基于AS-PCR技术pfdhps耐药位点快速检测平台的建立及评价  

A rapid and SNPs detection platform for Plasmodium falciparums ulfadoxine resistance based on allelespecific PCR

作　　者：朱辉银 李昱婷[1] 李芸 宋晓楠 程唯佳 李健 ZHU Huiyin;LI Yuting;LI Yun;SONG Xiaonan;CHENG Weijia;LI Jian(School of Basic Medical Sciences,Hubei University of Medicine,Shiyan 442000,Hubei,China;Department of Pediatrics,Taihe Hospital,Hubei University of Medicine)

机构地区：[1]湖北医药学院基础医学院,湖北十堰442000 [2]湖北医药学院附属十堰市太和医院

出　　处：《中国病原生物学杂志》2023年第8期899-904,共6页Journal of Pathogen Biology

摘　　要：目的磺胺多辛-乙胺嘧啶(SP)是WHO推荐的非洲地区间歇性预防治疗孕妇及儿童疟疾的主要药物。SP耐药性(SPR)的出现及传播给疟疾防控带来严峻挑战。恶性疟原虫二氢叶酸还原酶基因(pfdhfr)和二氢蝶酸合酶基因(pfdhps)作为SP耐药分子标记被广泛用于SPR监测。本研究旨在建立一种快速、低成本的pfdhps检测平台,为疟疾精准防控提供新的分子检测方法。方法基于等位基因特异性PCR(AS-PCR)原理,针对每个单核苷酸多态性(SNPs)位点设计野生型及突变型引物,对引物的倒数第三个碱基增加人工错配及硫代修饰。以人工构建的pfdhps重组质粒为模板,筛选最佳PCR体系及反应条件,建立pfdhps快速检测平台,以琼脂糖凝胶显影的方式呈现基因型检测结果。以梯度稀释的质粒为扩增模板,评估检测方法的灵敏度和特异性。结果针对pfdhps的不同位点,表现出不同的检测灵敏度及特异性,其中突变率最高的437位点质粒DNA灵敏度达10^(3)拷贝/μL,无非特异性扩增;540位点检测灵敏度为10^(4)拷贝/μL,但在质粒浓度大于10^(8)拷贝/μL时出现非特异性扩增;581位点检测灵敏度为10^(6)拷贝/μL,特异性良好。结论基于AS-PCR技术、辅以人工碱基错配及硫代修饰建立的快速基因分型检测方法具有较高的检测灵敏度及特异性,该方法不仅限于抗疟药物耐药基因检测,还可扩展至其他传染病的快速诊断以及遗传性疾病、肿瘤的快速诊断。Objective Sulfadoxine-pyrimethamine(SP)is the main drug recommended by theWHO for intermittent preventive treatment of malaria in pregnant women and children in Africa.The emergence and spread of SP resistance(SPR)poses a serious challenge to malaria control.Plasmodium falciparum dihydrofolate reductase(pfdhfr)and dihydropteroate synthase(pfdhps)genes are widely used as molecular markers for SPR surveillance.This study aim to establish a rapid and low-cost detection platform for pfdhps,providing a new molecular detection method for theprecise prevention and control of malaria.Methods Based on the principle of allele-specific PCR(AS-PCR),wild-type and mutant primers were designed for single nucleotide polymorphisms(SNPs)loci,with artificial mismatches and phosphorothioate modification added to the penultimate base of the primers to improve primer specificity.The pfdhps recombinant plasmids were used as templates,and the optimal PCR system and reaction conditions were selected to establish a rapid detection platform for pfdhps,and the genotype detection results were presented on agarose gel.The sensitivity and specificity of the assay were evaluated by using gradient diluted plasmids as amplification templates.Results For different loci of thepfdhps gene,the sensitivity and specificity were different.Among these loci,locus 437,with the highest mutation rate,had a detection sensitivity of 10^(3) copies/μL and no nonspecific amplification;locus 540 had a detection sensitivity of 10^(4)copies/μL,but nonspecific amplification occurred when the plasmid concentration was>10^(8) copies/μL;and locus 581 had a detection sensitivity of 10^(6)copies/μL with high specificity.Conclusion The rapid genotyping technique based on AS-PCR,supplemented with artificial base mismatches and phosphorothioate modification,has high detection sensitivity and specificity,and is not only limited to the detection of antimalarial drug resistance genes,but can alsobe extended to the rapid diagnosis of other infectious diseases,hereditary dise

关 键 词：等位基因特异性PCR 磺胺多辛 二氢蝶酸合酶基因 单核苷酸多态性 恶性疟原虫 耐药性 

分 类 号：R382.3[医药卫生—医学寄生虫学]