花鲈虹彩病毒交叉引物恒温扩增检测方法的建立

【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CPF1.0μmol/L，引物F3和B3均为0.4μmol/L，探针引物B1(FAM)和B2(Biotin)均为0.8μmol/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...

Establishment of a Cross Priming Amplification Detection Method of Lateolabrax maculatus Iridovirus

【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.