环带库蚊ISSR-PCR反应体系的建立及优化

Establishment and Optimization of an ISSR Reaction System for Culex annulus

目的:建立及优化适宜环带库蚊(Culex annulus)ISSR-PCR的反应体系,为环带库蚊遗传多样性研究奠定技术基础。方法:选用引物IS01,应用正交试验及单因素试验对的ISSR-PCR反应体系中的BSA、模板DNA、Mg2+、引物、dNTP、Taq DNA聚合酶浓度6个关键因素进行优化筛选。结果:20μL反应体系中最佳因素为BSA 2.0 g/L、DNA模板1.00×10-4mg/L、Mg2+2.0 mmol/L、引物1.0μmol/L、dNTP 0.15 mmol/L、Taq DNA聚合酶0.20 5U/μL。结论:优化后的反应体系适于环带库蚊的ISSR-PCR扩增。

Objective： To establish and optimize an ISSR-PCR amplification system which would pro- vide basis for genetic diversity analysis of Culex annulus. Methods： Primer IS01 were selected to es- tablish 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, Mg^2＋, 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, Mg^2＋ 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.