三疣梭子蟹微卫星多重PCR技术建立及条件的优化

Establishment and optimization of a microsatellite-labeling multiplex PCR system in Portunus trituberculatus

选择三疣梭子蟹22个多态性高的微卫星标记,经过引物间的搭配、组合测试以及反应条件的优化(退火温度、Mg2+含量、dNTPs的浓度等参数),构建了多重PCR扩增技术体系。本研究最终建立了47个二重PCR组合,并在47个二重引物的组合基础上建立15个三重PCR组合,3个四重PCR组合。该多重PCR技术体系的建立为三疣梭子蟹品种选育、种系评估提供快速、有效分子检测技术。

By selecting 22 pairs of polymorphic microsatellite markers of Portunus trituberculatus,primers matching,and combinatorial testing,the multiplex PCR amplification system was built and the reaction conditions were optimized,including annealing temperature,Mg2＋ concentration,dNTPs concentration and other parameters.Forty-seven combinations of double PCR,15 combinations of triple PCR,and 3 quadruple PCR combinations were established.The establishment of this technology will provide a fast,effective molecular detecting tool for the P.trituberculatus breeding and germ-line assessment.