| 摘要: |
| 【目的】建立一种基于重组酶介导恒温核酸扩增（RAA）技术快速检测犬传染性肝炎疾病的方法。【方法】以犬传染性肝炎病原体1型犬腺病毒（CAV-1）的E3基因序列作为靶标序列，设计合成引物和荧光探针，构建重组质粒作为标准品，进行RAA基础检测来筛选引物，确立RAA荧光反应体系。应用不同含量（105，104，103，102，10，1，0.1 COPY/μL）的质粒标准品作为模板，进行RAA检测，分析RAA检测的灵敏度；分别以CAV-1、2型犬腺病毒（CAV-2）、犬细小病毒（CPV）、犬瘟热病毒（CDV）、犬冠状病毒（CCV）、犬副流感病毒（CPIV）的基因组作为模板，进行RAA检测，分析RAA检测的特异性；选择同批次和不同批次的质粒标准品作为模板，进行RAA检测，分析RAA检测的重复性。选取经RT-qPCR检测为阳性和阴性的临床血液样本各40份，进行RAA检测，验证2种方法的符合情况。【结果】成功扩增到约419 bp的E3基因；构建了阳性质粒标准品p18TC1，其质量浓度为200 ng/μL，质粒含量为5.74×1010 COPY/μL。经筛选确立以CAV-F4/CAV-R3为引物、以CAV-P为探针的RAA荧光反应体系，反应温度恒定为39 ℃，反应时间为15 min，检测灵敏度为1 COPY/μL，与CAV-2、CPV、CDV、CCV、CPIV等病毒无任何交叉反应，且批内重复试验的变异系数小于1.00%，批间重复试验的变异系数小于2.00%。临床样本检测结果表明，建立的基于RAA的检测方法与RT-qPCR法的符合率为100%。【结论】建立了基于RAA技术的犬传染性肝炎快速检测方法，该方法具有检测快速、灵敏度高、特异性强、重复性好等优点。 |
| 关键词: 犬传染性肝炎 1型犬腺病毒 重组酶 恒温核酸扩增 |
| DOI：10.13207/j.jnwafu.2026.01.014 |
| 分类号: |
| 基金项目:江苏省高校“青蓝工程”优秀青年骨干教师项目（苏教师函［2021］11号）；江苏省高校自然科学基金项目（19KJB180030，23KJD180008）；企业委托横向课题（23WURD088） |
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| Establishment of a rapid detection method for canine infectious hepatitis based on recombinase aided isothermal nucleic acid amplification technology |
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SONG Shaozheng1, GU Leying1, WU Yingchao2, YU Kangying1, MENG Yaqin1
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1.Department of Basic Medicine,School of Health and Nursing,Wuxi Taihu University,Wuxi,Jiangsu 214064，China;2.Jiangyin Lingfeng Pet Hospital,Wuxi,Jiangsu 214400，China
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| Abstract: |
| 【Objective】This research aimed to establish a rapid detection method for canine infectious hepatitis disease based on recombinase aided isothermal nucleic acid amplification(RAA) technology.【Method】The E3 gene sequence of canine infectious hepatitis pathogen canine adenovirus 1 type(CAV-1) was used as the target sequence,and the primers and fluorescent probes were designed and synthesized.The recombinant plasmids were constructed as standard samples for RAA basic detection to screen primers and establish the RAA fluorescence reaction system.Plasmid standards with different concentrations (105,104,103,102,10,1,0.1 COPY/μL) were used as templates,to perform RAA detection and analyze the sensitivity of RAA detection.Virus genomes such as CAV-1,canine adenovirus 2 type(CAV-2), canine parvovirus(CPV),canine distemper virus(CDV),canine coronavirus(CCV) and canine parainfluenza virus(CPIV) were used as templates,to perform RAA testing and analyze the specificity of RAA detection. Plasmid standards from the same batch and different batches were used as templates to perform RAA testing and analyze the repeatability of RAA detection.40 clinical blood samples with positive and negative results detected by RT-qPCR were selected for RAA testing to verify the compliance of the two methods.【Result】E3 gene of approximately 419 bp was successfully amplified.A positive plasmid standard p18TC1 was constructed, with a mass concentration of 200 ng/μL and a plasmid content of 5.74×1010 COPY/μL.After screening,the RAA fluorescence reaction system was established with CAV-F4/CAV-R3 as primers and CAV-P as probes.The reaction temperature was constant at 39 ℃,the reaction time was 15 minutes,and the detection sensitivity was 1 COPY/μL.There was no cross reaction with viruses such as CAV-2,CPV,CDV,CCV,CPIV,etc.The coefficient of variation for intra-batch repeated trials was less than 1.00%,while the coefficient of variation for inter batch repeated trials was less than 2.00%.The clinical sample testing results showed that the established RAA based detection method had a 100% agreement rate with RT-qPCR method.【Conclusion】The rapid detection of canine infectious hepatitis based on RAA technology successfully established in this study can be used as a clinical rapid detection kit,with advantages such as fast detection,high sensitivity,strong specificity,and good repeatability. |
| Key words: canine infectious hepatitis canine adenovirus 1 type recombinase isothermal nucleic acid amplification |
