yuan.Conclusion Hemolytic protection method is an efficient,sensitive,rapid,and economical metho

关 键 词：疟原虫属 滤纸血样 DNA提取 溶血保护法 巢式PCR 

分 类 号：R382.31[医药卫生—医学寄生虫学]