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:

目的 建立一种能同步检测鉴定海分枝杆菌和溃疡分枝杆菌的二重PCR 方法，为手部及皮肤软组织感染海分枝杆菌和溃疡分枝杆菌早期诊断、早期治疗服务。方法 通过GeneBank 数据库和文献查找海分枝杆菌和溃疡分枝杆菌的特有基因序列，设计2 对能扩增海分枝杆菌和溃疡分枝杆菌特有序列的引物，建立能同步检测鉴定海分枝杆菌和溃疡分枝杆菌的二重PCR，特异性及敏感性。并初步应用该二重PCR 检测鉴定19 份临床考虑上肢感染NTM 的标本。结果 本研究成功地建立能同步检测鉴定海分枝杆菌和溃疡分枝杆菌的二重PCR，扩增产物的长度海分枝杆菌为429 bp，溃疡分枝杆菌为240 bp，检测其他非结核分枝杆菌时未出现阳性条带，敏感性试验最低可检测出0.508 pg/μl 海分枝杆菌，44.8 fg/μl 溃疡分枝杆菌。检测鉴定19 份临床标本，42.1%（8 份）为海分枝杆菌感染，15.8%（3 份）为溃疡分枝杆菌感染，42.1%（8 份）未检出病原菌。结论 本研究建立的二重PCR 可早期、快速地诊断手部及皮肤软组织感染海分枝杆菌和溃疡分枝杆菌，为临床制定最佳治疗方案提供一定依据。

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.