假丝酵母SSR-PCR反应体系的优化

The Optimization of SSR-PCR Reaction System of Candida

目的建立假丝酵母SSR-PCR反应体系,确立最佳反应条件,为将该技术用于临床假丝酵母感染的快速鉴定奠定基础。方法根据引物Tm值设立退火温度梯度,确定适宜的退火温度。利用正交实验,对影响SSR-PCR反应体系的Taq DNA聚合酶、模板DNA、dNTP、引物等4种主要因素,进行优化。结果引物最适退火温度为51℃。假丝酵母最佳的SSR-PCR反应体系为:在25μl的PCR反应体系中加入Taq DNA聚合酶1.0 U、模板DNA为25 ng、dNTP为0.15 mmol.L-1、引物为0.5μmol.L-1。结论采用正交实验优化的SSR-PCR反应体系,操作简便,电泳条带清晰,多态性好,重复性强,可为进一步建立临床假丝酵母感染的快速鉴定提供借鉴。

Objective To establish the reaction system of SSR-PCR and the optimal reaction conditions and lay the foundation for the rapid identification on the infection of clinical Candida.Methods The gradients of annealing temperature were set according to Tm value of primer in order to determine the appropriate annealing temperature.The Orthogonal Design was used to optimize SSR-PCR reaction system in the four factors including Taq DNA polymerase,DNA template,dNTP and Primer.Results The optimal annealing temperature was 51℃.The optimization of 25 μl SSR-PCR reaction system including Taq DNA polymerase 1.0 U,DNA template 25 ng,dNTP 0.15 mmol·L-1 and Primer 0.5 μmol·L-1 was established by Orthogonal Design.Conclusion It was simple and with clear electrophoretic bands and good repeatability and high polymorphism to optimize SSR-PCR reaction system by Orthogonal Design.This would provide a reference for the further development of rapid identification on the infection of clinical Candida.