猪流行性腹泻病毒可视化RT-LAMP检测方法的建立与初步应用  

Establishment of visual RT-LAMP assay for porcine epidemic diarrhea virus

作　　者：李林岳 李任峰 袁嘉康 秦保亮[2] 庞俊增 蔡琳琳 赵晓会 范国英 王自良 LI Lin-yue;LI Ren-feng;YUAN Jia-kang;QIN Bao-liang;PANG Jun-zeng;CAI Lin-lin;ZHAO Xiao-hui;FAN Guo-ying;WANG Zi-liang(College of Animal Science and Veterinary Medicine,Henan Institute of Science and Technology,Innovative Research Team for Animal Disease Prevention and Control in University of Henan Province,Xinxiang 453003,China;Animal Disease Prevention and Control Center of Xinxiang City,Xinxiang 453003,China;Sanquan College of Xinxiang Medical University,Xinxiang 453003,China)

机构地区：[1]河南科技学院动物科技学院河南省高校动物疫病防控创新团队,河南新乡453003 [2]新乡市动物疫病预防控制中心,河南新乡453003 [3]新乡医学院三全学院,河南新乡453003

出　　处：《中国预防兽医学报》2023年第8期814-821,828,共9页Chinese Journal of Preventive Veterinary Medicine

基　　金：河南省重点研发与推广专项(科技攻关)-农业领域项目(212102110098)。

摘　　要：为建立猪流行性腹泻病毒(PEDV)可视化反转录-环介导等温扩增(RT-LAMP)检测方法,本研究根据PEDV ORF1a/b基因的保守序列,设计了8组特异性引物,以PEDC cDNA为模板,采用试剂盒推荐的各反应条件经LAMP扩增,根据LAMP反应产物琼脂糖凝胶电泳结果筛选最佳引物组。以筛选的外引物PE2 F3/B3经PCR从PEDV cDNA中扩增ORF1a/b基因,构建重组质粒pPE-ORF1-2并分别经PCR和测序鉴定。结果显示,2号引物组(内引物PE2 FIP/PE2 BIP,外引物PE2 F3/PE2 B3)为最佳引物。PCR和测序鉴定结果表明重组质粒正确构建,经计算其浓度为1.56×1011拷贝/μL,将其稀释1000倍后作为质粒标准品。为建立检测PEDV的RT-LAMP方法,本研究对该方法的反应时间、反应温度、Mg2+浓度、内、外引物及dNTP浓度进行了优化,结果显示,RT-LAMP反应体系为:60℃反应50 min,Mg2+浓度80 mmol/L,内外引物终浓度分别为32μmol/L和5μmol/L,dNTP浓度10 mmol/L;通过在上述体系中加入甲酚红指示剂,初步建立了可视化RT-LAMP检测方法。特异性试验结果显示,该方法仅能检出PEDV,与猪德尔塔冠状病毒、猪繁殖与呼吸综合征病毒和猪传染性胃肠炎病毒均无交叉反应,且阳性管体系颜色由紫红色变为黄色,阴性反应管体系颜色保持紫红色,特异性较强;将重组质粒标准品pPE-ORF1-210倍倍比稀释(1.65×100拷贝/μL~1.65×108拷贝/μL)后作为模板,采用本研究建立的可视化RT-LAMP方法进行敏感性试验,结果显示,该方法对pPE-ORF1-2检测限为10拷贝/μL,比以重组质粒pPE-M-GB为模板,以引物PEGBF/PEGBR的PEDV国标RT-PCR方法的敏感性高1个数量级,敏感性较高;重复性试验结果显示,该方法对不同浓度质粒标准品的批内和批间重复性试验的变异系数均小于5%,重复性较好。对53份临床样品检测结果显示,RT-LAMP和RT-PCR对PEDV的检出率分别为32.1%(17/53)和28.3%(15/53)。二者的总符合率为96.2%(51/53)。本研究建立的PEDV RT-LAMP检To establish a visual reverse transcription loop-mediated isothermal amplification(RT-LAMP)detection method for porcine epidemic diarrhea virus(PEDV),we designed 8 sets of specific primers based on the conserved sequences of the PEDV ORF1a/b genes.Using PEDV cDNA as a template,LAMP amplification was performed using various reaction conditions recommended by the kit.The optimal primer set was selected through agarose gel electrophoresis of LAMP reaction products.The results showed that primer set 2(internal primers PE2 FIP/PE2 BIP,external primers PE2 F3/B3)was the best primer set.Amplification of ORF1a/b gene from PEDV cDNA using screened external primers PE2 F3/B3,recombinant plasmid pPE-ORF1-2 was constructed and confirmed to be correct by PCR and sequencing.The concentration of pPE-ORF1-2 was 1.56×1011 copies/μL,After diluting the plasmid 1000 times as the plasmid standard.The reaction time,reaction temperature,Mg2+concentration,inner and outer primer concentrations,and dNTP concentration were optimized to establish the initial PEDV RT-LAMP detection method.The optimized RT-LAMP reaction conditions are as follows:reaction temperature of 60℃for 50 minutes,Mg2+concentration of 80mmol/L,inner and outer primer concentrations of 32μmol/L and 5μmol/L,respectively,and dNTP concentration of 10mmol/L.Specificity tests showed that this method could only detect PEDV,and had no cross-reactivity with porcine deltacoronavirus,porcine reproductive and respiratory syndrome virus,and transmissible gastroenteritis virus.The positive reaction tube turned from purple-red to yellow,while the negative reaction tube remained purple-red,indicating strong specificity.The plasmid standard pPE-ORF1-2 was diluted by 10 times ratio(100 copies/μL-108 copies/μL)as the template and use the visualized RT-LAMP method established in this study for sensitivity detection.The results showed that the detection limit of this method for pPE-ORF1-2 was 10 copies/μL,which was one order of magnitude higher than the sensitivity of the PEDV natio

关 键 词：猪流行性腹泻病毒 RT-LAMP 快速检测 可视化 

分 类 号：S852.65[农业科学—基础兽医学]