摘要
目的应用微生物数据分析云平台筛选巴尔通体特异基因,并设计巴尔通体属(Bartonella spp.)细菌检测通用引物和探针。方法登录微生物数据分析云平台,选择“Special_Marker_Gene”工作流,上传目标菌基因组序列,设置参数Align(覆盖度)和E(相似度)。运行完成后,将得到1份特异基因序列表。为进一步确认筛选基因的特异性,可将筛选序列片段用美国国立生物技术信息中心-序列局部比对工具(NCBI-BLAST)进行验证,根据筛选片段设计实时荧光定量PCR引物或探针,并分析实验结果。结果应用上述方法筛选到巴尔通体特异基因49个,选择NCBI-BLAST验证后覆盖度和特异性水平高的基因序列设计引物和探针,经预实验分析,以格拉汉姆巴尔通体(Bartonella grahamii,Bg)基因组中的yfeC基因(ACS50472.1_79)为靶序列设计的Bar5引物和TaqMan探针检测效果良好:可以扩增12种巴尔通体,通用性较好,其他种属的生物核酸样品均未出现荧光信号;最低检出限为3.47×10拷贝/反应;经重复性分析,组内及组间变异系数分别为0.02%~2.27%和0.56%~0.83%;同时,标准曲线呈现良好的线性关系,相关系数R2为1.00,扩增效率(Amplification efficiency,E)为101%。结论运用微生物数据分析云平台筛选巴尔通体属水平的特异基因操作简便,检索高效,适用于快速查找筛选细菌鉴定、分型的备选基因位点。
Objective To establish a method of screening for Bartonella-specific genes based on the Microbial Data Analysis Cloud Platform,and to design universal primers and probes for detecting Bartonella spp.Methods On the Microbial Data Analysis Cloud Platform,the Special_Marker_Gene workflow was selected,the genome sequence of target bacteria was uploaded,and the parameters Align(coverage)and E(similarity)was set;when the operation was completed,a specific gene sequences list was obtained and validated by NCBI-BLAST.Real-time fluorescence quantitative PCR primers or probes were designed according to the screened fragments,and the experimental results were analyzed.Results Fortynine Bartonella-specific genes were screened out,and primers and probes were designed by selecting gene sequences with high coverage and specificity after NCBI-BLAST validation.According to pre-experimental analysis,the Bar5 primers and TaqMan probe for the target sequence of the yfeC gene(ACS50472.1_79)in the genome of B.grahamii had a good detection effect:twelve Bartonella species could be amplified with good generality,and no fluorescent signals were found in the nucleic acid samples of other species or genera;the minimum detection limit was 3.47×10 copies/reaction;the withingroup and between-group coefficients of variation were 0.02%-2.27%and 0.56%-0.83%respectively by repeatability analysis;at the same time,the standard curve revealed a good linear relationship,with a correlation coefficient(R2)of 1.00 and an amplification efficiency(E)of 101%.Conclusion It is simple and efficient to screen for Bartonella-specific genes using the Microbial Data Analysis Cloud Platform,which can be applied to quickly find alternative gene loci for bacterial identification and classification.
作者
康央
李庆多
苗娇娇
宋秀平
徐爱玲
张雯
栗冬梅
KANG Yang;LI Qing-duo;MIAO Jiao-jiao;SONG Xiu-ping;XU Ai-ling;ZHANG Wen;LI Dong-mei(State Key Laboratory of Infectious Disease Prevention and Control,Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases,National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;Tianjin Yaohua Middle School;Shandong First Medical University)
出处
《中国媒介生物学及控制杂志》
CAS
2020年第5期526-530,共5页
Chinese Journal of Vector Biology and Control
基金
国家科技重大专项(2017ZX10303404)。
关键词
巴尔通体
特异基因
引物设计
云平台
荧光定量PCR
Bartonella
Specific gene
Primer design
Microbial Data Analysis Cloud Platform
Fluorescent quantitative PCR
