Arnoldi J, Schlüter C, Duchrow M, Hübner L, Ernst M, Teske A, Flad H D, Gerdes J, Böttger E C
Department of Immunology and Cell Biology, Forschungsinstitut Borstel, Federal Republic of Germany.
Lab Invest. 1992 May;66(5):618-23.
Conventional histopathologic diagnosis of mycobacterial infections are limited to the determination of "acid-fast bacilli". A species-specific diagnosis is thus far impossible. In addition, routine microbiologic assessments of mycobacteria suffer from the major drawback that a species-specific diagnosis is extremely time-consuming and in several cases even impossible. As Mycobacterium leprae cannot be cultured in vitro, we tried to specifically target this obligate intracellular parasite by in situ hybridization and polymerase chain reaction (PCR) techniques. For this purpose we used a 22 mer oligonucleotide probe recognizing a species-specific sequence of the 16S rRNA of Mycobacterium leprae. Using an immunoenzymatic detection method for in situ hybridization we were able to specifically assess Mycobacterium leprae (a) in long-term cultured macrophages in vitro infected with different mycobacteria species and (b) in frozen sections of skin biopsies obtained from patients suffering from lepromatous leprosy. These results could be confirmed and extended by PCR experiments in which we used conserved oligonucleotide primers for 16S rRNA to amplify bacterial DNA isolated from different eubacterial species and from fresh-frozen as well as from formalin-fixed, paraffin-embedded and routinely processed mycobacteria-infected tissues. Upon Southern blot analysis, the Mycobacterium leprae-specific oligonucleotide probe exclusively hybridized with PCR products obtained from Mycobacterium leprae-containing samples (including paraffin sections), but not with PCR products obtained from samples containing other mycobacterial species. As species-specific oligonucleotide probes targeted at rRNA are described for a variety of mycobacterial species, these methods may be generally applied for a rapid species-specific assessment of mycobacteria in histologic material.
分枝杆菌感染的传统组织病理学诊断仅限于确定“抗酸杆菌”。因此，目前还无法进行种特异性诊断。此外，分枝杆菌的常规微生物学评估存在一个主要缺点，即种特异性诊断极其耗时，在某些情况下甚至无法进行。由于麻风分枝杆菌不能在体外培养，我们试图通过原位杂交和聚合酶链反应（PCR）技术特异性地靶向这种专性细胞内寄生虫。为此，我们使用了一种22聚体寡核苷酸探针，该探针识别麻风分枝杆菌16S rRNA的种特异性序列。使用免疫酶法进行原位杂交，我们能够特异性地评估（a）体外长期培养的、感染了不同分枝杆菌种类的巨噬细胞中的麻风分枝杆菌，以及（b）从瘤型麻风患者获取的皮肤活检冰冻切片中的麻风分枝杆菌。这些结果可以通过PCR实验得到证实和扩展，在PCR实验中，我们使用针对16S rRNA的保守寡核苷酸引物来扩增从不同真细菌种类以及从新鲜冰冻、福尔马林固定、石蜡包埋和常规处理的分枝杆菌感染组织中分离的细菌DNA。经过Southern印迹分析，麻风分枝杆菌特异性寡核苷酸探针仅与从含麻风分枝杆菌的样本（包括石蜡切片）中获得的PCR产物杂交，而不与从含其他分枝杆菌种类的样本中获得的PCR产物杂交。由于针对多种分枝杆菌种类描述了靶向rRNA的种特异性寡核苷酸探针，这些方法可能普遍适用于对组织学材料中的分枝杆菌进行快速种特异性评估。
