PCR-SSCP检测结核分枝杆菌利福平耐药相关基因rpoB突变

目的 建立聚合酶链反应-单链构象多态性(PCR-SSCP)技术快速检测结核分枝杆菌利福平(RFP)耐药相关基因rpoB突变.方法 设计结核分枝杆菌RFP耐药相关rpoB基因PCR引物,建立PCR-SSCP技术检测临床菌株rpoB基因的突变导致的运动变位,同时采用PCR直接测序(PCR-DS)技术检测rpoB基因突变,并对上述方法检测结果进行分析和比较.结果 84株临床菌株均含有rpoB基因;PCR-SSCP和PCR-DS检测结果显示,56株RFP敏感菌株中rpoB基因分别有3株和2株检测出突变,检测特异性分别为94.6％ (53/56)和96.4％(54/56);28株RFP耐药菌株中rpoB基因分别有27株和28株发生突变,检测灵敏度分别为96.4％和100％.结论 本研究建立的PCR-SSCP技术能快速、简便、特异、敏感地检测结核分枝杆菌利福平耐药基因rpoB突变,具有临床应用前景.     

非同位素PCR－单链构象多态性技术的建立和应用

ＰＣＲ－单链构象多态性技术问世以来,成为研究基因突变的工具,特别是在分子肿瘤学研究中,广泛应用于癌基因,抑癌基因突变的研究,常规ＰＣＲ－ＳＳＣＰ采用同位素标记ＰＣＲ产物,测序板电泳分离突变,在操作和费用上有种种局限,文章建立了一种非同位素ＰＣＲ－ＳＳＣＰ技术;通过不对称ＰＣＲ获得单链,普通ＰＡＧＥ分离,经银染检出突变,用这种方法,还研究了四株鼻咽癌细胞株ＣＮＥ１,ＣＮＥ２,ＨＫ１和ＳＵＮＥ１中肿瘤     

胃粘膜损伤中EB病毒感染与P53抑癌基因突变的研究

为了探讨EB病毒(EBV)感染与胃癌发生的关系,应用PCR技术对166例胃粘膜损伤标本的EBV DNA进行检测,其中66例应用银染PCR-SSCP分析技术检测了p53 exon5～8突变情况,10例应用直接法原位PCR技术检测了EBV在组织中的感染情况.结果166例标本中EBV感染率为30.1%;EBV在细胞中感染大体呈弥散型,主要存在于细胞核.66例标本中p53基因突变率为54.5%.对比分析,EBV阳性标本中p53基因突变率为75%(21/28),EBV阴性标本中p53基因突变率为39.5%(15/38).结果表明,EBV对胃粘膜组织细胞具有易感性,p53基因突变在胃粘膜病变中是一个常发事件,EBV感染与p53基因突变之间存在着高度相关性,对癌症的发生具有重要作用.     

PCR-SSCP技术在昆虫学研究中的应用

聚合酶链式反应-单链构象多态性(PCR-SSCP)技术是一种简便、灵敏的突变检测方法。随着该技术的不断发展和完善,其应用也越来越广泛。尤其是近年来该技术在昆虫学研究中涉及了:基因突变位点的检测、昆虫分类鉴定、多样性分析及连锁图谱的构建等多个领域。文章综述PCR-SSCP技术的发现、原理、在昆虫学研究中的应用及其优点和不足之处。     

宫颈癌组织中人乳头瘤病毒16型E5基因的变异

探讨了HPV16 E5基因突变与宫颈癌发病的关系.应用银染聚合酶链反应-单链构象多态性(PCR-SSCP)分析对50份人宫颈癌活检标本进行基因突变的筛查.PCR检测显示,50例宫颈癌组织中HPV16 E5的检出率为30%(15/50),其中6例宫颈癌HPV16 E5扩增片段在行SSCP分析时发现有泳动变位,突变率为40%(6/15).可见HPV16 E5基因的突变在宫颈癌的发生过程中具有重要作用.     

PCR-SSCP技术的研究及应用进展

阐述了SSCP技术的建立与发展过程;概述了PCR-SSCP技术的原理、方法、优缺点及突变技术研究进展;总结了PCR-SSCP技术在多个领域中(动物育种、基因突变、基因诊断、基因图谱和连锁分析等)的应用情况;分析了PCR-SSCP技术在分子生物学研究领域中的作用。     

DNA单链二级结构预测与单链构象多态性分析的一致性研究

单链构象多态性(SSCP)分析是一种简便,快速检测DNA突变的方法,它在基因突变检测、遗传分析、进化研究等领域有着广泛的应用价值.但是这种方法的突变检出率随DNA序列不同而变化,一般只能达到70%～80%.这主要是有的碱基突变对单链DNA的构象影响较小,不能通过SSCP检测出来.将计算机对DNA二级结构的预测结果和实验结果作了对比,发现二者有很高的一致性.这一结果表明计算机的DNA单链二级结构预测分析可用于PCR-SSCP分析的辅助设计,提高SSCP的突变检出率.     

卵巢子宫内膜异位症恶变过程中PTEN突变及微卫星不稳定的作用

目的:探讨卵巢子宫内膜异位症恶变过程中PTEN突变及微卫星不稳定的作用.方法:选取子宫内膜异位症相关性卵巢癌(endometriosis associated ovarian cancer,EAOC)26例、不典型子宫内膜异位症(atypical endometriosis,aEMs)7例、典型子宫内膜异位症(endometriosis,EMT)25例,应用PCR结合单链构象多态性(PCR-SSCP)技术及ABI测序系统对各组中PTEN基因第5和第8外显子突变情况检测,应用PCR-SSCP银染方法对5个微卫星监测点Bat25、Bat26、D2S123、D5S346及D17S250的微卫星不稳定(MSI)进行检测分析.结果:EAOC组、aEMs组及EMT组中PTEN的突变率分别为30.76%,14.29%及8%;MSI发生率分别为46.15%.28.67%及0.EAOC组中PTEN基因突变率和MSI发生率均明显高于aEMs和EMT组(P＜0.05),并且EAOC组中PTEN突变率与MSI发生率呈正相关.(r=0.409,P＜0.05).结论:PTEN突变及MSI可能参与了子宫内膜异位症的恶变过程.     

Detection of rifampin resistance patterns in Mycobacterium tuberculosis strains isolated in Iran by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing methods

Mutations in the rpoB locus confer conformational changes leading to defective binding of rifampin (RIF) to rpoB and consequently resistance in Mycobacterium tuberculosis. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) was established as a rapid screening test for the detection of mutations in the rpoB gene, and direct sequencing has been unambiguously applied to characterize mutations. A total of 37 of Iranian isolates of M. tuberculosis, 16 sensitive and 21 resistant to RIF, were used in this study. A 193-bp region of the rpoB gene was amplified and PCR-SSCP patterns were determined by electrophoresis in 10% acrylamide gel and silver staining. Also, 21 samples of 193-bp rpoB amplicons with different PCR-SSCP patterns from RIFr and 10 from RIFs were sequenced. Seven distinguishable PCR-SSCP patterns were recognized in the 21 Iranian RIFr strains, while 15 out of 16 RIFs isolates demonstrated PCR-SSCP banding patterns similar to that of sensitive standard strain H37Rv. However one of the sensitive isolates demonstrated a different pattern. There were seen six different mutations in the amplified region of rpoB gene: codon 516(GAC/GTC), 523(GGG/GGT), 526(CAC/TAC), 531(TCG/TTG), 511(CTG/TTG), and 512(AGC/TCG). This study demonstrated the high specificity (93.8%) and sensitivity (95.2%) of PCR-SSCP method for detection of mutation in rpoB gene; 85.7% of RIFr strains showed a single mutation and 14.3% had no mutations. Three strains showed mutations caused polymorphism. Our data support the common notion that rifampin resistance genotypes are generally present mutations in codons 531 and 526, most frequently found in M. tuberculosis populations regardless of geographic origin.     

Genomic organization and mutational analysis of KVLQT1, a gene responsible for familial long QT syndrome

To elucidate the role of the KVLQT1 gene in the pathogenesis of long QT syndrome (LQTS), we have established a screening system for detecting KVLQT1 mutations by the polymerase chain reaction-single strand conformation polymorphism technique (PCR-SSCP). We first determined exon/intron boundaries and flanking intronic sequences, and found that the KVLQT1 gene consists of 17 coding exons. Subsequently, we synthesized oligonucleotide primers to cover the coding region and the flanking intronic sequences, and searched for mutations in 31 Japanese LQTS families. When genomic DNA from each proband was examined by PCR-SSCP followed by direct DNA sequencing, mutations were detected in five families; two independent families carried the same mutation and three of the four were novel. Each mutation was present in affected relatives of the respective proband. This work will enable us to search more thoroughly for LQTS mutations associated with KVLQT1, and eventually will help us in finding genotype/phenotype relationships. Received: 20 March 1998 / Accepted: 30 April 1998     

结核分枝杆菌katG基因突变的研究

探讨编码过氧化氢-过氧化物酶的katG基因突变与结核分枝杆菌异烟肼(INH)耐药性的相关关系。根据结核分枝杆菌GenBank中的katG序列,自行设计特异性寡聚核苷酸引物,采用聚合酶链反应-单链构象多态性(PCR-SSCP)分析和直接测序法(DS)分析结核分枝杆菌中katG基因突变情况。以HR37Rv标准株为对照。所有23株敏感菌均未有SSCP结果异常;35株耐药菌中,有2株(5．7％)katG基因扩增阴性,且发生在高度耐药菌中。进一步分析发现,SSCP法突变检出23株(65．7％),测序法突变检出24株(68．6％),符合率为95．8％(23／24)。参照测序法对耐药菌突变序列的分析结果,PCR—SSCP敏感、特异,可快速检测结核分枝杆菌katG耐药基因突变,有利于耐药结核分枝杆菌耐药性的快速检测。     

PCR-SSCP技术在微生物检测中的应用

聚合酶链式反应-单链构象多态性(PCR-SSCP)是一种能够检测DNA突变的分子生物学分析技术,具有快速、简便、灵敏与适于大样本筛选的特点,近年来被广泛应用于生命科学领域的研究。对PCR-SSCP技术在微生物检测研究领域的应用和发展作简要的介绍。     

结核分枝杆菌rpoB基因突变的检测(简报)

结核病主要是由结核分枝杆菌(Mycobacterium tuberculosis)引起的一种慢性传染性疾病。利福平是结核病化疗方案中一个关键性的药物,它在结核病的短程化疗中起着重要的作用。但是,在我国结核菌对利福平的耐药发生率呈上升局势,而通过传统的依赖生物生长的药敏试验方法进行结核菌对利福平耐药性检测所需时间较长(4-8周),不能满足临床早期开展有效化疗的需要,所以迫切需要建     

Detection of mutation of the p53 gene with high sensitivity by fluorescence-based PCR-SSCP analysis using low-pH buffer and an automated DNA sequencer in a large number of DNA samples

Detection of mutations in genes responsible for hereditary diseases or tumors is important clinically. It is necessary to establish a simple technique for screening mutations in large numbers of samples. The polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) method has proved to be a useful technique for analyzing mutations or DNA polymorphisms. Non-radioisotopic versions using fluorescent dye and an automated DNA sequencer have also been exploited to extend this technique into the clinical field. We have examined mutations of exons 5-9 of the p53 gene in 112 colorectal, 28 esophageal and 33 hepatocellular carcinomas by fluorescence-based PCR-SSCP (F-SSCP) under various conditions. We found 64 types of mutations in 63, 17 and 12 cases of colon, esophageal and hepatocellular carcinomas by F-SSCP. We determined the sequence of all samples, and confirmed that all mutations were successfully detected by F-SSCP. With the low-pH buffer system, 61 types of mutants were detected, while 51 types were detected by TBE and 57 types were detected by TBE with glycerol gel. The polyacrylamide gel in TME or TBE without glycerol was tough and could be used repeatedly, but the glycerol containing gel was fragile and could not stand repeated use. Thus, use of a low-pH buffer in the electrophoresis of F-SSCP is simpler and better at detecting mutations than the conventional TBE buffer system. We believe that low-pH F-SSCP analysis is an efficient and powerful technique for examination of a large number of samples, in particular clinical specimens obtained by biopsy or surgery.     

结核分枝杆菌耐链霉素和乙胺丁醇分子机制的研究

目的:研究结核分枝杆菌耐链霉素和乙胺丁醇的rpsL和emb B基因突变情况,探讨耐药基因突变与耐药性的关系。方法:通过传统药敏实验和聚合酶链反应(PCR)--单链构象多态性(SSCP)技术初步鉴定62株临床分离株的药敏和rps L、emb B基因。结果:与结核菌标准株H37Rv对照,分析30例TB菌耐链霉素(SM)的rps L基因,发现其突变率为70.0%(21/30),分析29例耐乙胺丁醇(EMB)的emb B基因,该基因的突变率为65.5%(19/29)。结论:部分结核分枝杆菌耐SM和EMB是由于其rps L、emb B基因突变所致,PCR-SSCP银染技术可能成为测定部分结核分枝杆菌耐药的简便、快速的方法。     

Characterization of rpoB mutations associated with rifampin resistance in Mycobacterium tuberculosis from eastern China

Aims:  The aim of this study was to investigate the features of rpoB gene mutations associated with Rifampin (RIF) resistance in Mycobacterium tuberculosis ( M. tuberculosis ) in eastern China.
Methods and Results:  The mutations of rpoB gene in 56 clinical isolates of M. tuberculosis resisted to one to four first-line drugs (rifampin, isonicotinyl hydrazide, ethambutol and streptomycin) were analysed by polymerase chain reaction single strand conformation polymorphism analysis (PCR-SSCP) and DNA sequencing. The results of PCR-SSCP showed 52 isolates were positive (existing rpoB mutation) including 47 isolates resisted to RIF. Subsequent results of DNA sequencing showed that 54 isolates had rpoB gene mutation including 49 isolates resisted to RIF. The most frequently mutated sites were at codons 526 (73·2%), 513 (10·7%) and 531 (3·5%).
Conclusions:  The rpoB codon 526 was the most frequently mutated site of RIF-resistant M. tuberculosis strains in eastern China and its frequency is significantly higher ( P  < 0·0001) compared with that in other areas of China and in other geographic regions worldwide.
Significance and Impact of the Study:  Our results reveal that geographic variation is responsible for rpoB mutations in M. tuberculosis and the resulting information will be helpful to improve a novel rapid molecular drug resistance screening approach for MDR TB.