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首页> 外文期刊>Journal of Clinical Microbiology >Evaluation of Real-Time and Conventional PCR Targeting Complex 85 Genes for Detection of Mycobacterium leprae DNA in Skin Biopsy Samples from Patients Diagnosed with Leprosy
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Evaluation of Real-Time and Conventional PCR Targeting Complex 85 Genes for Detection of Mycobacterium leprae DNA in Skin Biopsy Samples from Patients Diagnosed with Leprosy

机译:实时和常规PCR靶向复合物85基因检测诊断为麻风病患者的皮肤活检样本中麻风分枝杆菌DNA的评估

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In spite of the decrease in the number of registered leprosy patients, the number of new cases diagnosed each year (400,000) has remained essentially unchanged. Leprosy diagnosis is difficult due to the low sensitivity of current methodologies to identify new cases. In this study, conventional and TaqMan real-time PCR assays for detection of Mycobacterium leprae DNA were compared to current classification based on clinical, bacteriological, and histological evaluation. M. leprae DNA was extracted from frozen skin biopsy specimens from 69 leprosy patients enrolled in the study and was amplified using specific primers for either the antigen 85B-coding gene or the 85A-C intergenic region by using conventional and real-time PCR. The detection rate was 100% among multibacillary (MB) patients and ranged from 62.5% to 79.2% among paucibacillary (PB) patients according to the assay used. The TaqMan system for 85B gene amplification showed the highest sensitivity, although conventional PCR using the 85A-C gene as a target was also efficient. The cycle threshold (CT) values obtained using the TaqMan system were able to statistically (P < 0.0001) differentiate MB (mean CT, 28.06; standard deviation [SD], 4.51) from PB (mean CT, 33.06; SD, 2.24) patients. Also, there was a correlation between CT values and the bacteriological index for MB patients (Pearson's r, ?0.444; P = 0.008). Within the PB patients' group, we tested normal skin from six patients exhibiting the pure neuritic form of leprosy (PNL). Five out of six PNL patients were positive for the presence of M. leprae DNA, even in the absence of skin lesions. In conclusion, the TaqMan real-time PCR developed here seems to be a useful tool for rapidly detecting and quantifying M. leprae DNA in clinical specimens in which bacilli were undetectable by conventional histological staining.
机译:尽管登记的麻风病患者人数有所减少,但每年确诊的新病例数(400,000)基本保持不变。由于目前鉴定新病例的方法敏感性较低,因此麻风病诊断困难。在这项研究中,基于临床,细菌学和组织学评估,将常规和TaqMan实时PCR检测Lempe分支杆菌DNA的方法与当前分类进行了比较。 M。从69名麻风患者的冷冻皮肤活检标本中提取麻风病DNA,并通过常规和实时PCR使用针对抗原85B编码基因或85A-C基因间区域的特异性引物进行扩增。根据所使用的检测方法,在多细菌性(MB)患者中的检出率为100%,而在脓疱性(PB)患者中的检出率为62.5%至79.2%。尽管使用85A-C基因作为靶标的常规PCR也有效,但用于85B基因扩增的TaqMan系统显示出最高的灵敏度。使用TaqMan系统获得的循环阈值( C T )值能够统计( P <0.0001)区分MB(均值 C T ,28.06;与PB的标准偏差[SD],4.51)(平均值 C T ,33.06; SD,2.24 ) 耐心。此外,MB患者的 C T 值与细菌学指标之间存在相关性(Pearson's r ,? 0.444; P < / em> = 0.008)。在PB患者组中,我们测试了6名表现出麻风病(PNL)的纯神经型患者的正常皮肤。 6名PNL患者中有5名阳性表达为 M。即使在没有皮肤病变的情况下,leprae DNA也是如此。总之,此处开发的TaqMan实时PCR似乎是快速检测和定量M的有用工具。常规组织学染色无法检测出细菌的临床标本中的麻风病DNA。

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