16S rDNA特异性引物设计优化及其在淞江鲈体表微生物鉴定中的应用

Improvement of Specific Primer Design on 16S rDNA and Its Application on Skin Microbes Detection of Trachidermus fasciatus

引物与模板的结合特异性是PCR反应成功与否的主要决定因素之一.在进行以16srDNA为目标序列的菌种鉴定或者各种检测应用中,对引物的特异性具有较普遍的要求.需扩增出完整的目标属,实现属内保守,同时避免有效扩增其他属的相应序列即实现属间特异.对此,我们对引物设计进行优化,通过分析所有已知细菌16srDNA序列,针对特定目标属筛选特异和最高效的引物对,并在淞江鲈的体表菌群样品中进行验证实验.结果表明,所设计的引物具有较好的属内保守性和特异性,而且引物在扩增中的效率也有一定的保证,能够很好地应用于菌群的定量PCR分析.

The specificity that primer pairs binding with template is one of main factor determining the success of PCR reaction.And general demands of primer specificity occur when dealing with microbe identification based on16 SrDNA and various detection.While high coverage of target genus is needed to amplify most species,the primer pairs is supposed to be special enough to avoid wrong amplification of other genus sequence,which refers to specificity among genus.This experiment programs a suited primer design software aimed at this problem.First,the analysis of all known bacteria 16 SrDNA are taken.And primer pairs are screened aimed at target genus.Moreover,an evaluation of primer efficiency is taken to gain efficient primers.A flora analysis of Trachidermus fasciatus＇skin microbes were done in order to check the validity of primer design.The result shows great coverage and specificity of primer with a relatively high efficiency.The primer pairs designed are suited for flora analysis.