中国现代医学杂志2018，Vol.28Issue(4)：45-50,6.DOI:10.3969/j.issn.1005-8982.2018.04.010
海分枝杆菌和溃疡分枝杆菌二重PCR检测方法的建立及应用
Establishment and application of a duplex PCR method for detection of Mycobacterium marinum and Mycobacterium ulcerans
摘要
Abstract
Objective To establish a duplex PCR method that can synchronously detect and identify Mycobacterium marinum and Mycobacterium ulcerans in order to detect and treat soft tissue infections of the arms and skin from these mycobacteria at an early stage. Methods The specific gene sequences for Mycobacterium marinum and Mycobaterium ulcerans were searched from the GeneBank and the available literature, and NCBI/Primer-BLAST software and MFEPrimer-2.0 software were utilized to design two pairs of primers that could amplify the specific gene sequences of Mycobacterium marinum and Mycobacterium ulcerans. A duplex PCR was to be established to synchronously detect and identify Mycobacterium marinum and Mycobacterium ulcerans, and its optimal annealing temperature, specificity and sensitivity were explored. Then this duplex PCR was preliminarily applied to identify 19 specimens of clinically-considered non-tuberculosis mycobacteria infections (NTM) of the upper limbs. Results This study successfully established a duplex PCR that can synchronously detect and identify Mycobacterium marinum and Mycobacterium ulcerans. The length of amplified product of Mycobacterium marinum was 429 bp and that of Mycobacterium ulcerans was 240 bp at an optimal annealing temperature of 62.7℃ . Positive bands did not appear when other NTM were detected. The lowest detectable level was 0.508 pg/μl for Mycobacterium marinum, and 44.8 fg/μl for Mycobacterium ulcerans. In the 19 clinical specimens, Mycobacterium marinum infection was detected and identified in 8 (42.1％), Mycobacterium ulcerans in 3 (15.8％) and pathogenic bacteria were not detected in 8 (42.1％). Conclusions The duplex PCR established in this study can quickly diagnose Mycobacterium marinum and Mycobacterium ulcerans infections of the soft tissue of the arms and skin at an early stage, providing a basis for clinical formulation of the optimal treatment plan.关键词
二重PCR/海分枝杆菌/溃疡分枝杆菌/同步检测/手/皮肤软组织Key words
duplex PCR/Mycobacterium marinum/Mycobacterium ulcerans/synchronous detection/hand/soft tissue of skin分类
医药卫生引用本文复制引用
武迪,陈世玖,王成,龙航,杨军,刘珑玲..海分枝杆菌和溃疡分枝杆菌二重PCR检测方法的建立及应用[J].中国现代医学杂志,2018,28(4):45-50,6.基金项目
贵州省科学技术基金No:[黔科合J字LKZ(2012)10] （2012）
