Establishment and Optimization of an ISSR Reaction System for Culex annulus

Objective: To establish and optimize an ISSR-PCR amplification system which would provide basis for genetic diversity analysis of Culex annulus.Methods: Primer IS01 were selected to establish the ISSR reaction system.Orthogonal design and single factor method were adopted to screen the optimal conditions for the six critical factors(BSA,DNA template,Mg2+,primer,dNTP,Taq DNA polymerase) of the ISSR-PCR reaction system.Results: The best reaction system was: 20 μL of reaction solution containing BSA 2.0 g/L,DNA template 1.00×10-4 mg/L,Mg2+ 2.0 mmol/L,primer 1.0 μmol/L,dNTP 0.15 mmol/L,and Taq DNA polymerase 0.20 5U/μl.Conclusion: The optimized reaction system is applicable to C.Annulus ISSR-PCR amplification.