OBJECTIVETo perform laboratory diagnosis and tracking source of a suspected tularemia patient in Beijing.

METHODSA 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.

RESULTSAll 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.

CONCLUSIONThis case was confirmed to be a tularemia patient, and a new subgroup of F. tularensis type B was found in China.