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。     

环介导等温扩增技术在快速检测炭疽芽胞杆菌中的应用

本文旨在建立适合国境口岸现场应用的生物恐怖防控快速检测方法,从而保障口岸安全.针对生物恐怖炭疽芽胞杆菌,选择目标菌种特异性基因片段,设计引物,运用环介导等温扩增(LAMP)技术建立一套简便、高效的检测方法,并模拟生物恐怖炭疽芽胞杆菌可能存在的基质条件,评价LAMP技术在快速筛查中的适用性.结果显示,LAMP技术排查生物恐怖炭疽芽胞杆菌简便、快速、特异,检测灵敏度为102～103 CFU/ml;且能有效检出在偏酸、偏碱及黏稠基质中的炭疽芽胞杆菌.而高盐环境对该反应影响较大,有必要采用能有效去除盐分的核酸抽提方法.     

A DNA microarray facilitates the diagnosis of Bacillus anthracis in environmental samples

Aims:  In order to improve the diagnosis of Bacillus anthracis in environmental samples, we established a DNA microarray based on the ArrayTube technology of Clondiag.
Methods and Results:  Total DNA of a bacterial colony is randomly biotinylated and hybridized to the array. The probes on the array target the virulence genes, the genomic marker gene rpoB , as well as the selective 16S rDNA sequence regions of B. anthracis , of the Bacillus cereus group and of Bacillus subtilis . Eight B. anthracis reference strains were tested and correctly identified. Among the analysed environmental Bacillus isolates, no virulent B. anthracis strain was detected.
Conclusions:  This array clearly differentiates B. anthracis from members of the B. cereus group and other Bacillus species in environmental samples by chromosomal ( rpoB ) and plasmid markers. Additionally, recognition of B. cereus strains harbouring the toxin genes or atypical B. anthracis strains that have lost the virulence plasmids is feasible.
Significance and Impact of the Study:  The array is applicable to the complex diagnostics for B. anthracis detection in environmental samples. Because of low costs, high security and easy handling, the microarray is applicable to routine diagnostics.     

A Bacillus thuringiensis strain producing a polyglutamate capsule resembling that of Bacillus anthracis

Bacillus thuringiensis serovar Monterrey strain BGSC 4AJ1 produced a microscopically visible capsule that reacted with a fluorescent antibody specific for the poly-gamma-d-glutamic acid (PGA) capsule of Bacillus anthracis. PGA capsule biosynthesis genes with 75%, 81%, 72%, 65% and 63% similarity, respectively, to those of the B. anthracis capBCADE cluster were present on a plasmid (pAJ1-1). Strain BGSC 4AJ1, together with five strains of Bacillus cereus that hybridized to a PGA cap gene probe, were analyzed phylogenetically using six housekeeping genes of a B. cereus multilocus sequence typing scheme. Bacillus thuringiensis BGSC 4AJ1 shared four identical alleles with B. anthracis and was the second most closely related to this bacterium of the 674 isolates in the multilocus sequence typing database. The other cap+ strains were distributed among various lineages of Clade 1 of the B. cereus group.     

炭疽杆菌致病性研究进展

炭疽杆菌是人类历史上第一个被发现的病原菌。炭疽杆菌的研究在近几年取得了较大进展 ,特别是本年度其基因组序列测定已完成并向全世界公布 ,进一步深化了对炭疽杆菌的研究。炭疽杆菌致病性的研究一直是炭疽杆菌研究的重点 ,近年来此方面的研究取得了很多新进展 ,从基因组、致病物质及致病机制 3个方面对此作一个简单的介绍。     

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。     

PlcR在炭疽芽胞杆菌A16R中的功能研究

炭疽芽胞杆菌(Bacillus anthracis)、蜡样芽胞杆菌(B. cereus)和苏云金芽胞杆菌(B. thuringiensis)均属于蜡样芽胞杆菌群,在遗传学上有很高的相似性。PlcR (Phospholipase C regulator)在蜡样芽胞杆菌中是十分重要的调控因子,但plcR基因在炭疽芽胞杆菌中发生一个无义突变导致在炭疽芽胞杆菌中产生一个截短PlcR蛋白。为了研究plcR基因对炭疽芽胞杆菌功能的影响,文章以蜡样芽胞杆菌CMCC6330基因组为模板,构建重组表达质粒pBE2A-plcR后导入炭疽芽胞杆菌疫苗株A16R中获得重组菌株,对其进行表型分析。结果显示,炭疽芽胞杆菌重组菌株的溶血活性基本没有恢复,但恢复了部分神经鞘磷脂酶活性,表明将蜡样芽胞杆菌的plcR基因导入炭疽芽胞杆菌后,可以直接激活神经鞘磷脂酶活性。     

Identification and characterization of Bacillus anthracis by multiplex PCR analysis of sequences on plasmids pXO1 and pXO2 and chromosomal DNA

Abstract Bacillus anthracis can be identified on the basis of the detection of virulence factor genes located on two plasmids, pXO1 and pXO2. Thus isolates lacking both pXO1 and pXO2 are indistinguishable from closely related B. cereus group bacteria. We developed a multiplex PCR assay for characterization of B. anthracis isolates, and simultaneous confirmation of the species identity independent of plasmid content. The assay amplifies lef, cya, pag (pXO1) and cap (pXO2) genes, and a B. anthracis specific chromosomal marker, giving an easy-to-read profile. This system unambiguously identified virulent (pXO1⁺/2⁺) and avirulent (pXO1⁺/2⁻, pXO1⁻/2⁺ and pXO1⁻/2⁻) strains of B. anthracis and distinguished 'anthrax-like' strains from other B. cereus group bacteria.     

Isolation of a specific chromosomic DNA sequence of Bacillus anthracis and its possible use in diagnosis

Abstract A 277-bp long DNA fragment, Ba813, was isolated from an avirulent Bacillus anthracis strain 7700 genomic library. Two oligonucleotides derived from the Ba813 sequence were used as primers in polymerase chain reaction tests on genomic DNA from 28 Bacillus anthracis and from 33 heterologous bacteria strains. A specific, 152-bp long DNA fragment was amplified only when Bacillus anthracis DNA was used as the target. The amplified product was analysed by non-radioactive sandwich hybridisation in microtiter plates using two oligonucleotides. The capture oligonucleotide C1 was covalently linked onto aminated wells of microtiter plates. The detection oligonucleotide D3 was labelled with biotine. The hybrid molecules were detected by avidine conjugated with alkaline phosphatase and chromogenic substrate. Amplification of Ba813 sequence may provide the basis for rapid and reliable assay for the detection and identification of Bacillus anthracis .     

Differential identification of Bacillus anthracis from environmental Bacillus species using microarray analysis

AIMS: To determine whether microarray analysis could be employed for the differential identification of a range of environmental Bacillus sp. from four strains of Bacillus anthracis. METHODS AND RESULTS: Oligonucleotide probes were designed that were specific to virulence factor genes of B. anthracis (pag, lef and cap), the variable number tandem repeat region of the B. anthracis vrrA gene and to the 16S-23S rRNA intergenic transcribed spacer region (ITS) and pleiotropic regulator (plcR) regions of the Bacillus cereus subgroup species. Generic probes were also designed to hybridize with conserved regions of the 16S rRNA genes of Bacillus (as a positive control), Neisseria sp., Pseudomonas sp., Streptococcus sp., Mycobacterium sp. and to all members of the Enterobacteriaceae to allow simultaneous detection of these bacteria. Identification of B. anthracis was found to rely entirely on hybridization of DNA specific to regions of the pag, lef and cap genes. Cross-reaction was observed between B. anthracis and other Bacillus species with all the other Bacillus probes tested. Results obtained using microarray hybridizations were confirmed using conventional microbiological techniques and found to have very high comparability. CONCLUSIONS: Microarray-based assays are an effective method for the identification of B. anthracis from mixed-culture environmental samples without problems of false-positivity that have been observed with conventional PCR assays. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of environmental Bacillus sp. by conventional PCR is prone to potential for reporting false-positives. This study provides a method for the exclusion of such isolates.     

Comparative analysis of 23S ribosomal RNA gene sequences of Bacillus anthracis and emetic Bacillus cereus determined by PCR-direct sequencing

The primary structures of the 23S ribosomal RNA genes of Bacillus anthracis and an emetic strain of Bacillus cereus were determined by direct sequencing of enzymatically amplified chromosomal DNA. The 23S rRNA gene sequences of B. anthracis and B. cereus were found to be almost identical and showed only two differences (a single nucleotide change, and a single base insertion in B. cereus). The feasibility of using PCR-direct sequencing for the rapid sequence determination of large-subunit rRNA genes is demonstrated.     

炭疽芽胞杆菌基因芯片探针文库的构建

为制备炭疽芽胞杆菌的基因芯片探针文库,以炭疽芽胞杆菌毒素质粒pX01和荚膜质粒pX02为原材料,用Sau3A I酶切pX01和pX02质粒DNA,Taq DNA聚合酶72℃补平加A,经AT克隆,PCR初步鉴定筛选出炭疽质粒片段的阳性克隆．DNA自动分析仪对克隆片段进行序列测定;用生物信息学方法对其片段进行同源性分析;并将克隆的探针打印于玻片上,制备成炭疽芽胞杆茵基因芯片,与炭疽杆茵质粒DNA样品进行初步芯片杂交的实验,杂交实验的阳性率达到了90％以上,证明大部分克隆探针属于炭疽芽胞杆菌．炭疽芽胞杆菌基因芯片探针文库的构建为基因芯片探针的制备摸索出一条简便、高效、可行的方法．     

Characterization of Zebu cattle breeds in Tanzania using random amplified polymorphic DNA markers

A total of 141 short primers, of arbitrary nucleotide sequence, were used singly in poly-merase chain reactions to amplify DNA fingerprints in pools of DNA representing three Zebu cattle breeds. Two primers, which discriminated between the breed-specific DNA pools were used further to amplify individual pool components in order to establish band frequencies of the amplified fingerprints. One of the primers (ILO 1127) amplified a RAPD fingerprint in 61%of TSZ animals but less than 6% in the other breeds, while another primer (ILO 1065) revealed a DNA sequence common to 89% of the Boran animals and less than 30% in the other two breeds. Bandsharing and mean average percentage difference calculated within and between the three breeds using RAPD fingerprint data showed a higher degree of homogeneity within than across the breeds and indicated measurable divergence between the three breeds. It is concluded that RAPD polymorphisms are useful as genetic markers for cattle breed differentiation.     

2-D reference map of Bacillus anthracis vaccine strain A16R proteins

Bacillus anthracis has always been an important pathogen because it can cause lethal inhalational anthrax, and may be used as a bioweapon or by bioterrorists. In this study, a 2-DE reference map and database of B. anthracis A16R was constructed. In total, 534 spots were processed, and 406 spots representing 299 proteins were identified. Gel-estimated pIs and molecular masses mostly matched well with their theoretical predictions, but some discrepancies also existed. Spot and protein corresponding analysis revealed that post-translational modifications might be common in B. anthracis. Through the MASCOT search, the similarity of B. anthracis, B. cereus and B. thuringiensis was further verified by protein level and a possible annotation error in B. anthracis strain Ames 0581 genome was found. Proteins of energy metabolism, fatty acid and phospholipid metabolism, protein synthesis, and cellular processes represented a large part of the most abundant proteins. At the same time, 27 hypothetical proteins were experimentally proved. There were 28 proteins also identified as spore composition in recently spore-related research, which indicated that they might play some roles in different phases such as growth, sporulation and outgrowth. Maps and information about all identified proteins are available on the Internet at http://www.mpiib-berlin.mpg.de/2D-PAGE and http://www.proteomics.com.cn.     

The extracellular and cytoplasmic proteomes of the non-virulent Bacillus anthracis strain UM23C1-2

The recently published genome sequence of Bacillus anthracis Ames has facilitated the prediction of proteins associated with the virulence of this bacterium. The aim of this study was to define reference maps for the extracellular and cytoplasmic proteomes of the avirulent B. anthracis strain UM23C1-2 that are useful for physiological studies and the development of improved vaccines. Using 2-DE and subsequent MALDI-TOF-TOF MS, 64 proteins were identified in the extracellular proteome, only 29 of which were predicted to be exported into the culture medium. The latter included chitinases, proteases, nucleotidases, sulfatases, phosphatases and proteins of unknown function. Of the remaining proteins in the culture medium, 18 were predicted to be associated with the cell wall or anchored on the trans side of the cytoplasmic membrane while 17 other proteins lacked identifiable export signals and were predicted to be cytoplasmic proteins. Among the S-layer proteins, Sap and Eag account for 10% of the total extracellular proteome. Many of the proteins are predicted to contribute to the virulence and antigenic signature of B. anthracis. We have also studied the composition of the cytoplasmic proteome, identifying 300 distinct proteins. The most abundant cytoplasmic proteins are primarily those involved in glycolysis, amino acid metabolism, protein translation, protein folding and stress adaptation. The presence of a variety of proteases, peptidases, peptide binding proteins, as well as enzymes required for the metabolism of amino acids, suggests that B. anthracis is adapted to life in a protein-rich environment rather than the soil. We therefore speculate that proteases and peptidases could be useful targets for the development of improved vaccines. In addition, both of these B. anthracis compartment-specific proteomes can be used as reference maps to monitor changes in the production of secreted and cytosolic proteins that occur, for example, during growth in macrophages.     

Destruction of Bacillus anthracis strain Sterne 34F2 spores in postal envelopes by exposure to electron beam irradiation

AIMS: To determine the irradiation dose necessary to reduce the populations of Bacillus anthracis spores in a dry medium in postal envelopes. METHODS AND RESULTS: Bacillus anthracis Sterne 34F2 spores were dispersed in non-fat dry milk and then placed into standard business postal envelopes. The spores were treated with a sequence of irradiation doses to determine the decimal reduction value (D10) in kiloGrays (kGy). The average D10 value was 3.35 +/- 0.02 kGy. CONCLUSIONS: An irradiation dose of 40.2 kGy would be required to result in a process equivalent to the thermal canning process (12 D10 reduction) to eliminate Clostridium botulinum spores. SIGNIFICANCE AND IMPACT OF THE STUDY: Irradiation is an effective means of reducing or eliminating B. anthracis spores in a dry medium in postal envelopes.     

Detection of section-specific random amplified polymorphic DNA (RAPD) markers in Lilium

Random amplified polymorphic DNA (RAPD) markers were utilized for the identification of Lilium species and inter-specific hybrids. The optimum annealing temperature of the polymerase chain reaction (PCR) for the RAPD assay in Lilium was 54 °C, which is relatively higher than the temperature used for other genera reported by previous researchers. Among 76 primers used to amplify genomic DNA by PCR, 18 primers (24%) generated polymorphic DNA fragments in Lilium species and hybrids. Cultivars were also identified by RAPD markers. Some amplified fragments were unique to species of each section and to hybrids derived from these species; that is, they were the section-specific DNA markers. Sections, Sinomartagon, Leucolirion b, Leucolirion a and Archelirion could be identified by 6 section-specific markers amplified with five primers. Seven inter-section hybrids showed the section-specific bands of both parental sections, indicating that these markers would be useful for identifying the parental sections of inter-section hybrids.     

Expression and secretion of the protective antigen of Bacillus anthracis in Bacillus brevis

We used the Bacillus brevis-pNU212 system to develop a mass production system for the protective antigen (PA) of Bacillus anthracis. A moderately efficient expression-secretion system for PA was constructed by fusing the PA gene from B. anthracis with the B. brevis cell-wall protein signal-peptide encoding region of pNU212, and by introducing the recombinant plasmid, pNU212-mPA, into B. brevis 47-5Q. The clone producing PA secreted about 300 microg of recombinant PA (rPA) per ml of 5PY-erythromycin medium after 4 days incubation at 30 degrees C. The rPA was fractionated from the culture supernatant of B. brevis 47-5Q carrying pNU212-mPA using ammonium sulfate at 70% saturation followed by anion exchange chromatography on a Hitrap Q, a Hiload 16/60 Superdex 200 gel filtration column and a phenyl sepharose hydrophobic interaction column, yielding 70 mg rPA per liter of culture. The N-terminal sequence of the purified rPA was identical to that of native PA from B. anthracis. The purified rPA exhibited cytotoxicity towards J774A.1 cells when combined with lethal factor. The rPA formulated in either Rehydragel HPA or MPL-TDM-CWS adjuvant (Ribi-Trimix) elicited the expression of a large amount of anti-PA and neutralizing antibodies in guinea pigs and completely protected them against a 100 LD50 challenge with fully virulent B. anthracis spores.     

Sexing birds using random amplified polymorphic DNA (RAPD) markers

We used random amplified polymorphic DNA (RAPD) markers to sex birds from small tissue (usually blood) samples. Arbitrarily chosen 10-mer PCR primers were screened with DNA from known-sex individuals for the production of a bright female-specific band. Suitable primers were found for seven bird species after screening about 30 primers (range 2–63), and no primer was found for three other species after screening about 50 primers for each species. Investigations into the reliability of RAPD markers for sexing great tits Parus major and oystercatchers Haematopus ostralegus show that: (i) when PCR reaction conditions for great tit DNA are varied, either the presence of the female-specific band correctly predicts the individual's sex or no DNA amplification occurs; (ii) the female-specific band in great tits can be sequenced, and subsequently amplified using specific PCR primers; (iii) null alleles of the female-specific fragment occur at an estimated frequency of 0% ( n = 241 females) in great tits and 0.6% ( n > 290 females) in oystercatchers; (iv) the female-specific fragment in great tits occurs in individuals from a wide geographical range encompassing two subspecies; and (v) the relative intensity of bands in great tit RAPD banding profiles is consistent across individual birds and scorers. The RAPD primers that we have identified are generally species specific, and the consequent time cost of screening for primers is the chief disadvantage of using RAPD markers to sex birds. However, with large sample sizes this disadvantage is outweighed by the relative technical simplicity and low cost of the technique.