基于重组酶聚合酶扩增技术快速检测间日疟原虫方法的建立及应用  

Establishment and application of a rapid detection method for Plasmodium vivax based on recombinase polymerase amplification

作　　者：罗鹏 孙菲 张荣 吕沁风 阮卫[4] 娄家怡 陈淑丹 杨国平 LUO Peng;SUN Fei;ZHANG Rong;LYU Qinfeng;RUAN Wei;LOU Jiayi;CHEN Shudan;YANG Guoping(Hangzhou International Travel Healthcare Center(Hangzhou Customs Port Clinic),Hangzhou,Zhejiang 310012,China;Hunan International Travel Healthcare Center(Changsha Customs Port Clinic),Changsha,Hunan 410114,China;Jiangsu International Travel Healthcare Center(Nanjing Customs Port Clinic),Nanjing,Jiangsu 210019,China;Zhejiang Provincial Center for Disease Control and Prevention,Hangzhou,Zhejiang 310009,China)

机构地区：[1]杭州国际旅行卫生保健中心(杭州海关口岸门诊部),浙江杭州310012 [2]湖南国际旅行卫生保健中心(长沙海关口岸门诊部),湖南长沙410114 [3]江苏国际旅行卫生保健中心(南京海关口岸门诊部),江苏南京210019 [4]浙江省疾病预防控制中心,浙江杭州310009

出　　处：《中华卫生杀虫药械》2025年第2期154-157,共4页Chinese Journal of Hygienic Insecticides and Equipments

基　　金：海关总署科技项目(2022HK094,2024HK267,2024HK167)。

摘　　要：目的建立一种用于快速检测间日疟原虫Plasmodium vivax的重组酶聚合酶扩增(recombinase polymerase amplification,RPA)方法。方法以间日疟原虫18 S rRNA基因为靶标,根据RPA技术原理设计引物和探针,构建18 S rRNA基因片段的质粒DNA为标准品,进行引物探针筛选和灵敏度检测;以间日疟原虫、恶性疟原虫、三日疟原虫、卵形疟原虫、人全血核酸、乙型脑炎病毒以及黄热病毒核酸评价方法的特异性;最后用实际样本来评估方法与PCR检测的一致性。结果所建立的方法在39℃20 min内,可以最低检测出10拷贝/反应的质粒DNA,并且与其他病原体核酸无交叉反应。样本检测结果显示,测试的结果同PCR检测具有一致性。结论建立的重组酶聚合酶扩增方法具有灵敏度高以及特异性强的特点,可应用于间日疟原虫的快速检测。Objective To establish a rapid detection method for Plasmodium vivax detecting using recombinase polymerase amplification(RPA)technology.Methods The 18 S rRNA gene of P.vivax was used as the target.Primers and probes were designed based on the principle of RPA technology.A plasmid containing 18S rRNA gene fragment was constructed for primers screening and sensitivity evaluating.The specificity of the method was evaluated using nucleic acids of P.vivax,P.falciparum,P.malariae and P.ovale.Finally,the consistency of the method with PCR detection was evaluated using actual samples.Results At 39℃ within 20 min,the method could detect as low as 10 copies/reaction of plasmid DNA and had no cross-reaction with P.falciparum,P.malariae and P.ovale.The test results of the samples were consistent with PCR detection.Conclusion The established RPA method can be used for rapid detection of P.vivax with high sensitivity and specificity.

关 键 词：间日疟原虫 重组酶聚合酶扩增 分子检测 

分 类 号：R382.31[医药卫生—医学寄生虫学]