Wang Yanhua, Qiao Fuyu, Cao Ju, Peng Yao, Xia Lianxu
Department of Plague, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China; Email:
Zhonghua Yu Fang Yi Xue Za Zhi. 2015 Nov;49(11):983-7.
To perform laboratory diagnosis and tracking source of a suspected tularemia patient in Beijing.
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 tularensis (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.
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 the fopA, 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.
This case was confirmed to be a tularemia patient, and a new subgroup of F. tularensis type B was found in China.
对北京一名疑似兔热病患者进行实验室诊断并追踪传染源。
2012年7月19日北京市报告了一名疑似兔热病患者。从患者血液样本中提取基因组DNA，然后使用土拉弗朗西斯菌（F.tularensis）的3个特异性基因（fopA、tul4和16S rRNA）以及2个基因分型引物（C1C4和RD1）进行常规PCR和扩增子测序。另外两个实验室同时平行重复fopA基因的PCR和测序。同时，使用4个靶标（fopA、ISFtul2、23kDa和tul4）进行实时荧光定量PCR，并使用11个典型单核苷酸多态性（SNP）和4个插入或缺失进行系统发育分析。
3个特异性基因均扩增为阳性，测序片段分别为409、407和1053 bp。与已发表的全基因组比较，该患者感染了土拉弗朗西斯菌。接下来，测序后两个分型引物分别获得了151和924 bp的扩增子。片段长度表明该患者感染了全北区亚种。另外两个实验室对fopA基因的PCR和测序均获得阳性结果。此外，实时荧光定量PCR检测的4个靶标对土拉弗朗西斯菌均呈阳性。fopA、ISFtul2、23kDa和tul4的Ct值分别为30、25、28和30。系统发育分析表明，该病例的全基因组属于来自俄罗斯的一个已知分支，即B3亚组。
该病例确诊为兔热病患者，在中国发现了土拉弗朗西斯菌B型的一个新亚组。
