摘要
目的对于北京市1例疑似土拉热病例，进行实验室诊断和溯源分析。方法2012年7月19日北京市报告1例疑似土拉热病例。从病例血液样本中提取基因组DNA后，采用土拉弗朗西斯菌（土拉菌）3种特异基因（fopA，tul4和16SrRNA）和两种基因分型引物（C1C4和RD1），进行PCR检测并对扩增子测序。另外两个实验室分别平行进行Tfopa的PCR扩增和测序。同时采用4个靶位点（fopA、ISFtul2、23kDa和tul4）进行荧光定量PCR检测，并采用11个规范的SNP和4个插入／缺失进行系统进化分析。结果3种特异基因均得到阳性扩增，测序后得到的片段大小分别为409、407和1053bp，经序列比对，显示病例感染的是土拉菌。两种基因分型引物扩增后分别得到了151和924bp的序列，根据片段长度可判定病例感染的土拉菌为B型亚种。另外两个实验室对TfopA的PCR扩增和测序，均得到了阳性结果。4个靶位点的荧光定量PCR检测也均得到了阳性结果，fopA、1SFtul2、23kDa和tul4位点的ct值分别为30、25、28和30。系统进化分析显示，本研究中土拉菌和俄罗斯来源的土拉菌聚在一个分支上，为B3亚型。结论该病例确定为土拉热病例，本研究在中国发现了土拉菌B型亚种的一个新亚型。
Objective To perform laboratory diagnosis and tracking source of a suspected tularemia patient in Beijing. Methods A suspected tularemia patient was reported in Beijing city on July 19, 2012. Genomic DNA was extracted from the blood sample of the patient, then general PCR and sequencing of amplieons were conducted using 3 specific genes （fopA, tul4 and 16S rRNA）Francisella tularensis（F.tularensis）, and 2 genotyping primers （C 1 C4 and RD1）. Two other laboratories repeated the PCR and sequencing of the fopA in parallel. At the same time, real-time PCR fluorescent ration was performed using 4 targets （fopA, lSFtul2, 23kDa, and tul4）, and phylogenetic analysis was carried out using 11 canonical single nucleotide polymorphisms （SNPs） and 4 insertions or deletions. Results All the 3 specific genes were amplified positively, and sequenced fragments were 409, 407 and 1 053 bp, respectively. The patient was infected by F. tularensis comparing with the whole genome published. Next, amplicons of 151 and 924 bp were obtained by the 2 typing primers after sequencing, respectively. The segment lengths suggested that the patient was infected by the subsp, holarctica, All of the two other laboratories obtained positive data for the PCR and sequencing of the fopA. In addition, all the 4 targets tested positive by real-time PCR for F. tularensis. The Ct value of thefopA, ISFtul2, 23kDa and tul4 were 30, 25, 28, and 30, respectively. The phylogenetie analysis indicated that the whole genome of this case was assigned to a known clade from Russia, which was subgroup B3. Conclusion This case was confirmed to be a tularemia patient, and a new subgroup of F. tularensis type B was found in China.
出处
《中华预防医学杂志》
CAS
CSCD
北大核心
2015年第11期983-987,共5页
Chinese Journal of Preventive Medicine
基金
国家自然科学基金（81473032）
关键词
弗朗西丝菌
土拉热
多态性
单核苷酸
系统进化
Franeisella tularemia
Polymorphism,single nueleotide
Phylogeography
