Nishimura M, Kwon K S, Shibuta K, Yoshikawa Y, Oh C K, Suzuki T, Chung T A, Hori Y
Department of Clinical Genetics, Kyushu University, Beppu, Japan.
Mod Pathol. 1994 Feb;7(2):253-6.
To improve the sensitivity of the previously reported polymerase chain reaction (PCR) for the detection of Mycobacterium (M.) leprae in the formaldehyde-fixed, paraffin-embedded tissues, we adapted the PCR designed to amplify an internal 372 bp fragment of a M. leprae-specific repetitive sequence to 39 skin biopsies taken from patients with leprosy of the lepromatous type and tuberculoid type. Crude DNA samples were prepared from tissue sections that were deparaffinized and subjected to proteinase-K digestion without any further treatment for DNA purification. Overcoming a false-negative reaction by an elongation of the period for enzymatic digestion and an appropriate dilution of the samples, an amplification of the target sequence was obtained as a single band with all 39 skin biopsies tested. The fragments specifically amplified by the PCR were subjected to direct sequencing and were confirmed to be identical with an internal 372 bp of M. leprae-specific repetitive sequence. Although in nine of 24 nonleprosy control samples, a false-positive amplification was observed as from one to several bands, they were distinguishable from the specific one by the electrophoretic pattern. This PCR makes up for the classic histological methods used in the diagnosis of leprosy.
为提高先前报道的用于检测甲醛固定、石蜡包埋组织中麻风分枝杆菌的聚合酶链反应（PCR）的灵敏度，我们将旨在扩增麻风分枝杆菌特异性重复序列内部372 bp片段的PCR方法应用于39份取自瘤型和结核型麻风患者的皮肤活检样本。从经过脱石蜡处理并经蛋白酶K消化的组织切片中制备粗DNA样本，无需进一步进行DNA纯化处理。通过延长酶消化时间和适当稀释样本克服假阴性反应，在所有检测的39份皮肤活检样本中均获得了作为单一条带的目标序列扩增产物。PCR特异性扩增的片段经直接测序，证实与麻风分枝杆菌特异性重复序列内部372 bp相同。虽然在24份非麻风对照样本中有9份观察到假阳性扩增，表现为1至数条条带，但通过电泳图谱可将它们与特异性条带区分开来。该PCR方法弥补了用于麻风诊断的经典组织学方法的不足。
