DNA指纹技术方法学的建立及其在结核菌株鉴定中的应用

目的探讨结核分支杆菌ＤＮＡ指纹技术的方法学及其在结核菌株水平鉴定中的应用。方法以结核分支杆菌染色体ＤＮＡ插入序列ＩＳ６ＩＩＯ为基础，根据不同来源的结核分支杆菌菌株ＤＮＡ有相异的ＩＳ６１１０拷贝数和分子量，应用增强化学发光标记技术对结核分支杆菌ＤＮＡ酶切产物进行杂交和检测。结果（１）不同来源的５０例结核病人临床分离株具有相异的ＤＮＡ指纹谱。（２）－病人肺部痰和膝关节脓培养阳性的结核菌株ＤＮＡ指纹图谱     

结核分支杆菌基因组DNA指纹技术在结核病流行病学中的应用

结核分支杆菌基因组ＤＮＡ指纹技术 ,可以在种、株水平对结核分支杆菌进行鉴定 ,因此在结核病学的多个领域 ,尤其是流行病学中有很大的应用价值。一、结核分支杆菌基因组ＤＮＡ指纹技术及标准方法的确立将结核分支杆菌的基因组ＤＮＡ采用特殊的技术切割成大小不同的片段 ,电泳分离 ,就可以清楚地看到像人的皮肤指纹一样具有特征性的条带。这就是结核分支杆菌的ＤＮＡ指纹。建立结核分支杆菌ＤＮＡ指纹的主要技术有 :限制性片段长度多态性 (ＲＦＬＰ)方法和聚合酶链反应 (ＰＣＲ)方法。ＲＦＬＰ方法为 :针对结核分支杆菌基因组ＤＮＡ上的特征…     

广东省肺结核临床分离菌株DNA指纹分析

目的 构建结核分支杆菌IS6110 DNA指纹图谱，从分子水平探讨广东省结核分支杆菌的特征。方法 参照Van Embden推荐的结核分支杆菌DNA指纹标准方法，构建标准菌株Mt14323和广东省的74株临床分离株的以RFLP为基础的结核分支杆菌的IS6110 DNA指纹图谱；经互联网与世界结核菌DNA指纹库进行相似性比较；应用Gel compar 4．1(Applied Maths，Kortrijk，Belgium)软件对上述菌株的指纹图谱进行聚类分析。结果 Mt14323标准菌株和74例临床分离菌株IS6110DNA指纹图谱结果与国外同类报道一致；其中24．3％(18／74)的结核菌株的IS6110 DNA指纹相似值在1-0．65之间，鉴定结果为它们均是北京家族结核分支杆菌；IS61100 1个拷贝和2个拷贝菌株较多(21／74)。结论 目前“北京家族”结核分支杆菌菌株一定程度上在广东地区流行。     

16S-23S rDNA间隔序列PCR与RFLP对分支杆菌临床分离株的鉴定价值

为阐明分支杆菌同种临床分离株不同菌株之间以及与标准株之间１６Ｓ－２３ＳｒＤＮＡ间隔序列是否有差异，以及结核分支杆菌耐药性和１６Ｓ－２３ＳｒＤＮＡ间隔序列有无关系。本文对５８株结核分支杆菌临床分离株（２０株敏感株，３８株耐药株）和淡黄、副偶然、母牛等分支杆菌的临床分离株的１６Ｓ－２３ＳｒＤＮＡ间隔序列进行了扩增，并对扩增产物进行了聚丙烯酰胺凝胶电和琼脂糖凝胶电泳及限制性内切酶ＨａｅⅢ、ＭａｐⅠ消化反应。同种分支杆菌各菌株之间扩增产物及限制性内切酶消化产物均无差异。不同种各株之间均不相同。结果说明同种分支杆菌不同临床分离株１６Ｓ－２３ＳｒＤＮＡ间隔序列没有差异，结核分支杆菌１６Ｓ－２３ＳｒＤＮＡ间隔序列与菌株耐药性没有关系。１６Ｓ－２３ＳｒＤＮＡ间隔序列ＰＣＲ扩增与ＲＦＬＰ分析对分支杆菌临床分离株的鉴定有价值。     

PCR指印技术对新疆结核病病原Bacter460液体培养标本进行 …

以结核分枝杆菌特异插入序列ＩＳ６１１０两端序列为模板,设计一对外向ＰＣＲ引物进行ＰＣＲ扩增,从而建立一种结核分枝杆菌的快速分子生物学分型技术,该试验的基础是利用ＩＳ６１１０在结核分支杆菌染色体ＤＮＡ中反复重复且ＩＳ６１１０序列之间相距较近,经ＰＣＲ扩增呈现多条带型构成ＤＮＡ指印。在对新疆结核病人的３１份液体培养标本的ＰＣＲ检测呈现６种指印。该ＰＣＲ分型技术对结核分枝杆菌的分型所需时间短,不需细菌再     

广东省肺结核临床分离菌株DNA指纹分析

目的　构建结核分支杆菌IS6 110DNA指纹图谱 ,从分子水平探讨广东省结核分支杆菌的特征。方法 参照VanEmbden推荐的结核分支杆菌DNA指纹标准方法 ,构建标准菌株Mt14 32 3和广东省的 74株临床分离株的以RFLP为基础的结核分支杆菌的IS6 110DNA指纹图谱 ;经互联网与世界结核菌DNA指纹库进行相似性比较 ;应用Gelcompar 4 .1(AppliedMaths ,Kortrijk ,Belgium )软件对上述菌株的指纹图谱进行聚类分析。结果 Mt14 32 3标准菌株和 74例临床分离菌株IS6 110DNA指纹图谱结果与国外同类报道一致 ;其中 2 4 .3% (18/74 )的结核菌株的IS6 110DNA指纹相似值在 1～ 0 .6 5之间 ,鉴定结果为它们均是北京家族结核分支杆菌 ;IS6 110 0 1个拷贝和 2个拷贝菌株较多 (2 1/74 )。结论 目前“北京家族”结核分支杆菌菌株一定程度上在广东地区流行。     

结核分支杆菌IS6110探针的克隆化

目的 制备克隆化结构分支杆菌ＩＳ６１１０探针。方法 将ＩＳ６１１０的ＰＣＲ扩增片段克隆至质粒载体ｐＣＲ２．１中。结果 得到了含有ＩＳ６１１０克隆化探针的质粒菌株。结论 结核分支杆菌克隆化的探针易操作具一致性较好。     

AmpliSensor—聚合酶链反应定量检测肺结核患者外周血结 …

目的 探讨ＡｍｐｌｉＳｅｎｓｏｒ－聚合酶链反应定量检测外周血中结核分支杆菌ＤＮＡ在肺结核的应用价值。方法 采用ＱｌＡａｍｐ和ＡｃｕＰｕｒｅ法提取，制备全血中模板ＴＢ－ＤＮＡ，应用ＡｍｐｌｉＳｅｎｓｏｒ－ＰＣＲ定量检测，并与ＩＳ６１１０－单管巢式聚合酶链反应（ＳＮ－ＰＣＲ）作比较。结果２００例肺结核患者的血液标本中，两种方法测得结核分支杆菌ＤＮＡ的阳性率分别为６０．５％、６３．５％。８５例非结核肺病     

应用多重聚合酶链反应法检测并鉴别结核分支杆菌与非结 …

目的 提高聚合酶链反应（ＰＣＲ）检测分支杆菌ＤＮＡ的特异性和敏感性、以检测和鉴别结核分支杆菌与非结核分支杆菌ＤＮＡ。方法 应用三对具有特异性的寡核苷酸引物，进行多重ＰＣＲ扩增。这三对引物分别对应于分支杆菌６５０００表面抗原、结核分支杆菌插入序列ＩＳ６１１０及人类β－珠蛋白基因的部分序列，其扩增产物分别为３８３ｂｐ、１２３ｂｐ和２６８ｂｐ。结果 此多重ＰＣＲ电泳检测的灵敏度为０．６ｐｇ。经多重ＰＣＲ     

16S—23S rDNA间隔序列PCR与RELP对分支杆菌临床分离株的鉴定价值

为阐明分支杆菌同种临床分离株不同菌株之间以及与标准株之间１６Ｓ－２３Ｓ ｒＤＮＡ间隔序列是否有差异，以及结核分支杆菌耐药性和１６Ｓ－２３Ｓ ｒＤＮＡ间隔序列有无关系。本文对５８株结核分支杆菌临床分离株（２０株敏感株，３８株耐药株）和淡黄、副偶然、母牛等分支杆菌的临床分离株的１６Ｓ－２３Ｓ ｒＤＮＡ间隔序列进行了扩增，并对扩增产物进行了聚丙烯酰胺凝胶电泳和琼脂糖凝胶电泳及限制性内切酶ＨａｅⅢ、Ｍｓｐ     

DNA指纹技术检测耐多药结核分枝杆菌的应用研究

目的应用结核分枝杆菌DNA指纹技术,了解耐多药结核分枝杆菌的基因型状况并探讨耐多药结核病的分子流行病学。方法以结核分枝杆菌插入序列IS6110序列为模板,设计一对特异外向引物,建立一种结核分枝杆菌DNA指纹技术方法,对临床标本中分离的220株耐多药结核分枝杆菌进行DNA指纹基因分析。同时对实验数据进行聚类分析,进行耐多药结核病分子流行病学研究。结果根据结核分枝杆菌DNA指纹图谱分析,220株耐多药结核分枝杆菌均产生特征DNA指纹图谱,DNA指纹图谱变异很大。每一菌株DNA指纹的拷贝数介于3至18之间。其中大部分菌株,即205株（93.2%）包含613个拷贝,平均为11个带。这205株DNA指纹图谱中,162株为特异的,提示它们在流行病学上是独立的。有58株（26.4%）指纹图谱组成了15个簇,每簇28株,它们的IDNA指纹图谱相同,可能代表了近期的传播。尤其是在此DNA指纹图谱中,其中分子量为200 bp扩增带频率最大,在220株耐多药结核分枝杆菌均出现。结论结核杆菌DNA指纹技术检测可应用耐多药结核分枝杆菌DNA指纹图谱分型,并可用于耐多药结核病分子流行病学调查。     

福建省畲族人群结核分支杆菌IS6110DNA指纹图谱特征分析

目的分析福建省畲族人群和汉族人群结核病患者分支杆菌DNA指纹特征,探讨畲族人群结核病的分子流行病学特征。方法采用基于IS6110的PCR分型方法,对我省10个畲族乡镇调查点分离的50例畲族人群结核菌株和50例汉族人群结核菌株进行检测,用Gel-Pro analyzer4.0软件和NTSYSpc2.10e软件对DNA指纹图谱进行聚类分析。结果100株结核分支杆菌共分为4个主要类型,其中Ⅰ型(北京基因型)59株占59%,Ⅱ型38株占38%,Ⅲ型2株占2%,IV型为1株占1%。汉族病例菌株指纹为Ⅰ、Ⅱ型,以I型为主,占80%,畲族为Ⅰ、Ⅱ、Ⅲ、Ⅳ型,以II型为主,占56%;经χ2分析,两族病例菌株指纹的型别分布差异有显著统计学意义(P<0.01)。Ⅱ型菌株耐药率(47.4%,18/38)与Ⅰ型菌株耐药率(20.3%,12/59)差异也有显著统计学意义(P<0.05)。结论畲、汉两族主要基因型不同,汉族人群以北京基因型(Ⅰ型)流行为主,而畲族人群则以非北京基因型(Ⅱ型)为主,该型菌耐药率明显高于Ⅰ型,提示与畲族人群结核病高疫情相关,有待于进一步研究。     

吉林市结核分支杆菌IS6110 DNA指纹图谱特征分析

目的　分析吉林市结核分支杆菌IS6110 RFLP图谱特征。方法 对53株结核分支杆菌分离株进行IS6110基因分型,得到IS6110 RFLP图谱;按菌株指纹特征的同源性高低予以分组,并进行分析。结果 吉林市结核分支杆菌IS6110 RFLP图谱特征为：(1)IS6110拷贝数目平均为13个;(2)A、B两组同源性很高,具有“北京基因型”特征,C组多态性较强;(3)IS6110 RFLP图谱特征分布无地域差异;(4)耐药菌株与敏感菌株图谱特征无明显差异,但初治耐药菌株成簇率较高。结论 吉林市结核分支杆菌IS6110RFLP图谱特征以“北京基因型”为主;但还具一些独有的特征。     

rpoB mutations as an epidemiologic marker in rifampin-resistant Mycobacterium tuberculosis.

SETTING: Cases of rifampin-resistant Mycobacterium tuberculosis from the prison population in Madrid and from the general population in Spain. OBJECTIVE: To identify the rpoB mutations associated with resistance to rifampin and to investigate rpoB genotyping as an epidemiological marker in rifampin-resistant M. tuberculosis. DESIGN: Twenty-nine rifampin-resistant clinical isolates of M. tuberculosis, 15 obtained from the prison population in Madrid and 14 from the general population in Spain, were characterized by sequence analysis of the 81-bp core region of the rpoB gene and IS6110 DNA fingerprinting. RESULTS: All the isolates had mutations in rpoB, with those in codon 531 accounting for 41% of the total. Twenty-three (79%) isolates were highly resistant to rifampin (minimum inhibitory concentration > or = 64 mg/L). Nineteen different IS6110 fingerprints were observed: one was shared by seven isolates, one by three, two by two, and 15 were unique. Two IS6110 clusters could be divided into subclusters on the basis of rpoB analysis. Epidemiologic links were identified among patients whose isolates had identical IS6110 patterns and rpoB genotypes, but not between those with identical IS6110 patterns and different rpoB genotypes. CONCLUSION: Characterization of rpoB mutations can provide information about susceptibility to rifampin and be a useful epidemiological tool for discrimination of rifampin-resistant strains of M. tuberculosis with identical IS6110 fingerprints.     

Genotypic patterns of multiple isolates of M. tuberculosis from tuberculous HIV patients

We investigated whether the recurrence of tuberculosis in HIV-infected patients is due to an exogenous reinfection or relapses after antituberculosis chemotherapy. We reviewed clinical information on 32 patients at a Rio de Janeiro hospital from whom multiple Mycobacterium tuberculosis isolates were taken. All isolates were analysed by DRE-PCR fingerprinting technique, and those with identical DRE-PCR patterns were analysed by the RFLP method. Twenty patients had M. tuberculosis simultaneously isolated from different organs. These patients and nine others with sequential positive cultures after 2 months of therapy showed stable DRE-PCR and RFLP patterns. One patient's isolate became resistant to isoniazid, but the molecular pattern remained unchanged despite the development of drug resistance. In three patients, the DRE-PCR patterns of the isolates changed dramatically. Clinical and microbiological evidence was consistent with active tuberculosis caused by a new strain of M. tuberculosis. The exogenous reinfection of the three patients was not due to an outbreak, but the isolates from each patient showed unique patterns.     

Molecular characterization of Mycobacterium tuberculosis isolated in the State of Parana in southern Brazil

Sequence IS6110 has been successfully used throughout the world for characterizing the Mycobacterium tuberculosis lineages. The aim of this study was to obtain data about circulating strains of M. tuberculosis in patients from the State of Parana in southern Brazil. Sixty-two clinical specimens obtained from sputum, bronchial aspirate, biopsy and urine from 62 patients clinically diagnosed with tuberculosis and admitted to the SUS-Brazil - The Brazilian Centralized Health Service System - were genotyped by the mixed-linker PCR DNA fingerprinting technique. The analysis demonstrated that the number of copies of the IS6110 sequence per isolates varied from four to 13 bands, with an average number of 8.5. From this, 93% of the isolates presented multiple copies. Isolates with no copies of the IS6110 element were not observed. The genetic analysis by UPGMA grouped the 62 isolates by similarity into three different groups: the first group contained two strains, the second was composed of 23, and the third, a more heterogeneous group, contained 37 isolates. Only two isolates (3.2%) formed a cluster; in other words, they presented a pattern of polymorphism with similarity above 95%. Such findings suggest that in the State of Parana, illness predominantly develops through reactivation of the latent infection as opposed to exogenous transmission. The methodology used (mixed-linker PCR DNA fingerprinting) allowed for 93.5% differentiation of the isolates tested, and proved to be a powerful tool for differentiation in the molecular genotyping of M. tuberculosis.     

Isolation of a single strain of Helicobacter pylori from the antrum and body of individual patients

OBJECTIVES: This study aimed to determine intra-patient colonization patterns of Helicobacter pylori strains based on DNA fingerprinting and antibiotic susceptibility. METHODS: Two biopsies, one from the antrum and one from the body of the stomach, were taken from 97 patients. Prior informed consent was obtained. The status of cagA gene of H. pylori strains was analysed by using the polymerase chain reaction (PCR) technique, while DNA fingerprints were generated by PCR-based, random amplified polymorphic DNA (RAPD) fingerprinting. The antibiotic susceptibility of the H. pylori isolates was examined by the disk diffusion method. RESULTS: A total of 51 pairs of H. pylori strains were isolated from both antrum and body specimens of 51 patients. This included two patients who were endoscoped twice because of treatment failure. All strains were positive for cagA gene by PCR. These 51 patients were found to harbour a single strain of H. pylori with identical or highly similar DNA profiles by PCR-based RAPD fingerprinting. In four of the 51 pairs, the DNA patterns of H. pylori from antrum and body showed minor differences, while three pairs of strains with different metronidazole sensitivities showed identical DNA fingerprints. Interestingly, the two treatment failure patients remained colonized with the strains that had the same RAPD fingerprinting patterns before and after treatment. CONCLUSION: The present study demonstrates that a single H. pylori strain colonizes a single stomach. However, this single genotypic strain may exhibit different metronidazole susceptibility in different parts of stomach.     

结核分支杆菌利用福平药物敏感性的直接快速检测

探讨利用分子生物 学方法直接快速检测临床标本结核分支杆菌利福平（ＲＦＰ）药物敏感性的可行性。方法自行设计针对ｒｐｏＢ基因特异扩增的引物（扩增产物为１８３ｂｐ片段）,运用聚合酶链反应－单链构象多态性（ＰＣＲ－ＳＳＣＰ）银染色法,检测４５株结核分支杆菌临床分离株,对其中部分菌株进行ｒｐｏＢ基因ＤＮＡ序列分析,运用ＰＣＲ－ＳＳＣＰ银染色法对７０份结核病患痰标本进行直接快速检测,并与药敏试验结果做对照分     

Reinfection and mixed infection cause changing Mycobacterium tuberculosis drug-resistance patterns

RATIONALE: Multiple infections with different strains of Mycobacterium tuberculosis may occur in settings where the infection pressure is high. The relevance of mixed infections for the patient, clinician, and control program remains unclear. OBJECTIVES: This study aimed to describe reinfection and mixed infection as underlying mechanisms of changing drug-susceptibility patterns in serial sputum cultures. METHODS: Serial M. tuberculosis sputum cultures from patients diagnosed with multi-drug-resistant (MDR) tuberculosis were evaluated by phenotypic drug-susceptibility testing and mutation detection methods. Genotypic analysis was done by IS6110 DNA fingerprinting and a novel strain-specific polymerase chain reaction amplification method. MEASUREMENTS AND MAIN RESULTS: DNA fingerprinting analysis of serial sputum cultures from 48 patients with MDR tuberculosis attributed 10 cases to reinfection and 1 case to mixed infection. In contrast, strain-specific polymerase chain reaction amplification analysis in 9 of the 11 cases demonstrated mixed infection in 5 cases, reinfection in 3 cases, and laboratory contamination in 1 case. Analysis of clinical data suggests that first-line therapy can select for a resistant subpopulation, whereas poor adherence or second-line therapy resulted in the reemergence of the drug-susceptible subpopulations. CONCLUSIONS: We have shown that, in some patients with MDR tuberculosis, mixed infection may be responsible for observations attributed to reinfection by DNA fingerprinting. We conclude that treatment and adherence determines which strain is dominant. We hypothesize that treatment with second-line drugs may lead to reemergence of the drug-susceptible strain in patients with mixed infection.