Ghossein R A, Ross D G, Salomon R N, Rabson A R
Department of Pathology, New England Medical Center, Boston, MA 02111.
Diagn Mol Pathol. 1992 Sep;1(3):185-91.
The sensitivity and specificity of the polymerase chain reaction (PCR) in the detection of mycobacteria in paraffin-embedded tissues and in crude lysates of mycobacterial cultures were assessed. Sections of formalin-fixed, paraffin-embedded tissues were deparaffinized and then subjected to a simple proteinase K and boiling lysis procedure. These preparations were used directly for PCR amplification of the 383 bp segment of the gene encoding the 65 kDa mycobacterial surface antigen. Crude lysates of mycobacteria were used as positive controls. The specificity of the PCR products was confirmed by Southern blot using a region-specific digoxigenin-labeled oligonucleotide probe and chemiluminescent detection. The 383 bp diagnostic fragment was visualized in 11 of 12 acid-fast bacilli (AFB) stain/culture-proven-positive blocks. Crude lysates of mycobacteria were detected to a sensitivity of approximately 80 organisms. Amplified fragments from paraffin-embedded tissues and mycobacterial cultures of M. tuberculosis, M. avium-intracellulare, and saprophytic mycobacteria were distinguished by digestion with Nar 1 restriction endonuclease. These results suggest that PCR amplification followed by restriction enzyme digestion of the PCR product is a rapid, specific, and highly sensitive technique for the detection and speciation of mycobacteria in paraffin-embedded tissues.
评估了聚合酶链反应（PCR）在检测石蜡包埋组织中的分枝杆菌以及分枝杆菌培养物粗裂解物中的分枝杆菌时的敏感性和特异性。将福尔马林固定、石蜡包埋组织的切片脱石蜡，然后进行简单的蛋白酶K和煮沸裂解程序。这些制备物直接用于编码65 kDa分枝杆菌表面抗原的基因的383 bp片段的PCR扩增。分枝杆菌粗裂解物用作阳性对照。使用区域特异性地高辛配基标记的寡核苷酸探针和化学发光检测通过Southern印迹确认PCR产物的特异性。在12个抗酸杆菌（AFB）染色/培养证实为阳性的组织块中的11个中可见383 bp诊断片段。检测到分枝杆菌粗裂解物的敏感性约为80个菌。通过用Nar 1限制性内切酶消化来区分来自结核分枝杆菌、鸟分枝杆菌-胞内分枝杆菌和腐生性分枝杆菌的石蜡包埋组织和分枝杆菌培养物的扩增片段。这些结果表明，PCR扩增后对PCR产物进行限制性酶切消化是一种用于检测石蜡包埋组织中分枝杆菌并进行菌种鉴定的快速、特异且高度灵敏的技术。
