炭疽芽胞杆菌中CRISPR位点

【目的】考察炭疽芽胞杆菌中规律成簇的间隔短回文序列(Clustered regularly interspaced short palindromic repeats,CRISPR)位点多态性情况及基于CRISPR位点多态性的分子分型方法是否在炭疽芽胞杆菌分型中适用。【方法】下载NCBI数据库中6株炭疽芽胞杆菌基因组并截取其中CRISPR位点片段序列。根据炭疽芽胞杆菌内CRISPR位点信息,设计相关引物,以193株炭疽芽胞杆菌基因组为模板PCR扩增CRISPR位点片段,测序。本地Blast比对截取序列及测序结果,查看CRISPR位点在炭疽芽胞杆菌中的多态性情况,并比较炭疽芽胞杆菌与蜡样芽胞杆菌和苏云金芽胞杆菌内CRISPR位点情况。【结果】炭疽芽胞杆菌内CRISPR位点不存在多态性。【结论】基于CRISPR位点多态性的分子分型方法不适用于炭疽芽胞杆菌分型,但可以用于区分炭疽芽胞杆菌与蜡样芽胞杆菌和苏云金芽胞杆菌。     

炭疽杆菌芽孢外壁胶原样蛋白(BclA)的多态性分析

炭疽杆菌芽孢外壁胶原样蛋白（BclA）是芽孢外壁发状菌丝的主要结构成分,也是芽孢的主要免疫原。从国内分离的3株炭疽杆菌中克隆出BclA基因并进行了序列分析,结果发现有2株（A16R和40048）的BclA与国外报道菌株长度不同,分别含有388个和322个氨基酸,72个和50个GXX三氨基酸重复序列,5个和3个含21个氨基酸的（GPT）5 GDTGTT重复序列（BclA重复）。另一株40022的BclA与国外报道的53169株完全一敛,含有370个氨基酸,66个GXX重复,5个BclA重复。对我国炭疽杆菌BclA蛋白多态性的分析为进行炭疽杆菌的基因分型以及研究炭疽芽孢的免疫原性和致病机理打下基础。     

用荧光抗体双重染色法快速检验炭疽芽孢杆菌

用FFTC标记的抗带荚膜的炭疽芽孢杆菌球蛋白和RB-200标记的抗无荚膜的炭疽芽孢杆菌和巨大芽孢杆菌菌体球蛋白进行双重染色,结果带荚膜的炭疽芽孢杆菌显示特异性染色反应,其荚膜发明亮的黄绿色荧光,荚膜内的菌体里黄红色。而蜡状芽孢杆菌、巨大芽孢杆菌(形成荚膜或不形成荚膜的)等都被染成红色,与带荚膜的炭疽芽孢杆菌形成鲜明的对比。这样就避免了交叉反应,成为一种特异性很高的快速检验炭疽芽孢杆菌的方法。     

利用扩增片段长度多态性分析鉴别炭疽和蜡样芽孢杆菌

目的:利用扩增片段长度多态性(AFLP)分析建立鉴别炭疽芽孢杆菌和蜡样芽孢杆菌的分子生物学方法。方法:3株炭疽芽孢杆菌和3株蜡样芽孢杆菌基因组经限制性内切酶EcoRⅠ和MseⅠ酶切后与对应接头连接,通过预扩增和选择性扩增获得特异性DNA片段,将片段进行毛细管电泳,并利用GeneScan和BioNumerics软件对电泳数据进行分析。结果:选择性扩增最佳引物组合为EcoRⅠ-G/MseⅠ-A,其扩增片段在100~500 bp范围内的有效数量为40~50条;比较炭疽芽孢杆菌和蜡样芽孢杆菌的AFLP特征峰值图和DNA指纹图谱,确定了5个有明显差异的片段区。结论:利用AFLP分析可对芽孢杆菌属中相近的炭疽芽孢杆菌和蜡样芽孢杆菌进行鉴别,该方法可作为炭疽芽孢杆菌传统鉴定方法的补充。     

炭疽芽孢杆菌检测鉴定技术研究进展

炭疽芽孢杆菌的感染,无论是自然感染,还是作为生物恐怖和生物战的手段。快速检验和鉴定疽芽孢杆菌是最为关键的,只有正确识别生物战剂的种类,才能为正确实施防治措施指明方向。本文讨论了炭疽芽孢杆菌的检验鉴定技术,包括常规的分析培养,免疫学技术,核酸分析技术,生物传感器,基因芯片技术的应用和新诊断分子,如肽核酸与适配子的应用。     

体外筛选炭疽芽孢适配子

用SELEX技术 ,寻获炭疽芽孢适配子 ,研究其亲和功能及是否作为炭疽芽孢的检测分子。化学合成长度为 35mer的随机DNA库 ,以炭疽杆菌疫苗株A .16R芽孢为靶标进行 18轮的筛选 ,筛选产物克隆、测序 ,利用生物素 亲和素 辣根过氧化物酶显色系统判断适配子与芽孢的结合活性 ;计算机软件分析测序适配子保守序列和二级结构 ;以兔抗炭疽芽孢抗体为捕获分子 ,适配子为检测分子混合夹心法检测炭疽芽孢。第 18轮筛选的适配子与芽孢结合后A值比第 1轮的提高了 3733 .33 %以上 ;测序 79个序列中 ,A值最高为 1.2 0 ,最低为 0 .2 0 ;混合夹心法检测表明 ,适配子量为 16 μg ,芽孢为 4× 10 7个时 ,显色信号强度最强。结果提示 ,其 5′端茎环及发夹结构是与芽孢结合的基础 ,远程高级结构对其结合功能有一定的影响 ;寡核苷酸适配子可以作为炭疽芽孢的检测分子     

抗体-适配子混合夹心法检测炭疽芽孢杆菌芽孢的研究

通过SELEX(SystemEvolutionofLigandsbyEXponentialenrichment)体外筛选技术寻获的能与炭疽芽孢杆菌芽孢特异结合的DNA适配子(aptamer)考察其作为检测分子的能力。利用抗体-适配子混合夹心法,根据显色反应强度,评价适配子检测炭疽芽孢杆菌芽孢的能力。结果表明,适配子可以作为检测分子检测靶物质的存在。适配子检测范围在2-16μg,芽孢的检测范围在4×104-4×107,当适配子16μg,芽孢量为4×107显色的指示强度最强。说明适配子作为检测用分子,具有潜在的实用价值。     

炭疽杆菌检测方法的研究现状与展望

炭疽是严重威胁人类健康的烈性传染病,其病原体为炭疽芽孢杆菌。炭疽芽孢杆菌在我国公布的《人间传染的病原微生物名录》中被列为第二类病原微生物(高致病性病原微生物),其芽孢可作为生物战剂和生物恐怖的原材料,因此,发展灵敏、高效的炭疽杆菌检测方法十分重要和紧迫。按检测的靶标分类,针对炭疽杆菌的检测方法主要有四大类:针对炭疽杆菌芽孢的检测方法,针对细菌繁殖体的检测方法,针对炭疽杆菌基因的检测方法和针对炭疽毒素蛋白的检测方法。其中,针对炭疽杆菌芽孢和细菌繁殖体的检测已经有比较成熟的方法,但其在特异性以及临床的实用性方面难以令人满意;针对炭疽杆菌基因的检测技术在特异性和灵敏度上有较大的提高,但在临床诊断等方面还有欠缺;而针对炭疽毒素蛋白的检测技术的发展,使得直接对炭疽杆菌的主要致病因子的检测成为可能,这对于临床诊断以及流行病学研究具有重要意义。本文对当前炭疽杆菌检测方法的最新进展做了简要的归纳,关注了不同检测方法的适用范围和检测能力,并展望了相关领域的发展趋势,希望能为从事炭疽杆菌检测方法研究的同行提供参考和帮助。     

炭疽杆菌与其他需氧芽孢杆菌鉴别方法的研究

炭疽杆菌与其他需氧芽孢杆菌间的鉴別至今尚无较可靠方法。本文乃报导43株标准炭疽杆菌,33株标准的其他需氧芽孢杆菌及212株新分离的需氧芽孢杆菌实验结果:（1）串珠试验,43株标准炭疽杆菌中95.3%阳性,80株新分离炭疽杆菌92.5%为阳性而165株其他需氧芽孢杆菌全部为阴性。（2）W噬菌体裂解试验,所有炭疽杆菌全部被裂解,其他的需氧芽孢杆菌中仅有1株新分离的蜡样杆菌被裂解,其余全不被裂解。（3）碳酸氢钠培养基上CO2培养试验,43株标准炭疽杆菌中除7株弱毒株外,均出现粘液菌落,80株新分离的炭疽杆菌中78株出现粘液菌落而其他的则均不出现粘液菌落。（4）青霉素抑制试验、水杨苷发酵试验、动力试验及溶血试验在炭疽杆菌为阴性,在其他需氧芽孢杆菌中则不一致。因此提出,串珠试验、W噬菌体裂解试验及碳酸氢钠培养基上CO2培养下菌落的观察可作为炭疽与非炭疽杆菌的主要鉴別方法;而普通培养基上菌落的观察、青霉秦抑制试验、水杨苷发酵试验、动力试验及溶血试验可作为辅助的鉴別方法。     

炭疽病:微生物学的一个老问题带来了新烦恼

炭疽病是由炭疽芽孢杆菌的芽孢通过皮肤、胃肠道及肺部侵入人体而造成的。芽孢在体内被巨噬细胞吞噬后萌发并繁殖。带有荚膜的炭疽芽孢杆菌进入血液并释放3种外毒素。外毒素侵入宿主细胞造成患者代谢紊乱而休克致死。根据国外有关炭疽病的最新报道,对炭疽芽孢杆菌的微生物学及炭疽病的发病机理进行了综述。     

Genotyping of Bacillus cereus strains by microarray-based resequencing

The ability to distinguish microbial pathogens from closely related but nonpathogenic strains is key to understanding the population biology of these organisms. In this regard, Bacillus anthracis, the bacterium that causes inhalational anthrax, is of interest because it is closely related and often difficult to distinguish from other members of the B. cereus group that can cause diverse diseases. We employed custom-designed resequencing arrays (RAs) based on the genome sequence of Bacillus anthracis to generate 422 kb of genomic sequence from a panel of 41 Bacillus cereus sensu lato strains. Here we show that RAs represent a "one reaction" genotyping technology with the ability to discriminate between highly similar B. anthracis isolates and more divergent strains of the B. cereus s.l. Clade 1. Our data show that RAs can be an efficient genotyping technology for pre-screening the genetic diversity of large strain collections to selected the best candidates for whole genome sequencing.     

Molecular characterisation of the haemolytic isolates of Bacillus anthracis originated in Poland

Bacillus anthracis is generally considered non-haemolytic, when cultured on the solid media. However, strains capable to lyse sheep erythrocytes have been reported. Anthrolysin O, an orthologue of cereolysin was proposed as a putative haemolysin of B. anthracis. AIM: to determine whether anthrolysin O, haemolytic enterotoxin HBL and the pleiotropic regulator PlcR that activates antrholysin O production are associated with a haemolytic activity of B. anthracis strains isolated in Poland. MATERIAL: in total 8 B. anthracis strains - the fully virulent BL1 and seven the pXO2 lacking strains including: a vaccine strain Sterne 34F2 together with three haemolytic and three non-haemolytic strains isolated from different samples of the same animal died from anthrax in Poland. METHODS: The haemolytic activity was detected using Columbia agar plates supplemented with 5% of sheep blood. Anthrolvsin O, cereolysin and gene hblA encoding the key subunit of the HBL were detected by PCR. In addition, the plcR gene fragment containing the B. anthracis specific non-sense mutation was analysed by the DNA sequencing. Ten marker loci based MLVA genotyping was performed to distinguish tested strains. RESULTS: The alo gene encoding anthrolysin O was detected in both the haemolytic and non-haemolytic strains while hblA was absent. The B. anthracis specific plcR non-sense mutation was detected in both the groups of tested strains, suggesting that the haemolysis in tested strains may rather be conferred by the PlcR-independent factors. Moreover, haemolytic and non-haemolytic strains were indistinguishable by the MLVA. Obtained results may argue the haemolytic and non-haemolytic strains are isogenic and most probably a single mutational event is responsible for the haemolytic phenotype induction.     

中国的炭疽杆菌DNA分型及其地理分布

炭疽广泛分布于中国各地,特别是西部地区,并经常造成人畜疾病,在一项合作研究中,用多位点VNTR分析（MLVA）对从1952-1998年自中国主要地理流行区域分离的病人,病畜和土壤等来源的炭疽杆菌进行了基因分型,MLVA分析结果揭示了21种新的基因型,其等位基因组合在以前世界范围分离物的研究中未曾发现,此外,分离物的分群显示,A3b组是地理上最广泛分布的基因组,说明该组可能是中国的“地方流行株”。而来自古丝绸之路重要贸易中心新疆的大量分离株其基因型特别分散。     

Markers for species-specific detection of bacteria from Bacillus cereus group

rpoB and gyr genes (and their fragments) of chromosomal DNA of bacteria from Bacillus cereus group - B. anthracis, B. cereus, and B. thuringiensis - which are the potential markers for their genotyping were sequenced and phylogenetic trees were constructed. Sets of primers for species-specific detection of B. anthracis, B. cereus, and B. thuringiensis by multiplex polymerase chain reaction were designed. Also primers sets, which allow to differentiate strains of B. anthracis with various plasmid profiles (containing both plasmids (pXO1+, pXO2+), and without one (pXO1+, pXO2- or pXO1-, pXO2+) or both plasmids (pXO1-, pXO2-), determining pathogenic characteristics of the strains, were developed. For multiplex PCR primer sets were optimized on the annealing temperature of primers and amplicon length. Itwas shown that phylogenetic tree can be applied as an indicator of reliability and accuracy of taxonomical classification of microorganisms' species and subspecies. Comparison of pXO1 and pXO2 plasmid sequences of B. anthracis showed that these plasmids contain 18 and 4 palindrome sequences respectively which can potentially form thermodynamically stable hairpin-loop structures.     

Denaturing HPLC for identifying bacteria

Denaturing HPLC (DHPLC) is used in a wide variety of genetic applications. Here we introduce a new application for this technique, the identification of bacteria. We combined the capability of DHPLC to detect sequence variation with the principles of rRNA genotyping analysis to develop a high-throughput method of identifying microorganisms. Thirty-nine bacterial species from a broad spectrum of genera were tested to determine if DHPLC could be usedfor identification. Most (36 of 39) species of bacteria had a unique peak profile that could be used as a molecular fingerprint. Furthermore, a blind panel of 65 different bacterial isolates was analyzed to demonstrate the diagnostic capability of this method to specifically identify Yersinia pestis and Bacillus anthracis. All the Y. pestis samples (10 of 10) and the majority of B. anthracis samples (12 of 14) were correctly identified. The procedure had an overall specificity of 100%, overall sensitivity of 91.7%, and a predictive value of 96.9%. The data suggest that DHPLC of products spanning regions of genetic variability will be a useful application for bacterial identification.     

Meso-scale ecology of anthrax in southern Africa: a pilot study of diversity and clustering

It has only recently been possible to detect sufficient genetic diversity among anthrax isolates to allow genotype grouping (Keim et al. 1997). Early results of such grouping suggest that the southern African subcontinent may be the geographical origin of Bacillus anthracis. This report describes a pilot investigation of the genetic diversity of a study group of isolates from the Kruger National Park, South Africa, and efforts to detect spatio-temporal clustering within the study group. This study has also served as further validation for the newly developed Multi-Locus VNTR Analysis (MLVA), designed to simplify genotyping of B. anthracis isolates. The results reveal a diverse range of genotypes within the park allied with three genotype reference groups, and show that the MLVA procedure is robust for rapid analysis of B. anthracis genotypes. We also observed multiple genotype groups within epidemics and between geographically and temporally close epidemic episodes. This is in contrast to earlier characterizations of anthrax epidemics. The result of a Mantel test for time-space clustering indicates clustering of the anthrax isolates selected for the study.     

制备炭疽芽胞杆菌检测基因芯片的初步研究

建立制备炭疽芽胞杆菌检测基因芯片的技术,并探讨研制检测炭疽芽胞杆菌基因芯片的方法。酶切炭疽芽胞杆菌的毒素质粒和荚膜质粒,通过建立质粒DNA文库的方法获取探针,并打印在经过氨基化修饰的玻片上,制成用于炭疽芽胞杆菌检测的基因芯片。收集了290个阳性克隆探针,制备了检测炭疽芽胞杆菌的基因芯片。提取炭疽芽胞杆菌质粒DNA与基因芯片杂交,经ScanArray Lite芯片阅读仪扫描得到初步的杂交荧光图像。通过分析探针的杂交信号初步筛选出273个基因片段作为芯片下一步研究的探针。     

Species-specific peptide ligands for the detection of Bacillus anthracis spores

Currently available detectors for spores of Bacillus anthracis, the causative agent of anthrax, are inadequate for frontline use and general monitoring. There is a critical need for simple, rugged, and inexpensive detectors capable of accurate and direct identification of B. anthracis spores. Necessary components in such detectors are stable ligands that bind tightly and specifically to target spores. By screening a phage display peptide library, we identified a family of peptides, with the consensus sequence TYPXPXR, that bind selectively to B. anthracis spores. We extended this work by identifying a peptide variant, ATYPLPIR, with enhanced ability to bind to B. anthracis spores and an additional peptide, SLLPGLP, that preferentially binds to spores of species phylogenetically similar to, but distinct from, B. anthracis. These two peptides were used in tandem in simple assays to rapidly and unambiguously identify B. anthracis spores. We envision that these peptides can be used as sensors in economical and portable B. anthracis spore detectors that are essentially free of false-positive signals due to other environmental Bacillus spores.     

Identification of the amino acid residues critical for specific binding of the bacteriolytic enzyme of gamma-phage, PlyG, to Bacillus anthracis

Bacillus anthracis causes anthrax, a lethal disease affecting humans, which has attracted attention due to its bioterrorism potential. gamma-Phage specifically infects B. anthracis, and is used for its detection. gamma-Phage lysin, PlyG, specifically lyses B. anthracis. Mutational analysis of PlyGB (PlyG binding domain; residues 156-233) indicated that positions 190-199 are necessary for binding to B. anthracis. This region is the central part of PlyGB and is predicted to form a beta-sheet. The amino acid residues of this region are also conserved in other lysins specific for B. anthracis. Alanine substitution at position 190 or 199 within this region resulted in significantly reduced binding, suggesting that L190 and Q199 play key roles in binding of PlyGB to B. anthracis. Our observations provide new insight into the mechanism of specific binding of lysin to B. anthracis, and may be useful in establishing new methods for detection of B. anthracis.     

Use of long-range repetitive element polymorphism-PCR to differentiate Bacillus anthracis strains.

The genome of Bacillus anthracis is extremely monomorphic, and thus individual strains have often proven to be recalcitrant to differentiation at the molecular level. Long-range repetitive element polymorphism-PCR (LR REP-PCR) was used to differentiate various B. anthracis strains. A single PCR primer derived from a repetitive DNA element was able to amplify variable segments of a bacterial genome as large as 10 kb. We were able to characterize five genetically distinct groups by examining 105 B. anthracis strains of diverse geographical origins. All B. anthracis strains produced fingerprints comprising seven to eight bands, referred to as "skeleton" bands, while one to three "diagnostic" bands differentiated between B. anthracis strains. LR REP-PCR fingerprints of B. anthracis strains showed very little in common with those of other closely related species such as B. cereus, B. thuringiensis, and B. mycoides, suggesting relative heterogeneity among the non-B. anthracis strains. Fingerprints from transitional non-B. anthracis strains, which possessed the B. anthracis chromosomal marker Ba813, scarcely resembled those observed for any of the five distinct B. anthracis groups that we have identified. The LR REP-PCR method described in this report provides a simple means of differentiating B. anthracis strains.