基于无性期18S rDNA特异性引物检测5种疟原虫qPCR的建立和应用  

Establishment and application of a specific primers-based qPCR targeting asexual stage 18S rDNA for detecting five Plasmodium species

作　　者：李美 肖宁 夏志贵 LI Mei;XIAO Ning;XIA Zhigui(National Institute of Parasitic Diseases,Chinese Center for Disease Control and Prevention(Chinese Center for Tropical Diseases Research),NHC Key Laboratory of Parasite and Vector Biology,WHO Collaborating Centre for Tropical Diseases,National Center for International Research on Tropical Diseases,Shanghai 200025,China)

机构地区：[1]中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心),国家卫生健康委员会寄生虫病原与媒介生物学重点实验室,世界卫生组织热带病合作中心,国家级热带病国际联合研究中心,上海200025

出　　处：《中国寄生虫学与寄生虫病杂志》2023年第1期36-43,共8页Chinese Journal of Parasitology and Parasitic Diseases

摘　　要：目的建立基于疟原虫无性期(A期)18S rDNA检测感染人的5种疟原虫的qPCR,并评价其检测效果。方法从GenBank中下载5种疟原虫A期、子饱子期(S期)、卵囊期(O期)和人的18S rDNA序列进行比对,划分保守区和变异区,在保守区设计疟原虫属特异性引物,在变异区设计种特异性引物。以5种疟原虫DNA、田鼠巴贝虫和婴儿利什曼原虫DNA为模板进行qPCR扩增,筛选特异性引物对。应用qPCR法筛选特异性引物对的最佳退火延伸温度和引物浓度。分别构建5种原虫A期18S rDNA短片段(筛选出的引物对扩增产物)和长片段(属特异性引物扩增产物)质粒,以连续浓度梯度的短片段质粒DNA[(5×10^(1))~(5×10^(8))拷贝/μl]、长片段质粒DNA[(5×10^(4))~(5×10^(9))拷贝/μl]为模板分别进行qPCR扩增,测定筛选出的引物对的扩增效率、最低检出限[包括最低质粒浓度、循环数阈值(Ct值)、变异系数]、无扩增时非目标DNA的最高浓度、可检出的混合DNA中不同虫种间的最大拷贝浓度比值。以5倍稀释的18份疟原虫血样DNA[恶性疟原虫(Pf)6份、间日疟原虫(Pv)6份、卵形疟原虫(Po)4份、三日疟原虫(Pm)2份、诺氏疟原虫(Pk)1份]为模板,应用筛选出的特异性引物分别进行qPCR、一步反转录qPCR和巢式PCR扩增,计算最低检出限时的原虫密度。用筛选出的特异性引物进行应用验证,对70份疟疾患者血样DNA(Pf 26份、Pv 14份、Pm 14份、Po 9份、Pk 3份、混合感染4份)、65份疟原虫阴性人血样DNA以及阿米巴(1份)、田鼠巴贝虫(1份)和杜氏利什曼原虫DNA(3份)进行qPCR扩增,以样品提供者的检测结果为标准,计算qPCR检测的敏感度、特异度、符合率。不同PCR方法检测结果的比较采用卡方检验、t检验、Mann-Whitney检验或Wilcoxon秩和检验。结果序列比对结果显示,5种疟原虫A期18S rDNA序列的一致性为89.3%~96.7%,高于S期的60.0%~92.1%,可分为9个保守区和8个变异区。除Pv O期和Pm Objective To establish a qPCR method for detecting the 18S rDNA of the asexual stage(stage A)of 5 Plasmodium species and to evaluate its detection efficacy.Methods The 18S rDNA sequences of stage A,sporozoite stage(stage S)and oocyst(stage O)of five human malaria parasite species and the sequence from human were downloaded from GenBank and aligned to confirm the distribution pattern of the conserved and variable regions.Specific primers of Plasmodium genus were designed in the conserved regions and the specific primers of species were designed in the variable regions.Specific primer pairs were screened by the qPCR amplifying the DNA of the five Plasmodium species,Microtus babesi and Leishmania infantis as templates,and the optimal annealing extension temperature and primer concentration of the specific primer pairs were screened.The plasmids of short 18S rDNA fragments(amplified with screened primers)and long 18S rDNA fragments(amplified DNA fragments with conserved primers)of the five Plasmodium species of stage A were constructed respectively.The qPCR amplification was carried out with plasmid DNA[(5×10^(1))-(5×10^(8))copy/μl]of the short fragments and plasmid DNA[(5×10^(4))-(5×10^(9))copy/μl]of the long fragment as templates,and the amplification efficiency,the minimum detection limit[including the minimum plasmid concentration and the corresponding cycle threshold(Ct)value,the coefficient of variation of repeated detection],the maximum concentration of non-target DNA when there was no amplification,and the maximum copy concentration ratio between the different Plasmodium species in the detectable mixed DNA were determined respectively.Using the DNA of the 18 blood samples infected with Plasmodium parasites(6 samples of P.falciparum,6 samples of P.vivax,2 samples of P.malariae,4 samples of P.ovale,and 1 sample of P.knowlesi),which were diluted by 5 folds as the template,the selected specific primers were used for qPCR and one-step reverse transcription qPCR amplification respectively,and nested PCR ampli

关 键 词：疟原虫 染料法qPCR 一步反转录qPCR 最低检出限 

分 类 号：R382.31[医药卫生—医学寄生虫学]