烟草野火病菌特异性检测引物筛选及应用

摘要

Tobacco wildfire,caused by Pseudomonas syringae pv.tabaci,is an important leaf tobacco bacterial disease.In order to quickly and accurately detect this disease,we downloaded the genome sequences from NCBI,including 25 Pseudomonas genus and 127 pathovars of P.syringae,and made a blast with Mauve 2.3.1 to find the specific fragments for P.syringae pv.tabaci.Based on this result,13 primer pairs were designed with Primer Premier 6.0 and study their specificity for P.syringae pv.tabaci.One primer pair,40429, was picked out and verified with wildfire strains of different provinces. These results showed that primer 40429 could separate P.syringae pv.tabaci from other Pseudomonas genus and other pathovars of P.syringae by a single PCR product of 203 bp. It is indicated that the primer has a good applicability to the detection of P.syringae pv.tabaci and can be used in molecular identification.%烟草野火病是山东烟区主要的细菌性病害之一,为了能够对该病害进行精确、快速的分子鉴定,本研究从 NCBI基因组数据库中下载了假单胞菌属25个不同菌种和127个丁香假单胞菌属不同致病变种的全基因组数据,利用Mauve 2.3.1进行全基因组比对,获得丁香假单胞菌烟草致病变种特异性区段.在此基础上利用Primer Premier 6.0进行引物设计,筛选出了一对丁香假单胞菌烟草致病变种的特异性检测引物40429.利用7株山东不同烟区的烟草野火病菌和4株分别来自于贵州、湖北、福建及云南的烟草野火病菌进行了该引物的适用性验证,结果表明该引物均可扩增得到大小为203 bp的单一目的条带.说明该引物对于烟草野火病菌的检测具有良好的适用性,可以用于野火病菌的分子鉴定.