一种用于高变异细菌检测的PCR引物设计新方法  

A novel method for PCR primer design for the detection of highly variable bacteria

作　　者：户东正 屈武斌 郑翔文 佟凡 李江域 赵东升 HU Dong-zheng;QU Wu-bin;ZHENG Xiang-wen;TONG Fan;LI Jiang-yu;ZHAO Dong-sheng(Information Center,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China;iGeneTech Bioscience Co.,Ltd,Beijing 102206,China)

机构地区：[1]军事科学院军事医学研究院信息中心,北京100850 [2]艾吉泰康生物科技(北京)有限公司,北京102206

出　　处：《军事医学》2023年第4期297-301,F0003,共6页Military Medical Sciences

基　　金：装备军内科研项目(JK20202A060425)。

摘　　要：目的 建立一种引物设计新方法,提升高变异细菌检测引物的特异性和敏感性。方法 通过基因命名实体识别技术标注文献中的基因,构建高变异细菌保守基因知识库用于指导引物设计;通过MAFFT、Clustal Omega、Gblocks、Primer3、MFEprimer3.0进行多序列比对、引物设计、引物评价和迭代设计单重引物,并利用改进的索引算法和并行计算实现高性能特异性检测;利用贪心算法组合单重引物得到多重引物。结果 对大肠杆菌、志贺菌、单核细胞增生李斯特菌、金黄色葡萄球菌、沙门菌等5种高变异细菌进行了引物设计。经比较,根据该法设计的单重引物比文献中检测同一保守基因的引物特异性和敏感性更强;设计的针对各细菌的多重引物具有强特异性,敏感性方面除大肠杆菌引物的覆盖率达到93.14%,其余细菌引物的覆盖率均超过99%。结论 该法能够适用于高变异细菌检测的PCR引物设计。Objective To establish a novel primer design method to improve the specificity and sensitivity of primers for the detection of highly variable bacteria.Methods Genes in literature were annotated via the gene named entity recognition technology,and a knowledge base of highly variable bacterial conserved genes was constructed to guide primer design.MAFFT,Clustal Omega,Gblocks,Primer3 and MFEprimer3.0 were used to perform multiple sequence alignment,primer design,primer evaluation and iterative design of single repeat primers.The improved indexing algorithm and parallel computing were used to achieve high-performance specificity detection.Finally,multiple primers were obtained by combining single repeat primers using the greedy algorithm.Results Primers were designed for five highly variable bacteria,including Escherichia coli,Shigella,Listeria monocytogenes,Staphylococcus aureus,and Salmonella.In comparison,the single primers designed using our method were more specific and sensitive than primers in literature for detecting the same conserved gene.The multiple primers designed for each bacterium had strong specificity,and the coverage rate of Escherichia coli primers was 93.14% in terms of sensitivity,and the coverage rate of other bacterial primers was more than 99%.Conclusion This method can be applied to the design of PCR primers for the detection of highly variable bacteria.

关 键 词：高变异细菌 引物设计 特异性 敏感性 高性能计算 

分 类 号：R446.5[医药卫生—诊断学]