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1. 发明公开

KR1020010094219A 역전사 중합효소 연쇄반응을 이용한 마이코박테리움레프레의 약제 감수성 측정방법 失效
标题翻译：使用反向转录 - 聚合酶链反应（RT-PCR）的MYCOBACTERIUM LEPRAE的药物敏感性测试方法
公开(公告)号：KR1020010094219A
公开(公告)日：2001-10-31
申请号：KR1020000017727
申请日：2000-04-04
申请人： 유지창
发明人： 유지창 , 김민주 , 김세곤 , 김상헌
IPC分类号： C12Q1/00
摘要： PURPOSE: Provided is a drug sensitivity test method for Mycobacterium leprae(hereafter referred to as M.leprae), using reverse transcription polymerase chain reaction(RT-PCR), thereby assaying drug efficacy in a simple, fast, reliable, accurate and sensible manner compared with other existing tests which need costly equipment. The method can be used in developing therapeutic agents in Hansen's disease and deciding the effectiveness of chemotherapeutic treatment in Hansen's disease. CONSTITUTION: The test method comprises steps of: (i) infecting M.leprae to a host cell in a ratio of cell number to M.leprae number being 1:10 to 1:30, for 8 hours, where the host cell is characteristically mouse macrophage cell line IC-21, J77rA.l, RAW 264.17 or THP-1 and treating an antibiotics for Hansen's disease for 12 to 24 hours to test sensitivity, where antibiotics is selected from rifampin, Dapsone(4,4-diamino diphenyl sulfone), clofazimine, mimocin, ethonamide, fluoroquinolones, macrolide antibiotics, minocycline and fusidic acid; (ii) separating M.leprae from the host cell, extracting RNA from separated M.leprea and obtaining cDNA by reverse transcription from the extracted RNA as a template; (iii) amplifying the obtained cDNA by polymerase chain reaction(PCR) using M.leprae-specific 18kDa gene primer 1(SEQ NO ID:1) and 2(SEQ ID NO:2) and the reaction target base sequence selected from the group consisting of the heat shock protein, hsp 10, 14, 34, 45, 65 or 70 gene, Dmak gene, GroEl gene, 16S rRNA, 16S rRNA, M.leprae-specific 18kDa gene, and M.leprae specific repetitive sequence and finally performing agarose gel electrophoresis.
摘要翻译：目的：提供使用逆转录聚合酶链反应（RT-PCR）的麻风分枝杆菌药物敏感性试验方法（以下简称M.leprae），从而以简单，快速，可靠，准确，合理的方式测定药物疗效与其他需要昂贵设备的现有测试相比。该方法可用于开发汉森氏病的治疗药物，并确定汉森氏病中化学治疗的有效性。构成：测试方法包括以下步骤：（i）将宿主细胞以细胞数与M.leprae数的比例感染M.leprae为1:10至1:30，持续8小时，其中宿主细胞特征性小鼠巨噬细胞细胞系IC-21，J77rA.l，RAW 264.17或THP-1，并将抗生素用于汉森氏病12-24小时，以检测敏感性，其中抗生素选自利福平，氨苯砜（4,4-二氨基二苯砜），clofazimine，mimocin，乙酰胺，氟喹诺酮类，大环内酯类抗生素，米诺环素和夫西地酸; （ii）从宿主细胞中分离出M.leprae，从分离的M.leprea提取RNA，通过逆转录从提取的RNA作为模板获得cDNA; （iii）使用M.leprae特异性18kDa基因引物1（SEQ ID NO：1）和2（SEQ ID NO：2）通过聚合酶链反应（PCR）扩增获得的cDNA，并且选自下组的反应靶基序列由热休克蛋白，hsp 10,14,44,45,65或70基因，Dmak基因，GroE1基因，16S rRNA，16S rRNA，M.leprae特异性18kDa基因和M.leprae特异性重复序列组成，最后进行琼脂糖凝胶电泳。

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