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

Abstract

【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 importantrole 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.

Keywords

• lateolabrax maculatus iridovirus
• cross priming amplification
• specificity
• sensitivity
• nucleic acid strip
• visualization