CAJ | 학술논문

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16S rRNA)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了fopA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1 053 bp,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924 bp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.另外两个实验室对于opA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、ISFtul2、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.
목적 대우북경시1례의사토랍열병례,진행실험실진단화소원분석.방법 2012년7월19일북경시보고1례의사토랍열병례.종병례혈액양본중제취기인조DNA후,채용토랍불랑서사균(토랍균)3충특이기인(fopA,tul4화16S rRNA)화량충기인분형인물(C1C4화RD1),진행PCR검측병대확증자측서.령외량개실험실분별평행진행료fopA적PCR확증화측서.동시채용4개파위점(fopA、ISFtul2、23kDa화tul4)진행형광정량PCR검측,병채용11개규범적SNP화4개삽입/결실진행계통진화분석.결과 3충특이기인균득도양성확증,측서후득도적편단대소분별위409、407화1 053 bp,경서렬비대,현시병례감염적시토랍균.량충기인분형인물확증후분별득도료151화924 bp적서렬,근거편단장도가판정병례감염적토랍균위B형아충.령외량개실험실대우opA적PCR확증화측서,균득도료양성결과.4개파위점적형광정량PCR검측야균득도료양성결과,fopA、ISFtul2、23kDa화tul4위점적Ct치분별위30、25、28화30.계통진화분석현시,본연구중토랍균화아라사래원적토랍균취재일개분지상,위B3아형.결론 해병례학정위토랍열병례,본연구재중국발현료토랍균B형아충적일개신아형.
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 amplicons were conducted using 3 specific genes (fopA, tul4 and 16S rRNA)Francisella tutarensis(F.tularensis), and 2 genotyping primers (C1C4 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, ISFtul2, 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 phylogenetic 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.