PCR-LDR-核酸试纸条检测法在结核分枝杆菌katG基因315位密码子突变检测中的应用
摘要
目的:改良优化一项新的分子生物学方法PCR-LDR-核酸试纸条检测法,并将其应用于结核分枝杆菌katG基因315位密码子单碱基突变的检测。为临床检测结核分枝杆菌耐药相关单碱基突变提供一种新方法,以指导临床用药。
方法:设计与合成用于检测结核分枝杆菌katG基因315位密码子单碱基突变的引物和探针,通过LDR反应,使用核酸检测试纸条检测,并与PCR-RFLP法及直接测序法的检测结果相比较。
结果:成功检测各单碱基突变类型及野生型,与PCR-RFLP法及直接测序法的检测结果呈高度一致性,符合率分别为97.5%和100%。
结论:该方法可有效检测结核分枝杆菌katG基因315位密码子的单碱基突变,并为临床检测结核分枝杆菌耐药相关单碱基突变提供一种新的快速简便廉价的分子诊断方法。
Objective:This study aims to meliorate and optimize a novel molecular diagnositic method(PLNA), prove the feasibility of this method for detecting the site specific mutations of katG gene condon 315 of Mycobacterium tuberculosis , and apply this method to the distruction of clinical drug use .
Methods:Primers and probes were designed and synthesized for the detection of mutations in katG gene codon315 of Mycobacterium tuberculosis , performed the LDR reaction , and then detected use the nucleic acid detection strip , the results were compared with PCR-RFLP and direct sequencing .
Results:The wildtype and mutants were successfully diagnosed , a high consistency was found compared with PCR-RFLP and direct sequencing ,the concordance rate was 97.5% and 100% respectively .
Conclusion:This study provided a viable low-cost quick molecular diagnosis method for the clinical detection of mutations at katG gene codon 315 of Mycobacterium tuberculosis.
方法:设计与合成用于检测结核分枝杆菌katG基因315位密码子单碱基突变的引物和探针,通过LDR反应,使用核酸检测试纸条检测,并与PCR-RFLP法及直接测序法的检测结果相比较。
结果:成功检测各单碱基突变类型及野生型,与PCR-RFLP法及直接测序法的检测结果呈高度一致性,符合率分别为97.5%和100%。
结论:该方法可有效检测结核分枝杆菌katG基因315位密码子的单碱基突变,并为临床检测结核分枝杆菌耐药相关单碱基突变提供一种新的快速简便廉价的分子诊断方法。
Objective:This study aims to meliorate and optimize a novel molecular diagnositic method(PLNA), prove the feasibility of this method for detecting the site specific mutations of katG gene condon 315 of Mycobacterium tuberculosis , and apply this method to the distruction of clinical drug use .
Methods:Primers and probes were designed and synthesized for the detection of mutations in katG gene codon315 of Mycobacterium tuberculosis , performed the LDR reaction , and then detected use the nucleic acid detection strip , the results were compared with PCR-RFLP and direct sequencing .
Results:The wildtype and mutants were successfully diagnosed , a high consistency was found compared with PCR-RFLP and direct sequencing ,the concordance rate was 97.5% and 100% respectively .
Conclusion:This study provided a viable low-cost quick molecular diagnosis method for the clinical detection of mutations at katG gene codon 315 of Mycobacterium tuberculosis.
引文
[1]Ferguson LA,Rhoads J.Multidrug-rresistant and extensively drug-resistant tuberculosis:The new face of an old disease. J Am Acad Nurse Prac,2009,21(11):603-609.
[2]WHO.Multidrug and extensively drug-resistant TB (M/XDR-TB):2010 global report on surveillance and response.WHO,2010.
[3]Riordan PO,Schwab U,Logan S,et al.Rapid molecular detection of rifampicin resietance facilitates early diagnosis and treatment of multi-drug resistant tuberculosis:case control study.PLoS ONE,2008,3(9):e3173.
[4]Luo T,Zhao M,Li X,et al.Selection of mutations to detect multidrug-resistant Mycobacterium tuberculosis strains in Shanghai,China.Antimicrob Agents Chemother,2010, 54(3):1075-1081.
[5]Balabanova Y,Drobniewski F,Nikolayevskyy V,et al.An integrated approach to rapid diagnosis of Tuberculosis and multidrug resistance using liquid culture an molecular methods in Russia.PLoS ONE,2009,4(9):e7129.
[6]Caws M,Tho DQ,Duy PM,et al.PCR-Restriction fragment length polymorphism for rapid,low-cost identification of isoniazid-resistant Mycobacterium tuberculosis.J Clin Microbiol,2007,45(6):1789-1793.
[7]Sekiguchi J,Tohru MA,Ewa A K,et al.Detection of multidrug resistance in Mycobacterium tuberculosis.J Clin Microbiol,2007,45(1):179-192.
[8]Abdelaal A,El-Ghaffar HA,Zaghloul MHE,et al.Genotypic detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis strains by DNA sequencing:a randomized trial.Ann Clin Microbiol and Antimicrob,2009,8:4.
[9]Forbes B A,Hicks KES.Direct Detection of Mycobacterium tuberculosis in respiratory specimens in a clinical laboratory by polymerase chain reaction.J Clin Microbiol,1993,31(7):1688-1694.
[10]石小华,景奉香,胡忠义,等.DNA芯片快速检测结核分支杆菌对链霉素和乙胺丁醇的耐药性方法的建立.南京医科大学学报(自然科学版), 2009,29(4):435-440.
[11]单万水,单金岚,詹能勇,等.DNA芯片快速检测耐利福平结核分支杆菌rpoB基因突变.中华检验医学杂志,2003,26(11):680-682.
[12]曾涛,朱中元.结核分枝杆菌耐药分子机制及检测方法的研究进展.中国热带医学,2008,8(3):481-484.
[13]孙冰梅,车志宏.结核分枝杆菌耐药的分子机制及耐药基因检测方法的研究进展.山西医药杂志,2005,34(4):301-303.
[14]谢明章,陈昌杰.结核分支杆菌耐药的分子机制及分子生物学检测方法进展.分子诊断与治疗杂志,2010,2(5):348-352.
[15]Loddenkemper R,Hauer B.Drug-resistant Tuberculosis:a worldwide epidemic pose a new challenge. Dtsch Arztebl Int, 2010, 107(1-2):10-9.
[16]Cohen T,Becerra MC,Murray MB.Isoniazid resistance and the future of drug-resistant Tuberculosis. Microb Drug Resist, 2004 ,10(4): 280–285.
[17] WHO. WHO Address TB/HIV, MDR/XDR-TB and other challenges. WHO, 2007.
[18]WHO.The Global MDR-TB & XDR-TB Response Plan 2007-2008. WHO, 2007.
[19]吴铮,许建中.基因芯片技术在结核耐药检测中的应用进展.中国脊柱脊髓杂志,2009,19 (11):863-866.
[20]Bwanga F,Hoffer S,Haile M,et al.Direct susceptibility testing for multi drug resistant Tuberculosis:a meta-analysis.BMC InfectDis ,2009, 9:67.
[21]Kant S,Maurya AK,Kushwaha RAS,et al. Multi-drug resistant tuberculosis: An iatrogenic problem. BioScience Trends,2010, 4(2):48-55.
[22]张敏,彭建涛,顾世民.结核分枝杆菌耐药检测方法的研究进展.医学综述,2010,16(18):2846-2849.
[23]刘旭红,陈旭庚,张眉眉.PCR-SSCP技术快速检测结核分支杆菌三种基因突变.中国防痨杂志,2005,27(2):130-131.
[24]吴建林,曹慧.耐药结核病流行现状与控制策略.寄生虫病与感染性疾病,2009,7(1):51-54.
[25]Lavender C,Globan M,Sievers A,et al.Molecular characterization of isoniazid-resistant Mycobacterium tuberculosis isolates collected in Australia. Antimicrob Agents Ch,2005,49(10):4068-4074.
[26]Cardoso RF,Cooksey RC,Morlock GP,et al.Screening and characterization of mutations in isoniazid-resistant Mycobacterium tuberculosis isolates obtained in Brazil.Antimicrob Agents Ch,2004,48(9):3373-3381.
[27]Marrakchi H,Laneelle G,Quemard A,et al.InhA,a target of the antituberculous drug isoniazid,is involved in a mycobacterial fatty acid elongation system,FAS-Ⅱ.Microbiology,2000, 146:289-296.
[28]Ranaswamy SV,Reich R,Dou SJ,et al.Single nucleotide polymorphisms in genes associated with isoniazid resistance in Mycobacterium tuberculosis. Antimicrob Agents Ch,2003,47(4):1241-1250.
[29]Doorn HRV,Claas ECJ,Templeton KE,et al.Detection of a point mutation associated with high-level isoniazid resistance in Mycobacterium tuberculosis by using real-time PCR technology with 3’-minor groove binder-DNA probes.J Clin Microbiol,2003,41(10):4630-4635.
[30]Chiyoji A,Ikuo K,Satoshi M,et al.Biological and molecular characteristics of Mycobacteriumtuberculosis clinical isolates with low-level resistance to isoniazid in Japan . J Clin Microbiol,2008, 46(7):2263-2268.
[31]Sreevatsan S,Pan X,Zhang Y,et al.Analysis of the oxyR-ahpC region in isoniazid-resistant and–susceptible Mycobacterium tuberculosis complex organisms recovered from diseased humans and animals in diverse localities. Antimicrob Agents Ch,1997,41(3):600-606.
[32]Baker LV,Brown TJ,Maxwell O,et al.Molecular analysis of isoniazid-resistant Mycobacterium tuberculosis isolates from England and Wales reveals the phylogenetic significance of the ahpC -46A polymorphism. Antimicrob Agents Ch,2005,49(4):1455-1464.
[33]Costa ERD,Ribeiro MO,Silva MSN,et al.Correlations of mutations in katG,oxyR-ahpC and inhA genes and in vitro susceptibility in Mycobacterium tuberculosis clinical strains segregated by spoligotype families from tuberculosis prevalent countries in South America.BMC Micobiology,2009,9(39):1471-2180.
[34]Lee ASG,Teo ASM,Wong SY.Novel mutations in ndh in isoniazid-resistant Mycobacterium tuberculosis isolates.Antimicrob Agents Ch,2001,45(7):2157-2159.
[35]Payton M,Auty R,Delgoda P,et al.Cloning and characterization of arylamine N-acetyltransferase genes from Mycobacterium smegnatis and Mycobacterium tuberculosis:increased expression results in isoniazid resistance. J Bacteriol,1999,181(4):1343-1347.
[36]Zahrt TC,Song J,Siple J,et al.Mycobacterial FurA is a negative regulator of catalase-peroxidase gene katG.Mol Microbiol,2001,39(5):1174-1185.
[37]Pym AS,Domenech P,Honore N,et al.Regulation of catalase-Peroxidase(KatG) expression,isoniazid sensitivity and virulence by furA of Mycobacterium tuberculosis. Mol Microbiol,2001,40(4):879-889.
[38]Zhang M,Yue J,Yang YP,et al.Detection of mutations associated with isoniazid resistance in Mycobacterium tuberculosis isolates from China. J Clin Microbiol,2005,43(11):5477-5482.
[39]Xu GL,You QM,Pickerill S,et al.Application of PCR-LDR-Nucleic acid detection strip in detection of YMDD mutation in Heptitis B patients treated with Lamivudine[J].J Med Virol,2010,82(7):1143-1149.
[40]Niederhauser C,Kaempf L,Heinzer I.Use of the ligase detection reaction-polymerase chain reaction to identify point mutations in extended-spectrum beta-lactamases. Eur J Clin Microbiol Infect Dis,2000, 19 :477-480.
[41]Mcnamara DT,Thomson JM,Kasehagen LJ,et al. Development of a multiplex PCR-ligase detection reaction assay for diagnosis of infection by the four parasite species causing malaria in humans.J.Clin. Microbiol,2004,42(6):2403-2410.
[42]张世扬,肖振贤,赵建龙等.基于连接酶检测反应的并行分型系统检测AGT M235T和ACE I/D基因多态性.华东理工大学学报(自然科学版),2006,32(9):1050-1054.
[43]张莹,束婧婷,韩威等.基于连接酶检测反应的NPY基因多态与清远麻鸡繁殖性状的相关性研究.中国农学通报,2010,26(17):9-12.
[44]Chen X.N. , Kenneth J.L. and Pui Y.K. . A homogeneous , Ligase-Medizted DNA diagnostic test . Genome Res.1998(8) : 549-556 .
[45]Wilson M,Derisi J,Kristensen HH,et al. Exploring drug-induced alterations in gene expression in Mycobacterium tuberculosis by microarray hybridization. Proc Natl Acad Sci USA,1999,96(22):12833-12838.
[46]Kibili GS,Mulder B,Dolmans WM,et al. M. tuberculosis genotypic diversity and drug susceptibility pattern in HIV-infected and non-HIV-infected patients in northern Tanzania. BMC Microbiol,2007,7: 51.
[47]Ming Z,Xia L,Peng X,et al. Transmission of MDR and XDR Tuberculosis in Shanghai,China. PLoS ONE,2009,4(2):e4370.
[48]Weiwei J, Igor M,Guizhi S,et al. Molecular characteristics of rifampin and isoniazid resistant Mycobacterium tuberculosis strains from Beijing , China . Chin Med J 2007 ; 120 (9) : 814-819 .
[2]WHO.Multidrug and extensively drug-resistant TB (M/XDR-TB):2010 global report on surveillance and response.WHO,2010.
[3]Riordan PO,Schwab U,Logan S,et al.Rapid molecular detection of rifampicin resietance facilitates early diagnosis and treatment of multi-drug resistant tuberculosis:case control study.PLoS ONE,2008,3(9):e3173.
[4]Luo T,Zhao M,Li X,et al.Selection of mutations to detect multidrug-resistant Mycobacterium tuberculosis strains in Shanghai,China.Antimicrob Agents Chemother,2010, 54(3):1075-1081.
[5]Balabanova Y,Drobniewski F,Nikolayevskyy V,et al.An integrated approach to rapid diagnosis of Tuberculosis and multidrug resistance using liquid culture an molecular methods in Russia.PLoS ONE,2009,4(9):e7129.
[6]Caws M,Tho DQ,Duy PM,et al.PCR-Restriction fragment length polymorphism for rapid,low-cost identification of isoniazid-resistant Mycobacterium tuberculosis.J Clin Microbiol,2007,45(6):1789-1793.
[7]Sekiguchi J,Tohru MA,Ewa A K,et al.Detection of multidrug resistance in Mycobacterium tuberculosis.J Clin Microbiol,2007,45(1):179-192.
[8]Abdelaal A,El-Ghaffar HA,Zaghloul MHE,et al.Genotypic detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis strains by DNA sequencing:a randomized trial.Ann Clin Microbiol and Antimicrob,2009,8:4.
[9]Forbes B A,Hicks KES.Direct Detection of Mycobacterium tuberculosis in respiratory specimens in a clinical laboratory by polymerase chain reaction.J Clin Microbiol,1993,31(7):1688-1694.
[10]石小华,景奉香,胡忠义,等.DNA芯片快速检测结核分支杆菌对链霉素和乙胺丁醇的耐药性方法的建立.南京医科大学学报(自然科学版), 2009,29(4):435-440.
[11]单万水,单金岚,詹能勇,等.DNA芯片快速检测耐利福平结核分支杆菌rpoB基因突变.中华检验医学杂志,2003,26(11):680-682.
[12]曾涛,朱中元.结核分枝杆菌耐药分子机制及检测方法的研究进展.中国热带医学,2008,8(3):481-484.
[13]孙冰梅,车志宏.结核分枝杆菌耐药的分子机制及耐药基因检测方法的研究进展.山西医药杂志,2005,34(4):301-303.
[14]谢明章,陈昌杰.结核分支杆菌耐药的分子机制及分子生物学检测方法进展.分子诊断与治疗杂志,2010,2(5):348-352.
[15]Loddenkemper R,Hauer B.Drug-resistant Tuberculosis:a worldwide epidemic pose a new challenge. Dtsch Arztebl Int, 2010, 107(1-2):10-9.
[16]Cohen T,Becerra MC,Murray MB.Isoniazid resistance and the future of drug-resistant Tuberculosis. Microb Drug Resist, 2004 ,10(4): 280–285.
[17] WHO. WHO Address TB/HIV, MDR/XDR-TB and other challenges. WHO, 2007.
[18]WHO.The Global MDR-TB & XDR-TB Response Plan 2007-2008. WHO, 2007.
[19]吴铮,许建中.基因芯片技术在结核耐药检测中的应用进展.中国脊柱脊髓杂志,2009,19 (11):863-866.
[20]Bwanga F,Hoffer S,Haile M,et al.Direct susceptibility testing for multi drug resistant Tuberculosis:a meta-analysis.BMC InfectDis ,2009, 9:67.
[21]Kant S,Maurya AK,Kushwaha RAS,et al. Multi-drug resistant tuberculosis: An iatrogenic problem. BioScience Trends,2010, 4(2):48-55.
[22]张敏,彭建涛,顾世民.结核分枝杆菌耐药检测方法的研究进展.医学综述,2010,16(18):2846-2849.
[23]刘旭红,陈旭庚,张眉眉.PCR-SSCP技术快速检测结核分支杆菌三种基因突变.中国防痨杂志,2005,27(2):130-131.
[24]吴建林,曹慧.耐药结核病流行现状与控制策略.寄生虫病与感染性疾病,2009,7(1):51-54.
[25]Lavender C,Globan M,Sievers A,et al.Molecular characterization of isoniazid-resistant Mycobacterium tuberculosis isolates collected in Australia. Antimicrob Agents Ch,2005,49(10):4068-4074.
[26]Cardoso RF,Cooksey RC,Morlock GP,et al.Screening and characterization of mutations in isoniazid-resistant Mycobacterium tuberculosis isolates obtained in Brazil.Antimicrob Agents Ch,2004,48(9):3373-3381.
[27]Marrakchi H,Laneelle G,Quemard A,et al.InhA,a target of the antituberculous drug isoniazid,is involved in a mycobacterial fatty acid elongation system,FAS-Ⅱ.Microbiology,2000, 146:289-296.
[28]Ranaswamy SV,Reich R,Dou SJ,et al.Single nucleotide polymorphisms in genes associated with isoniazid resistance in Mycobacterium tuberculosis. Antimicrob Agents Ch,2003,47(4):1241-1250.
[29]Doorn HRV,Claas ECJ,Templeton KE,et al.Detection of a point mutation associated with high-level isoniazid resistance in Mycobacterium tuberculosis by using real-time PCR technology with 3’-minor groove binder-DNA probes.J Clin Microbiol,2003,41(10):4630-4635.
[30]Chiyoji A,Ikuo K,Satoshi M,et al.Biological and molecular characteristics of Mycobacteriumtuberculosis clinical isolates with low-level resistance to isoniazid in Japan . J Clin Microbiol,2008, 46(7):2263-2268.
[31]Sreevatsan S,Pan X,Zhang Y,et al.Analysis of the oxyR-ahpC region in isoniazid-resistant and–susceptible Mycobacterium tuberculosis complex organisms recovered from diseased humans and animals in diverse localities. Antimicrob Agents Ch,1997,41(3):600-606.
[32]Baker LV,Brown TJ,Maxwell O,et al.Molecular analysis of isoniazid-resistant Mycobacterium tuberculosis isolates from England and Wales reveals the phylogenetic significance of the ahpC -46A polymorphism. Antimicrob Agents Ch,2005,49(4):1455-1464.
[33]Costa ERD,Ribeiro MO,Silva MSN,et al.Correlations of mutations in katG,oxyR-ahpC and inhA genes and in vitro susceptibility in Mycobacterium tuberculosis clinical strains segregated by spoligotype families from tuberculosis prevalent countries in South America.BMC Micobiology,2009,9(39):1471-2180.
[34]Lee ASG,Teo ASM,Wong SY.Novel mutations in ndh in isoniazid-resistant Mycobacterium tuberculosis isolates.Antimicrob Agents Ch,2001,45(7):2157-2159.
[35]Payton M,Auty R,Delgoda P,et al.Cloning and characterization of arylamine N-acetyltransferase genes from Mycobacterium smegnatis and Mycobacterium tuberculosis:increased expression results in isoniazid resistance. J Bacteriol,1999,181(4):1343-1347.
[36]Zahrt TC,Song J,Siple J,et al.Mycobacterial FurA is a negative regulator of catalase-peroxidase gene katG.Mol Microbiol,2001,39(5):1174-1185.
[37]Pym AS,Domenech P,Honore N,et al.Regulation of catalase-Peroxidase(KatG) expression,isoniazid sensitivity and virulence by furA of Mycobacterium tuberculosis. Mol Microbiol,2001,40(4):879-889.
[38]Zhang M,Yue J,Yang YP,et al.Detection of mutations associated with isoniazid resistance in Mycobacterium tuberculosis isolates from China. J Clin Microbiol,2005,43(11):5477-5482.
[39]Xu GL,You QM,Pickerill S,et al.Application of PCR-LDR-Nucleic acid detection strip in detection of YMDD mutation in Heptitis B patients treated with Lamivudine[J].J Med Virol,2010,82(7):1143-1149.
[40]Niederhauser C,Kaempf L,Heinzer I.Use of the ligase detection reaction-polymerase chain reaction to identify point mutations in extended-spectrum beta-lactamases. Eur J Clin Microbiol Infect Dis,2000, 19 :477-480.
[41]Mcnamara DT,Thomson JM,Kasehagen LJ,et al. Development of a multiplex PCR-ligase detection reaction assay for diagnosis of infection by the four parasite species causing malaria in humans.J.Clin. Microbiol,2004,42(6):2403-2410.
[42]张世扬,肖振贤,赵建龙等.基于连接酶检测反应的并行分型系统检测AGT M235T和ACE I/D基因多态性.华东理工大学学报(自然科学版),2006,32(9):1050-1054.
[43]张莹,束婧婷,韩威等.基于连接酶检测反应的NPY基因多态与清远麻鸡繁殖性状的相关性研究.中国农学通报,2010,26(17):9-12.
[44]Chen X.N. , Kenneth J.L. and Pui Y.K. . A homogeneous , Ligase-Medizted DNA diagnostic test . Genome Res.1998(8) : 549-556 .
[45]Wilson M,Derisi J,Kristensen HH,et al. Exploring drug-induced alterations in gene expression in Mycobacterium tuberculosis by microarray hybridization. Proc Natl Acad Sci USA,1999,96(22):12833-12838.
[46]Kibili GS,Mulder B,Dolmans WM,et al. M. tuberculosis genotypic diversity and drug susceptibility pattern in HIV-infected and non-HIV-infected patients in northern Tanzania. BMC Microbiol,2007,7: 51.
[47]Ming Z,Xia L,Peng X,et al. Transmission of MDR and XDR Tuberculosis in Shanghai,China. PLoS ONE,2009,4(2):e4370.
[48]Weiwei J, Igor M,Guizhi S,et al. Molecular characteristics of rifampin and isoniazid resistant Mycobacterium tuberculosis strains from Beijing , China . Chin Med J 2007 ; 120 (9) : 814-819 .