应用膜芯片快速检测石蜡包埋组织中的结核杆菌
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摘要
研究背景及目的随着近年结核病发病率上升,耐药性难治性结核病的增多以及艾滋病在全球范围内迅速漫延,传统的结核杆菌检测手段如抗酸染色、分枝杆菌培养、药敏试验与菌种鉴定技术以及抗体检测等已经远远不能满足临床诊断需求,而一些新兴的分子检测方法如PCR、基因芯片等又由于种种原因目前尚不能在临床上推广使用,因此建立一种简洁而敏感的诊断、鉴别诊断方法成为防治结核病迫切需要解决的关键性问题之一。本研究通过使用膜芯片技术检测石蜡包埋组织中的结核杆菌,并与PCR及抗酸染色技术进行比较分析,寻求一种敏感、快速、有效的实验方法,从而进一步探讨膜芯片技术在临床上推广应用的可能性。
方法(1)对120例经临床和病理拟诊为结核病的石蜡包埋组织样本进行抗酸染色。(2)提取120例拟诊为结核病的石蜡包埋组织样本DNA,经酚-氯仿-异戊醇纯化后用PCR技术扩增结核分枝杆菌特异序列IS6110片段。(3)用膜芯片技术将样本PCR产物与膜芯片上的探针杂交,通过统计学方法分析比较抗酸染色、PCR以及膜芯片三种方法检测石蜡包埋组织中结核杆菌的阳性率。(4)27例相应组织来源的非结核病石蜡包埋组织为阴性对照。
结果1、形态学结果:
(1)苏木素-伊红染色镜下观察:全部病例镜下看到结核结节,包括结节中央的干酪样坏死,坏死周围的朗格汉斯巨细胞,上皮样细胞以及外部集聚的淋巴细胞和反应性增生的成纤维细胞。
(2)抗酸染色镜下观察:400至1000倍高倍镜下抗酸染色阳性菌呈鲜红色,纤细,长3μm左右、红染、不折光、稍弯的微细杆状物,或为稍弯的念珠样微细杆状物,与组织切片位于同一成像水平面上,位于巨噬细胞胞质内或位于巨噬细胞之外。120例拟诊为结核病的石蜡包埋组织经抗酸染
Background and Objective Tuberculosis is one of infectious diseases and the main threats for mankind. Tuberculosis is becoming more and more critical since the emergence and spread of AIDS and multidrug resistant (MDR) strains of M.tuberculosis. Conventional techniques, such as clinical features, histopathology, demonstration of acid fast bacilli (AFB) and isolation of Mycobacterium tuberculosis from the clinical specimens, are limited for diagnosis of tuberculosis in clinically because of their speed, sensitivity and specificity. Gene chips are demonstrably highly sensitive and specific but expensive. To develop a novel diagnostic method for tuberculosis, membrane-based gene chip, a newly developed molecular biological technique, was used for rapid detection of Mycobacterium tuberculosis.
Methods (1) Acid-fast staining was used to detect the presence of acid fast bacillus in 120 paraffin-embedded tissues which were clinically and histologically suspected tuberculosis infection. (2) PCR technique was used to detect the IS6110 specific insertion sequence DNA of Mycobacterium tuberculosis complex in the 120 paraffin-embedded tissues. (3) Membrane-based gene chip technique was used to detect the Mycobacterium tuberculosis in the 120 paraffin-embedded tissues. (4) Twenty seven paraffin-embedded tissues of non-tuberculosis were used for negative controls.
Result 1. Histological observation:
(1) HE staining: Microscopically, all samples showed epithelioid granulomas, which were composed of epithelioid macrophages, Langhans giant cells, lymphocytes, a few fibroblasts with collagen, and characteristic caseous necrosis
方法(1)对120例经临床和病理拟诊为结核病的石蜡包埋组织样本进行抗酸染色。(2)提取120例拟诊为结核病的石蜡包埋组织样本DNA,经酚-氯仿-异戊醇纯化后用PCR技术扩增结核分枝杆菌特异序列IS6110片段。(3)用膜芯片技术将样本PCR产物与膜芯片上的探针杂交,通过统计学方法分析比较抗酸染色、PCR以及膜芯片三种方法检测石蜡包埋组织中结核杆菌的阳性率。(4)27例相应组织来源的非结核病石蜡包埋组织为阴性对照。
结果1、形态学结果:
(1)苏木素-伊红染色镜下观察:全部病例镜下看到结核结节,包括结节中央的干酪样坏死,坏死周围的朗格汉斯巨细胞,上皮样细胞以及外部集聚的淋巴细胞和反应性增生的成纤维细胞。
(2)抗酸染色镜下观察:400至1000倍高倍镜下抗酸染色阳性菌呈鲜红色,纤细,长3μm左右、红染、不折光、稍弯的微细杆状物,或为稍弯的念珠样微细杆状物,与组织切片位于同一成像水平面上,位于巨噬细胞胞质内或位于巨噬细胞之外。120例拟诊为结核病的石蜡包埋组织经抗酸染
Background and Objective Tuberculosis is one of infectious diseases and the main threats for mankind. Tuberculosis is becoming more and more critical since the emergence and spread of AIDS and multidrug resistant (MDR) strains of M.tuberculosis. Conventional techniques, such as clinical features, histopathology, demonstration of acid fast bacilli (AFB) and isolation of Mycobacterium tuberculosis from the clinical specimens, are limited for diagnosis of tuberculosis in clinically because of their speed, sensitivity and specificity. Gene chips are demonstrably highly sensitive and specific but expensive. To develop a novel diagnostic method for tuberculosis, membrane-based gene chip, a newly developed molecular biological technique, was used for rapid detection of Mycobacterium tuberculosis.
Methods (1) Acid-fast staining was used to detect the presence of acid fast bacillus in 120 paraffin-embedded tissues which were clinically and histologically suspected tuberculosis infection. (2) PCR technique was used to detect the IS6110 specific insertion sequence DNA of Mycobacterium tuberculosis complex in the 120 paraffin-embedded tissues. (3) Membrane-based gene chip technique was used to detect the Mycobacterium tuberculosis in the 120 paraffin-embedded tissues. (4) Twenty seven paraffin-embedded tissues of non-tuberculosis were used for negative controls.
Result 1. Histological observation:
(1) HE staining: Microscopically, all samples showed epithelioid granulomas, which were composed of epithelioid macrophages, Langhans giant cells, lymphocytes, a few fibroblasts with collagen, and characteristic caseous necrosis
引文
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