Background: Pyrazinamide (PZA) is one of the most important drugs for tuberculosis (TB) treatment, however, its susceptibility is not routinely tested. High-resolution melting (HRM) curve analysis has been widely used...Background: Pyrazinamide (PZA) is one of the most important drugs for tuberculosis (TB) treatment, however, its susceptibility is not routinely tested. High-resolution melting (HRM) curve analysis has been widely used for many applications. In this study, HRM assay was developed and evaluated for the detection of PZA resistance in Mycobacterium tuberculosis clinical isolates. Methods: Ninety five M. tuberculosis clinical isolates with different susceptibility patterns to anti-TB drugs were used to evaluate this assay. Isolates were phenotypically (Bactec MGIT 960) and genotypically (HRM and pncA gene sequencing) analysed for PZA resistance. Results: Bactec MGIT 960 analysis revealed that 29 of the 95 M. tuberculosis isolates were PZA resistant. In comparison to the Bactec MGIT 960, HRM showed a sensitivity of 47.7% and specificity of 74.6%, and the overall agreement between the two methods was 68.4%. Based on DNA sequencing, a correlation of 0.67 (significant at p-value pncA mutations was observed. PZA resistance was strongly associated with multi-drug resistant (MDR)-TB as it was shown in 79.3% of the MDR isolates included in the study. Conclusion: HRM is simple and useful for screening clinical M. tuberculosis isolates for PZA resistance, however, further modifications to improve its performance are required.展开更多
建立高分辨熔解曲线技术同时快速检测鸡肉中沙门氏菌、单增李斯特菌、金黄色葡萄球菌和空肠弯曲菌。本研究分别以上述4种菌的fimY、hly、nuc和orfC特异性基因为靶基因,建立能同时检测多种食源性致病菌的多重HRM-real time PCR检测体系,...建立高分辨熔解曲线技术同时快速检测鸡肉中沙门氏菌、单增李斯特菌、金黄色葡萄球菌和空肠弯曲菌。本研究分别以上述4种菌的fimY、hly、nuc和orfC特异性基因为靶基因,建立能同时检测多种食源性致病菌的多重HRM-real time PCR检测体系,并对反应的特异性、灵敏度、重复性以及在人工染菌样品进行了评价。所建立的HRM-real time PCR检测体系对沙门氏菌、单增李斯特菌、金黄色葡萄球菌和空肠弯曲菌均产生特异性熔解曲线,Tm值分别为82.2℃、78.2℃、75.5℃和73.5℃;多重HRM-real time PCR反应体系检测单一致病菌菌液的灵敏度均为102拷贝数/mL,检测多种致病菌菌液的灵敏度为102拷贝数/mL;该反应体系重复性的试验内CV值在0.03%~0.32%之间,试验间CV值在0.45%~2.12%;人工染菌样本对4种菌的检测限均能达到5 CFU/25 g。结果表明该多重HRM-real time PCR检测体系可对上述4种禽肉中常见致病菌进行有效的检测与区分,特异性强,灵敏度高,反应重复性好,有望在食源性致病菌检测分析中发挥作用。展开更多
目的建立甲基化特异高分辨率溶解曲线(Methylation sensitive high resolution melting curve,MS-HRM)定量检测胶质瘤MGMT基因启动子甲基化的方法,用于指导胶质瘤患者术后化疗及预后判断。方法从标本库随机选取胶质瘤组织37例,提取DNA...目的建立甲基化特异高分辨率溶解曲线(Methylation sensitive high resolution melting curve,MS-HRM)定量检测胶质瘤MGMT基因启动子甲基化的方法,用于指导胶质瘤患者术后化疗及预后判断。方法从标本库随机选取胶质瘤组织37例,提取DNA进行甲基化修饰,应用MS-HRM方法和甲基化特异性PCR(methylation specific PCR,MSP)进行MGMT基因启动子区甲基化检测。结果 MSP方法检测显示部分甲基化标本29例(78.4%),较甲基化(13.5%)和未甲基化(8.1%)差异显著。MS-HRM发现MGMT基因启动子甲基化水平在10%~25%和25%~50%的标本分别有14例(37.8%)和17例(49.5%)。结论成功建立MS-HRM检测胶质瘤MGMT启动子甲基化的方法,该方法特异性高、灵敏度强和可重复性,有望应用于临床胶质瘤MGMT启动子甲基化高通量定量检测,以指导个体化治疗。展开更多
文摘Background: Pyrazinamide (PZA) is one of the most important drugs for tuberculosis (TB) treatment, however, its susceptibility is not routinely tested. High-resolution melting (HRM) curve analysis has been widely used for many applications. In this study, HRM assay was developed and evaluated for the detection of PZA resistance in Mycobacterium tuberculosis clinical isolates. Methods: Ninety five M. tuberculosis clinical isolates with different susceptibility patterns to anti-TB drugs were used to evaluate this assay. Isolates were phenotypically (Bactec MGIT 960) and genotypically (HRM and pncA gene sequencing) analysed for PZA resistance. Results: Bactec MGIT 960 analysis revealed that 29 of the 95 M. tuberculosis isolates were PZA resistant. In comparison to the Bactec MGIT 960, HRM showed a sensitivity of 47.7% and specificity of 74.6%, and the overall agreement between the two methods was 68.4%. Based on DNA sequencing, a correlation of 0.67 (significant at p-value pncA mutations was observed. PZA resistance was strongly associated with multi-drug resistant (MDR)-TB as it was shown in 79.3% of the MDR isolates included in the study. Conclusion: HRM is simple and useful for screening clinical M. tuberculosis isolates for PZA resistance, however, further modifications to improve its performance are required.
文摘建立高分辨熔解曲线技术同时快速检测鸡肉中沙门氏菌、单增李斯特菌、金黄色葡萄球菌和空肠弯曲菌。本研究分别以上述4种菌的fimY、hly、nuc和orfC特异性基因为靶基因,建立能同时检测多种食源性致病菌的多重HRM-real time PCR检测体系,并对反应的特异性、灵敏度、重复性以及在人工染菌样品进行了评价。所建立的HRM-real time PCR检测体系对沙门氏菌、单增李斯特菌、金黄色葡萄球菌和空肠弯曲菌均产生特异性熔解曲线,Tm值分别为82.2℃、78.2℃、75.5℃和73.5℃;多重HRM-real time PCR反应体系检测单一致病菌菌液的灵敏度均为102拷贝数/mL,检测多种致病菌菌液的灵敏度为102拷贝数/mL;该反应体系重复性的试验内CV值在0.03%~0.32%之间,试验间CV值在0.45%~2.12%;人工染菌样本对4种菌的检测限均能达到5 CFU/25 g。结果表明该多重HRM-real time PCR检测体系可对上述4种禽肉中常见致病菌进行有效的检测与区分,特异性强,灵敏度高,反应重复性好,有望在食源性致病菌检测分析中发挥作用。
文摘目的建立甲基化特异高分辨率溶解曲线(Methylation sensitive high resolution melting curve,MS-HRM)定量检测胶质瘤MGMT基因启动子甲基化的方法,用于指导胶质瘤患者术后化疗及预后判断。方法从标本库随机选取胶质瘤组织37例,提取DNA进行甲基化修饰,应用MS-HRM方法和甲基化特异性PCR(methylation specific PCR,MSP)进行MGMT基因启动子区甲基化检测。结果 MSP方法检测显示部分甲基化标本29例(78.4%),较甲基化(13.5%)和未甲基化(8.1%)差异显著。MS-HRM发现MGMT基因启动子甲基化水平在10%~25%和25%~50%的标本分别有14例(37.8%)和17例(49.5%)。结论成功建立MS-HRM检测胶质瘤MGMT启动子甲基化的方法,该方法特异性高、灵敏度强和可重复性,有望应用于临床胶质瘤MGMT启动子甲基化高通量定量检测,以指导个体化治疗。
