| 马艳平 1，覃宝田 1，梁 曦 2，王 刚 1，郝 乐1，周东来 3，刘振兴 1.花鲈虹彩病毒交叉引物恒温扩增检测方法的建立[J].广东农业科学,2024,51(3):148-156 |
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花鲈虹彩病毒交叉引物恒温扩增检测方法的建立 |
| Establishment of a Cross Priming Amplification Detection Method of Lateolabrax maculatus Iridovirus |
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| DOI：10.16768/j.issn.1004-874X.2024.03.014 |
| 中文关键词: 花鲈虹彩病毒 交叉引物恒温扩增 特异性 灵敏度 核酸试纸条 可视化 |
| 英文关键词: Lateolabrax maculatus iridovirus cross priming amplification specificity sensitivity nucleic acid strip visualization |
| 基金项目:广东省自然科学基金（2021A1515010498）；广东省农业科学院协同创新中心项目（XT202305）；广东省畜禽疫病防治研究重点实验室项目（2023B1212060040） |
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| 中文摘要: |
| 【目的】花鲈虹彩病毒（Lateolabrax maculatus iridovirus，LMIV）严重威胁花鲈养殖业安全，无特效防控药物，早期诊断在 LMIV 防控中发挥极其重要的作用。建立一种简便、快捷、准确的现场快速诊断方法，可为 LMIV 的基层诊断提供技术支撑。【方法】利用交叉引物恒温扩增技术（Cross priming amplification，CPA），针对 LMIV ATPase 基因高保守区设计 1 套单交叉引物。以构建的 ATPase 重组质粒作为阳性模板，对反应体系中的引物浓度比，Bst DNA 聚合酶、Betaine、MgSO4、dNTP 浓度，以及反应温度和反应时间进行优化；结合一次性核酸试纸条，建立可视化检测 LMIV-CPA 的方法。【结果】最优引物浓度比组合为交叉引物 CPF1.0 µmol/L，引物 F3 和B3 均为 0.4 μmol/L，探针引物 B1（FAM）和 B2（Biotin）均为 0.8 μmol/L；MgSO4 浓度为 6 mmol/L、Betaine 浓度为0.4 mol/L、dNTP 浓度为 0.6 mmol/L、Bst DNA 聚合酶浓度为 0.256 U/µL；最佳反应温度为 62 ℃，最佳反应时间为45 min。扩增产物经凝胶电泳检测呈梯形条带，带有探针的反应产物采用一次性核酸试纸条检测装置进行检测，在3~5 min 内即可通过是否出现特征性条带而使反应结果可视化。该方法可特异性地检测出 LMIV，不与其他水生常见病毒和常见细菌发生交叉反应。使用 LMIV-CPA 方法和常规 PCR 方法共同检测 156 份临床样品，LMIV-CPA 的阳性检出率为 93.30%，常规 PCR 方法的阳性检出率为 85.83%；在比较二者的灵敏度差异时，LMIV-CPA 的检测限为 102 copies/µL，灵敏度为常规 PCR 的 10 倍，综合结果显示 LMIV-CPA 优于 PCR。【结论】LMIV-CPA 检测方法不依赖昂贵的仪器设备与专业技术人员，可应用于 LMIV 的现场快速检测，为花鲈 LMIV 的准确快速诊断和有效防控提供技术支撑。 |
| 英文摘要: |
| 【Objective】Lateolabrax maculatus iridovirus (LMIV) is a serious threat to the safety of Lateolabrax maculatus aquaculture industry, and there are no specific prevention and control drugs. Early diagnosis plays an important role in the prevention and control of LMIV. The study intends to establish a simple, fast and accurate on-site rapid diagnosis method to provide technical support for the primary layer diagnosis of LMIV. 【Method】Cross priming amplification (CPA) was used to design CPA primers for the highly conserved region of ATPase gene of LMIV. The constructed ATPase recombinant plasmid was used as a positive template to optimize the primer concentration ratio, Bst DNA polymerase, Betaine, MgSO4, dNTPs, concentration reaction temperature and time in the reaction system. Combined with the disposable nucleic acid test strip technology, the visual detection of LMIV was established by LMIV-CPA. 【Result】The results showed that the optimal primer concentration ratio was 1.0 μmol/L for the cross-primer CPF, 0.4 μmol/L for the stripping primers F3 and B3, and 0.8 μmol/L for the probe primers B1 (FAM) and B2 (Biotin); Concentrations of MgSO4, Betaine, dNTPs and Bst DNA polymerase were 6 mmol/L, 0.4 mol/L, 0.6 mmol/L and 0.256 U/μL; The reaction temperature was 62 ℃ and the optimum reaction time was 45 min. The amplified product of this experiment was trapezoidal band by gel electrophoresis, the reaction product with probe was detected by a disposable nucleic acid test strip detection device, and the reaction result could be visualized by the presence or absence of a characteristic band within 3 to 5 minutes. LMIV could be detected specifically by this method without cross-reaction with other aquatic viruses and pathogenic bacteria. A total of 156 clinical samples were detected by the LMIV-CPA method and the conventional PCR method. The positive detection rate of LMIV-CPA was 93.30%, and that of the conventional PCR method was 85.83%. The detection limit of LMIV-CPA was 102 copies/µL and the sensitivity was 10 times that of conventional PCR, revealing that LMIV-CPA was better than PCR.【Conclusion】LMIV-CPA detection method does not rely on expensive instruments and professional technicians. It can be applied to the on-site rapid detection of LMIV, which provides technical support for accurate and rapid diagnosis as well as effective prevention and control of LMIV. |
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