基于量子点微球和RPA技术的ASFV抗体-核酸快速现场联检方法的建立  

Establishment of Rapid Field Co-Detection Method of ASFV Antibody and Nucleic Acid Based on Quantum Dot Microspheres and RPA Technology

作　　者：赵伊然 单衍可 李嘉豪 何昭群 王欣艺 温墩 王米拉 储蕊 赵东明 刘斐[1] ZHAO YiRan;SHAN YanKe;LI JiaHao;HE ZhaoQun;WANG XinYi;WEN Dun;WANG MiLa;CHU Rui;ZHAO DongMing;LIU Fei(Single Molecule Biochemistry and Biomedical Laboratory,College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095;State Key Laboratory of Veterinary Biotechnology/Harbin Institute of Veterinary Medicine,Chinese Academy of Agricultural Sciences,Harbin 150009)

机构地区：[1]南京农业大学动物医学院单分子生物化学与生物医学实验室,南京210095 [2]兽医生物技术国家重点实验室/中国农业科学院哈尔滨兽医研究所,哈尔滨150009

出　　处：《中国农业科学》2024年第24期4990-5002,共13页Scientia Agricultura Sinica

基　　金：国家重点研发计划(2023YFD1800502);湖南省重点研发计划(2023NK2017)。

摘　　要：【背景】非洲猪瘟(African swine fever,ASF)作为世界动物卫生组织(world organization for animal health,WOAH)规定必须通报的疫病之一,同时也是我国一类动物疫病,因缺乏有效疫苗和治疗方法,早期、有效、快速和敏感的检测对防控ASF至关重要。目前,非洲猪瘟病毒(African swine fever virus,ASFV)常用临床检测方法主要包括荧光定量PCR(quantitative real-time PCR,qPCR)、酶联免疫吸附测定试验(enzyme linked immunosorbent assay,ELISA)等,但这些技术均需要复杂的操作、昂贵的仪器以及较长的检测时间,不适用于现场快速检测。此外,ASFV感染后宿主体内的抗体和核酸含量在不同时间段有明显的差异,因此单独检测ASFV核酸或抗体可能会导致假阴性的存在。【目的】通过研发一种基于量子点微球和重组酶聚合酶扩增技术(recombinase polymerase amplification,RPA)的ASFV抗体-核酸联检试纸条,实现ASFV抗体和核酸的现场同时检测,以提高检测的灵敏度、特异性,降低检测成本,节约检测时间。【方法】通过结合量子点荧光免疫层析技术及RPA技术建立了同时检测ASFV抗体及核酸的侧向流层析试纸条,配套便携式荧光分析仪可实现ASFV抗体-核酸的快速现场联检。本研究优化了量子点偶联蛋白量及检测线包被浓度等条件。在此基础上,确定了该检测方法的cutoff值、敏感性、特异性及重复性,并将所建立的ASFV联检试纸条与市售ELISA试剂盒、qPCR检测试剂盒对临床样品进行检测,比较检测结果,验证该方法的符合率,评价该试纸条的实用性。【结果】联检试纸条使用方便、快速,整个检测过程可在30 min内完成。且与其他6种常见猪传染性病毒之间不存在交叉反应,特异性良好。核酸检测灵敏度可达10 copies/μL,抗体检测灵敏度可达1:3 200。抗体和核酸检测批内变异系数均小于10%,批间变异系数均小于15%,重复性良好。经市售ELISA试剂盒、q【Background】African swine fever(ASF),recognized as a notifiable disease by the World Organization for Animal Health(WOAH) and categorized as a Class I animal disease in China,presents significant challenges due to the absence of effective vaccines and treatment options.Therefore,early,effective,rapid,and sensitive detection on ASF is crucial for controlling ASF.Current clinical diagnostic methods for African swine fever virus(ASFV) primarily include quantitative real-time PCR(qPCR) and enzyme-linked immunosorbent assay(ELISA).However,these techniques require complex procedures,expensive equipment,and extended detection times,rendering them unsuitable for rapid field testing.Moreover,the levels of antibodies and nucleic acids in the host exhibit significant variations over time following ASFV infection,which can result in false negatives if only ASFV nucleic acids or antibodies are tested in isolation.【Objective】This study aimed to develop a test strip that allowed for the simultaneous detection of ASFV antibodies and nucleic acids based on quantum dot microspheres and recombinase polymerase amplification(RPA)technology.The objective was to enable on-site detection of both ASFV antibodies and nucleic acids,thereby enhancing the sensitivity and specificity of the tests,reducing detection costs,and saving time.【Method】In this study,a lateral flow immunoassay strip for the simultaneous detection of ASFV antibodies and nucleic acids was developed by combining quantum dot fluorescence immunochromatography technology with recombinase polymerase amplification(RPA).The corresponding portable fluorescence analyzer enables rapid on-site dual detection of ASFV antibodies and nucleic acids.The study optimized key parameters,such as the quantum dot-protein conjugation amount and the coating concentration of the test line.Based on these optimizations,the cutoff value,sensitivity,specificity,and reproducibility of the detection method were determined.Furthermore,the developed ASFV dual detection strip was compared wi

关 键 词：非洲猪瘟 核酸检测 抗体检测 侧向流免疫试纸条 

分 类 号：S858.28[农业科学—临床兽医学]