Rapid diagnosis of extrapulmonary tuberculosis by PCR:: Impact of sample preparation and DNA extraction
Rapid diagnosis of extrapulmonary tuberculosis by PCR:: Impact of sample preparation and DNA extraction
复制标题
DOI：
10.1128/jcm.41.6.2323-2329.2003
复制
发表时间：
2003-06-01
影响因子：
9.4
通讯作者：
Herrmann, JL
中科院分区：
文献类型：
--
作者：
Honoré-Bouakline, S;Vincensini, JP;Herrmann, JL
在疑似肺外结核病病例中，快速和准确的实验室诊断至关重要，因为传统的抗酸杆菌检测技术存在局限性。分枝杆菌的主要困难是实现最佳的细胞裂解。商业试剂盒中使用的缓冲液不允许在许多临床标本中进行这种完全溶解。对120例患者的144份肺外标本进行了PK-Roche和完全DNA纯化(CTAB-Roche)两种制备方法的比较，以评价两种制备方法对Cobas-Amplicor法的影响。30例确诊为肺结核，其中15例结核分枝杆菌培养阳性。大量抑制性样本(39%至52%)影响了扩增和扩增产物的检测。与PK-罗氏相比，CTAB-罗氏可以检测到更多的培养阳性标本。与结核病的最终诊断相比较，CTAB-Roche的敏感性(53.8%)高于培养(43.3%)、PK-Roche(16%)和涂片(13%)。然而，CTAB-Roche提取液的特异性(78.8%)始终低于培养(100%)和PK-Roche(96.5%)。假阳性标本为肺活检材料、淋巴结活检材料和抽吸物，或骨髓抽吸物，主要来自免疫功能低下的患者。尽管完整的DNA提取在快速诊断肺外结核方面是有效的，但假阳性结果挑战了我们对PCR结果的理解。
In cases of suspected extrapulmonary tuberculosis, rapid and accurate laboratory diagnosis is of prime importance, since traditional techniques of detecting acid-fast bacilli have limitations. The major difficulty with mycobacteria is achieving optimal cell lysis. Buffers used in commercial kits do not allow this complete lysis in a number of clinical specimens. A comparison of two sample preparation methods, pretreatment with proteinase K (PK-Roche) and complete DNA purification (cetyltrimethylammonium bromide [CTAB] -Roche), was conducted on 144 extrapulmonary specimens collected from 120 patients to evaluate the impact on the Cobas-Amplicor method. Thirty patients were diagnosed with tuberculosis, with 15 patients culture positive for Mycobacterium tuberculosis. Amplification and detection of the amplicons were impaired by a high number of inhibitory specimens (39 to 52%). CTAB-Roche allowed the detection of more culture-positive specimens by PCR than PK-Roche. Comparison with the final diagnoses of tuberculosis confirmed that CTAB-Roche produced the best sensitivity (53.8%) compared to culture (43.3%), PK-Roche (16%), and smear (13%). However, the specificity of the PCR assay with CTAB-Roche-extracted material was always lower (78.8%) than those with culture (100%) and PK-Roche (96.5%). False-positive specimens were lung biopsy material, lymph node biopsy material and aspirate, or bone marrow aspirate, mainly from immunocompromised patients. Despite the efficiency of complete DNA extraction for the rapid diagnosis by PCR of extrapullmonary tuberculosis, the false-positive results challenge our understanding of PCR results.