人结核分枝杆菌特异性核酸探针制备及结核病PCR检测技术的初步临床应用

采用人结核分枝杆菌(Mycobacterium tuberculosis TB)染色体DNA为模板,选择位于插入片段IS6110中884～865和568～588碱基对处的两个片段为引物,扩增出317bp的特异性片段．将其克隆进pUCl9载体。酶切图谱分析和DNA序列测定证实为目的片段。该片段经DIG标记,分别与11种分枝杆菌DNA进行Southern杂交,结果证明只与人型复合分枝杆菌发生杂交反应。利用该对引物建立的PcR检测拄术对74份结核病痰液标本进行检测,并与临床细菌快速培养结果相比较,发现48份临床阳性均为PcR阳性,在26份临床阴性标本中亦发现11份PCR检测阳性。将标本PCR产物与克隆探针进行杂交,显示两者结果完全一致。说明PCR检测体系结果可靠,其灵敏度明显高于目前临床所采用的方法,可作为一种常规技术用于结核病的临床检测。     

建立液相芯片方法检测鉴别结核分枝杆菌复合群、鸟分枝杆菌与副结核分枝杆菌

运用液相芯片技术原理,以分枝杆菌菌种(群)特异基因序列IS6110、IS1081、IS1245和F57为目标基因,设计筛选4套扩增引物和杂交探针,建立同时检测鉴别结核分枝杆菌复合群、鸟分枝杆菌和副结核分枝杆菌的四重液相基因芯片检测方法。对13种共54株分枝杆菌菌株以及23种常见微生物样品的检测结果显示,四重液相芯片方法可特异检测鉴别目标菌种(群),与其它分枝杆菌菌种或微生物无非特异交叉反应;检测敏感性达2.1×101-2.5×102基因拷贝或0.06-0.74 fg DNA;组内检测变异系数和组间检测变异系数均<10%。采用四重液相芯片方法从临床结核疑似人痰样和牛组织样品中检出结核致病菌,检出率分别达75.6%(99/131)和94.9%(37/39),显著高于培养法(38.9%和53.8%)。对副结核疑似临床样品的检测试验结果显示,四重液相芯片方法与荧光PCR方法的阳性符合率为83%(24/29)。对四重混合模板的检测试验结果显示该液相芯片方法可鉴别不同菌种混合感染。四重液相芯片方法的检测周期<1 d,其中对纯化DNA模板的检测时间可在2-3 h内完成。     

六株成团肠杆菌 (Enterobacteragglomerans)接合子的分子生物学分析

对 6株成团肠杆菌 (Enterobacteragglomerans)接合子的分子生物学进行了分析 .6株菌与nifHDK基因有杂交 .菌株总DNA经BamHⅠ酶切后与pEA9 DNA进行Southern杂交 ,只有 2株菌具有完整的质粒DNA ,其余菌株质粒DNA发生了 15 3～ 137 7kb不同程度的缺失 .用切割位点较少的限制性内切酶XbaⅠ酶切 6株菌的总DNA ,经脉冲场凝胶电泳 (PFGE)后用pEA9 DNA为探针进行Southern杂交 ,每株菌的pEA9 DNA明显大于用BamHⅠ酶切后的杂交结果 ,表明质粒与染色体发生了整合 .转座子Tn5或插入序列IS 12 2 2和IS 12 71可能参与质粒与染色体的整合过程 .     

PCR 扩增人型结核杆菌 DNA 及结核杆菌属 DNA 探针的制备

用多聚酶链反应(PCR)方法扩增人型、牛型结核杆菌基因组 DNA,获得特异的158 bpDNA 片段,而从另外十三种分枝杆菌未见到特异的扩增产物.回收158 bpDNA 片段作探针,它除与人型、牛型结核杆菌有特异的杂交信号外,与金黄色葡萄球菌、绿脓杆菌及一些分枝杆菌皆没有杂交反应.结果表明,PCR 可用于检测结核杆菌基因组 DNA,扩增产物158 bp DNA 片段可作为探针用于检测人型、牛型结核杆菌并鉴别结核杆菌与其它分枝杆菌.     

两种DNA探针杂交检测结核分支杆菌方法的研究

为改进结核杆菌DNA探针的特异性与实用性,研制了以生物素标记的两种对结核分支杆菌特异的DNA探针:一个5’端标记的20bp的寡核苷酸探针和一个采用PCR方法合成的188bp长链探针。两种探针分别与结核分支杆菌的全染色体DNA,以及基因组上IS6110序列的一段317bp的PCR扩增产物进行斑点杂交,以碱性磷酸酶(AP)催化的染色反应检测,测试了两个探针的敏感性和特异性。系统地比较研究了两种探针杂交检测条件:探针的浓度选择,杂交温度与洗膜温度的选择,以及杂交与洗膜温度对检测的敏感性与特异性的影响。寡核苷酸探针和188bp探针杂交检测纯化结核分支杆菌基因组DNA的敏感性分别为100ng与6ng,杂交检测PCR产物的敏感性分别是400pg与50pg。两探针的最佳杂交浓度均为40～160ng/ml,最佳杂交温度分别是42℃与68℃,最佳洗膜温度分别是60℃与60～68℃之间。两种探针均仅与结核分支杆菌及BCG有杂交信号,而与其它受试分支杆菌及非分支杆菌杂交结果都呈阴性。它们的特异性都很强,但188bp探针的敏感性约是寡核苷酸探针的7～16倍,而且188bp探针检测本底较低,是检测结核分支杆菌的较佳选择     

利用rpoB基因芯片技术进行快速分枝杆菌菌种鉴定

利用rpoB基因芯片技术快速进行分枝杆菌菌种鉴定。以分枝杆菌rpoB基因编码序列为靶基因, 用基因芯片技术检测21种分枝杆菌标准株;8种其它细菌标准株;126株临床分离株。分枝杆菌与其它细菌标准株经PCR扩增后, 分枝杆菌标准株均扩增出360 bp DNA片段, 在其它细菌中, 除甲型溶血性链球菌和假白喉棒状杆菌出现同样片段外, 其它细菌均未见扩增。21种寡核苷酸探针除海分枝杆菌与偶然分枝杆菌的探针有交叉杂交外, 其余均为特异性杂交。对126株临床分离株进行鉴定, 89株为结核分枝杆菌, 占70.6％(89/126), 非结核分枝杆菌(NTM)占9.2％(9/98)。应用rpoB基因芯片技术鉴定分枝杆菌菌种, 是一种快速、准确的方法, 具有较高的临床应用价值。     

利用rpoB基因芯片技术进行快速分枝杆菌菌种鉴定

利用rpoB基因芯片技术快速进行分枝杆菌菌种鉴定.以分枝杆菌rpoB基因编码序列为靶基因,用基因芯片技术检测21种分枝杆菌标准株;8种其它细菌标准株;126株临床分离株.分枝杆菌与其它细菌标准株经PCR扩增后,分枝杆菌标准株均扩增出360 bp DNA片段,在其它细菌中,除甲型溶血性链球菌和假白喉棒状杆菌出现同样片段外,其它细菌均未见扩增.21种寡核苷酸探针除海分枝杆菌与偶然分枝杆菌的探针有交叉杂交外,其余均为特异性杂交.对126株临床分离株进行鉴定,89株为结核分枝杆菌,占70.6%(89/126),非结核分枝杆菌(NTM)占9.2%(9/98).应用rpoB基因芯片技术鉴定分枝杆菌菌种,是一种快速、准确的方法,具有较高的临床应用价值.     

联合使用放射检测和核酸探针方法快速检出和鉴定致病性分枝杆菌

<正>由于与AIDS病流行有关的结核分枝杆菌和复合鸟型分枝杆菌感染的发病率骤然增加以及市政收容所中无家可归人群中结核病的大量发生,放射检测和探钟技术在分枝杆菌实验室中的应用恰好适应了这种势头。出于临床和流行病学的考虑,快速诊断和迅速投入治疗在这两类病人中是极端重要的。 最先描述分枝杆菌的放射检测技术是在1971年。使用含确~(14)C标记的棕榈酸盐液体培养基自动检出痰液中分枝杆菌报导于1977年,使用BACTEC放射检测方法从临床标本中快速检由分枝杆菌,通过大量研究已经证实。 近年来,DNA杂交技术己应用于分枝杆菌的检测与鉴定,包括特异性探针对结核分枝杆菌,鸟型分枝杆菌和细胞内分枝杆菌培养物的鉴定。 本研究讨论了两个大医院的实验室对不同病人联合使用放射检测和DNA探针分析,以求迅速检出和鉴定分枝杆菌。     

El Tor型霍乱弧菌及其细胞壁缺陷型分子遗传学背景的研究

El Tor 型霍乱弧菌(以下简称 El Tor 弧菌)可以在人工培养条件下长期存活。当微环境改变时可形成细胞壁有不同程度缺陷的菌株如抗噬菌体突变株或 L 型菌株。我们以 DNA 酶切图谱和 El Tor 弧菌溶血素、神经氨酸酶基因探针杂交图谱为参数对 El Tor 弧菌的野生型及其细胞壁缺陷型变异株在遗传背景上进行了比较分析研究。结果提示细胞壁缺陷型菌株与其野生型在DNA 水平上高度同源。此外,文中还介绍了一种从 L 型菌株中制备 DNA 的方法。     

中国东部栗疫病菌群体的遗传分化

本实验采用RFLP技术,对中国东部栗疫病菌(Cryphonectria parasitica)进行了群体遗传结构的研究。313个参试菌株来自10个省(市)的16个群体(子群体),样本分布在北纬24°N—41°N。各菌株的DNA分别用限制性内切酶Pst Ⅰ和EcoR Ⅰ酶切,先后以10个低拷贝DNA探针和1个DNA指纹图谱探针进行了杂交和检测。结果表明,两个探针(pCB29和pMS29.1)的杂交图谱呈单态性;探针pCB19的杂交图谱显示,菌株DNA以PstⅠ酶切的为单态性,以EcoR Ⅰ酶切的则呈多态性;其他7个低拷贝探针的杂交图谱都呈多态性(Pst Ⅰ酶切)、指纹图谱探针的检测结果显示,辽宁凤城群体的菌株与中国东部其他群体的菌株相比,具有更多的限制性杂交片段,菌株间的遗传变异性也更大。     

Use of the insertion element IS6110 for DNA fingerprinting of Mycobacterium tuberculosis isolates presenting various profiles of drug susceptibility

Abstract IS 6100 is an insertion sequence of the IS3 family and it is present in multiple copies in the chromosome of Mycobacterium tuberculosis . Four to 15 copies are present in various strains of M. tuberculosis . In this study, the value of IS 6110 as an epidemiological marker of tuberculosis was examined. Unrelated clinical strains from Greek patients presented, in restriction fragment length polymorphism analysis, a high degree of polymorphism, whereas patterns of related clinical strains from familial outbreaks were identical. Since RFLP analysis with acetylaminofluorene labeled IS 6110 as the probe gave satisfactory results, it is suggested that this non-radioactive probe can be used in hospitals and health centres for the epidemiological survey of M. tuberculosis infections.     

A genomic library-based amplification approach (GL-PCR) for the mapping of multiple IS6110 insertion sites and strain differentiation of Mycobacterium tuberculosis

Evidence suggests that insertion of the IS6110 element is not without consequence to the biology of Mycobacterium tuberculosis complex strains. Thus, mapping of multiple IS6110 insertion sites in the genome of biomedically relevant clinical isolates would result in a better understanding of the role of this mobile element, particularly with regard to transmission, adaptability and virulence. In the present paper, we describe a versatile strategy, referred to as GL-PCR, that amplifies IS6110-flanking sequences based on the construction of a genomic library. M. tuberculosis chromosomal DNA is fully digested with HincII and then ligated into a plasmid vector between T7 and T3 promoter sequences. The ligation reaction product is transformed into Escherichia coli and selective PCR amplification targeting both 5' and 3' IS6110-flanking sequences are performed on the plasmid library DNA. For this purpose, four separate PCR reactions are performed, each combining an outward primer specific for one IS6110 end with either T7 or T3 primer. Determination of the nucleotide sequence of the PCR products generated from a single ligation reaction allowed mapping of 21 out of the 24 IS6110 copies of two 12 banded M. tuberculosis strains, yielding an overall sensitivity of 87,5%. Furthermore, by simply comparing the migration pattern of GL-PCR-generated products, the strategy proved to be as valuable as IS6110 RFLP for molecular typing of M. tuberculosis complex strains. Importantly, GL-PCR was able to discriminate between strains differing by a single IS6110 band.     

Analysis of sequence diversity among IS6110 sequence of Mycobacterium tuberculosis: possible implications for PCR based detection

The IS6110 belongs to the family of insertion sequences (IS) of the IS3 category. This insertion sequence was reported to be specific for Mycobacterium tuberculosis complex and hence is extensively exploited for laboratory detection of the agent of tuberculosis and for epidemiological investigations based on polymerase chain reaction. IS6110 is 1361-bp long and within this sequence different regions have been utilized as targets in the identification of M. tuberculosis by PCR. However, the results are not always consistent, specific and sensitive. In recent years, a few clinical investigations raised concerns over IS6110 specificity and sensitivity in the diagnosis of tuberculosis due to false-positive (homology with other target DNA besides M. tuberculosis) or false negative (due to absence of copies of IS6110) results with IS6110 specific primers. To unravel the variations in IS6110 sequences, an insilico analysis of IS6110 sequence of different strains of M. tuberculosis was carried out. Our results of comparative analysis of IS6110 insertion sequences of M. tuberculosis complex suggests that, IS6110 insertion sequences harbored variations in its sequence, which is evident from the phylogenetic analysis. Importantly, IS6110 sequence has divergence within the copies of same strain and formed different clusters. A list of IS6110 specific primers used in various clinical investigation of tuberculosis was obtained from the literature and their performance scrutinized. Our study emphasizes the need to develop PCR assays (multiplex format) targeting more than one region of the genome of M. tuberculosis.     

Mapping of IS6110 flanking regions in clinical isolates of Mycobacterium tuberculosis demonstrates genome plasticity

Southern hybridization was used in combination with IS6110 insertion-locus-specific probes in a comparative study to determine the structure of chromosomal domains flanking IS6110 elements in clinical isolates of Mycobacterium tuberculosis. The resulting restriction fragment length polymorphism (RFLP) data demonstrated three mutational mechanisms responsible for the polymorphisms observed: IS6110 insertion, chromosomal mutation and deletion. The frequency of IS6110 insertion within many of the chromosomal regions demonstrates that preferential integration regions are common in M. tuberculosis. Mapping the IS6110 insertion positions and chromosomal deletions in relation to the M. tuberculosis H37Rv and M. bovis BCG genome sequences reveals numerous disruptions of predicted open reading frames (ORFs). A phylogenetic tree, based on the mutational data, showed a number of independently evolving lineages of M. tuberculosis, while analysis of the mutational events occurring at each branch point suggests both divergent and convergent evolution. A significant positive correlation was demonstrated between the mutation rate and the frequency of occurrence of different isolates in families of strains, suggesting that evolution may impact on strain 'fitness' or that strain proliferation may increase the chance of mutation. We conclude that the genome of clinical isolates of M. tuberculosis continues to evolve.     

Highly polymorphic variable-number tandem repeats loci for differentiating Beijing genotype strains of Mycobacterium tuberculosis in Shanghai, China

The PCR-based variable-number tandem repeats (VNTR) typing method is a very promising tool for the molecular epidemiological study of Mycobacterium tuberculosis. The discriminatory power of the VNTR loci that were optimized in many previous studies has not been evaluated in Shanghai, an area where Beijing genotype strains dominate. In the present study, we first performed a literature search to identify VNTR loci that were at least 45 bp in length. Second, we determined the Hunter-Gaston discriminatory index (HGI) values of each of the 45 VNTR loci that we identified, using Beijing genotype strains from a 'test set' of isolates from a population with low migration in Chongming Island, Shanghai, China. Third, we optimized two sets of VNTR loci, which we named VNTR-7 and VNTR-16. The HGI value of VNTR-7 was slightly lower than that of IS6110 restriction fragment length polymorphisms (RFLP), and the HGI values of VNTR-16 and IS6110 RFLP were comparable. Our results suggest that VNTR-7, followed by VNTR-16 and IS6110 RFLP, can be used routinely as a tool to discriminate between M. tuberculosis isolates in population-based epidemiologic studies of M. tuberculosis transmission in Shanghai, China.     

Evolutionary relationships among strains of Mycobacterium tuberculosis with few copies of IS6110

Molecular typing of Mycobacterium tuberculosis by using IS6110 shows low discrimination when there are fewer than five copies of the insertion sequence. Using a collection of such isolates from a study of the epidemiology of tuberculosis in London, we have shown a substantial degree of congruence between IS6110 patterns and both spoligotype and PGRS type. This indicates that the IS6110 types mainly represent distinct families of strains rather than arising through the convergent insertion of IS6110 into favored positions. This is supported by identification of the genomic sites of the insertion of IS6110 in these strains. The combined data enable identification of the putative evolutionary relationships of these strains, comprising three lineages broadly associated with patients born in South Asia (India and Pakistan), Africa, and Europe, respectively. These lineages appear to be quite distinct from M. tuberculosis isolates with multiple copies of IS6110.     

Molecular evidence for independent occurrence of IS6110 insertions at the same sites of the genome of Mycobacterium tuberculosis in different clinical isolates

Several characteristics of Mycobacterium tuberculosis (e.g., conserved genome and low growth rate) have severely restricted the study of the microorganism. The discovery of IS6110 raised hopes of overcoming these obstacles. However, our knowledge of this IS element is relatively limited; even its two basic characteristics (transposition mechanism and target site selection) are far from well understood. In this study, IS6110 insertions in ipl loci (iplA and iplB) in two collections of clinical isolates of M. tuberculosis from different geographic locations, one from Scotland and the other from Thailand, were investigated. Five different IS6110 insertions in the loci were identified: ipl-4::IS6110, ipl-5::IS6110, ipl-11::IS6110, ipl-12::IS6110, and ipl-13::IS6110. An attempt to establish the phylogenetic relationship of the isolates containing these insertions was unsuccessful, suggesting that some of these insertions may have arisen from more than one event. This possibility is further supported by the observation that IS6110 copies existed in the same site but with different orientations in different isolates, and the insertion site of ipl-1::IS6110 harbored IS6110 copies in both iplA and iplB in different strains. All these suggest the independent occurrence of IS6110 insertions at the same sites of the genome of M. tuberculosis in different clinical isolates. The implications of this finding are discussed.     

Identification of an insertion sequence, IS1081, in Mycobacterium bovis

Abstract: An insertion sequence, IS1081, in the genome of Mycobacterium bovis has been identified and sequenced. It is 1324 bp long with 15 bp inverted repeat ends and contains a large ORF. There are six copies of IS1081 in the genome of M. bovis and the element is also present in Mycobacterium tuberculosis . IS1081 is not closely related to other DNA elements described in actinomycetes but its putative transposase bears some resemblance to that of IS256 from Staphylococcus aureus . IS1081 may be useful for genetic manipulations and for developing a diagnostic test for bovine tuberculosis based on the polymerase chain reaction.     

Identification of an insertion sequence, IS1081, in Mycobacterium bovis.

An insertion sequence, IS1081, in the genome of Mycobacterium bovis has been identified and sequenced. It is 1324 bp long with 15 bp inverted repeat ends and contains a large ORF. There are six copies of IS1081 in the genome of M. bovis and the element is also present in Mycobacterium tuberculosis. IS1081 is not closely related to other DNA elements described in actinomycetes but its putative transposase bears some resemblance to that of IS256 from Staphylococcus aureus. IS1081 may be useful for genetic manipulations and for developing a diagnostic test for bovine tuberculosis based on the polymerase chain reaction.