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
The high phenotypic plasticity in the shell of oysters presents a challenge during taxonomic and phylogenetic studies of these economically important bivalves.However,because DNA can exhibit marked differences among m...The high phenotypic plasticity in the shell of oysters presents a challenge during taxonomic and phylogenetic studies of these economically important bivalves.However,because DNA can exhibit marked differences among morphologically similar species,DNA barcoding offers a potential means for oyster identifi cation.We analyzed the complete sequences of the cytochrome oxidase subunit I(COI)of fi ve common Crassostrea species in China(including Hong Kong oyster C.hongkongensis,Jinjiang oyster C.ariakensis,Portuguese oyster C.angulata,Kumamoto oyster C.sikamea,and Pacifi c oyster C.gigas)and screened for distinct fragments.Using these distinct fragments on a high-resolution melting analysis platform,we developed an identifi cation method that does not rely on species-specifi c PCR or fragment length polymorphism and is effi cient,reliable,and easy to visualize.Using a single pair of primers(OysterCOI-1),we were able to successfully distinguish among the fi ve oyster species.This new method provides a simple and powerful tool for the identifi cation of oyster species.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.
基金Supported by the National Basic Research Program of China(973 Program)(No.2010CB126402)the National Natural Science Foundation of China(Nos.40730845,41206149)+4 种基金the Shandong Provincial Natural Science Foundation(No.ZR2010DQ024)the National High Technology Research and Development Program of China(863 Program)(No.2012AA10A405)the Earmarked Fund for Modern Agro-Industry Technology Research System(No.CARS-48)the Taishan Scholar Program of Shandong Provincethe Taishan Scholar Climbing Program of Shandong Province
文摘The high phenotypic plasticity in the shell of oysters presents a challenge during taxonomic and phylogenetic studies of these economically important bivalves.However,because DNA can exhibit marked differences among morphologically similar species,DNA barcoding offers a potential means for oyster identifi cation.We analyzed the complete sequences of the cytochrome oxidase subunit I(COI)of fi ve common Crassostrea species in China(including Hong Kong oyster C.hongkongensis,Jinjiang oyster C.ariakensis,Portuguese oyster C.angulata,Kumamoto oyster C.sikamea,and Pacifi c oyster C.gigas)and screened for distinct fragments.Using these distinct fragments on a high-resolution melting analysis platform,we developed an identifi cation method that does not rely on species-specifi c PCR or fragment length polymorphism and is effi cient,reliable,and easy to visualize.Using a single pair of primers(OysterCOI-1),we were able to successfully distinguish among the fi ve oyster species.This new method provides a simple and powerful tool for the identifi cation of oyster species.
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。
