副溶血性弧菌重复序列-PCR分型研究

利用基因外重复回文序列-PCR(REP-PCR)和肠细菌基因间共有重复序列-PCR(ERIC-PCR)技术,对副溶血性弧菌进行了分子分型研究和亲缘关系的探讨,并使用Hunter和Gaston方法计算分辨力指数.结果显示40株副溶血性弧茵分离株均可扩增产生可重复的DNA指纹图谱,并且不同菌株基因组DNA的扩增条带具有多态性.根据SPSS10.0软件得出的树状图结果,REP-PCR可以把40株茵分为21个型,分辨力指数可达到0.953,优势菌型为G1型;ERIC-PCR可将40株菌分成4个型,分辨力指数为0.5.研究显示重复序列-PCR方法可以用于该菌分型分析,REP-PCR具有较好的分型能力.在两种PCR的DNA指纹图谱中,血清型O1群与O3群主条带均非常相似,表明它们之间亲缘关系密切.     

中国人常见HLA—DPB1不明确杂合子SSP分型解决方案

目的：人类染色体是二倍体,这使得以测序方法研究多态性区域时会遇到不明确杂合子,这个问题在HLA-DPB1的分型上尤为突出。试图寻找一个来解决HLA-DPB1分型中不明确杂合子比例高给分型带来的困难的方案。方法：对946例样品进行HLA-DPB1分型,其中不明确杂合子有353例,共30种,占总例数的37％。建立了一套SSP分型方法,设计上游引物6条,下游引物15条,每一个样品同时用两对特异性引物进行扩增,两对引物分别代表两种不同的杂合模式。再从30种不明确杂合子类型中各挑2个样品进行克隆测序,结果与SSP分型结果比较。结果：SSP分型方法采用同一套扩增体系与两套循环反应条件,只需一次PCR扩增,就可以方便快捷地实现不明确杂合子的分辨,其结果与克隆测序结果相符。结论：与以往的克隆测序、SSCP方法相比,本研究中SSP分型方法具有高效率、高通量、省时省力的特点。     

Study of the influence of plasmids on the arbitrary primer polymerase chain reaction fingerprint of Escherichia coli strains

Abstract To study the effect of plasmids on the arbitrary primer-polymerase chain reaction fingerprint of bacterial strains, the Escherichia coli strains DH5, Top10, and W3110 were transformed with plasmids of different sizes: respectively, pUC19, pCEP and two clinically important plasmids carrying resistance to several antibiotics. Total DNA, i.e. both chromosomal and plasmid DNA, was prepared from transformed cells by boiling the cell suspensions and by phenol-chloroform extraction; chromosomal DNA was prepared by the same methods from the non-transformed, plasmid-free strains; plasmid DNA of pUC19 was purchased; plasmid DNA of pCEP was purified from the transformed strains by caesium chloride density gradient centrifugation. Arbitrarily primed polymerase chain reaction was carried out for all of these preparations. Amplification carried out independently with three different primers resulted in similar patterns for the chromosomal preparations whether or not plasmid was present. Amplification of plasmid DNA gave different patterns, characterized by fragments larger than those obtained when total or chromosomal DNA were used as the target. These data illustrate that the plasmids studied here do not influence the chromosomal arbitrarily primed PCR fingerprint, although plasmids alone are amplified in the absence of chromosomal DNA. Experiments comparing different relative concentrations of plasmid and chromosomal DNA indicate that under natural conditions the amount of chromosomal DNA per cell is sufficient to inhibit observable amplification of the plasmid(s) present.     

粤蓝链霉菌榴菌素生物合成基因orf20的功能

【目的】在大肠杆菌中,转录因子SoxR作为胞内氧化还原感应器,参与抗氧化胁迫的全局性调控。粤蓝链霉菌榴菌素生物合成基因簇内存在一个类soxR基因orf20,但其生理功能仍不清楚。【方法】将orf20基因在大肠杆菌中进行表达,分析携带重组质粒的大肠杆菌对百草枯抗性的变化。同时通过修改后的PCR-targeting方法构建粤蓝链霉菌orf20删除的突变株,分析突变株的表型变化和对百草枯抗性水平的变化。【结果】重组ORF20在羧基端含有组氨酸标签,并在大肠杆菌中获得可溶性表达,携带重组质粒pET28b-orf20的大肠杆菌对百草枯的抗性水平显著提高。粤蓝链霉菌orf20删除突变株仍具有产孢能力,生长特性没有改变,对百草枯的抗性水平也没有变化,但榴菌素的产量大幅提高,是野生株的3.3倍。【结论】在大肠杆菌中,orf20基因的编码产物能够被百草枯激活,替代SoxR参与抗氧化胁迫的调控。在粤蓝链霉菌中,orf20基因不参与抗氧化胁迫,而对榴菌素的产生有负调控效应。     

广州地区嗜肺军团菌环境分离株的基因序列分型分析

摘要:【目的】研究广州市嗜肺军团菌的基因特征,对来自不同水域环境的嗜肺军团菌进行分子分型研究。【方法】选择嗜肺军团菌的7个基因flaA、asd、mip、pilE、mompS、proA和neuA 作为目的基因, 对在2006-2009年间广州地区分离的44株嗜肺军团菌进行PCR扩增和测序,并将核苷酸序列上传至欧洲军团菌病感染工作组（EWGLI）数据库进行比对,得到基因型别（Sequence type, ST）,对结果进行基因序列分型（Sequence-Based Typing, SBT）和系统进化分析。【结     

单纯疱疹病毒分子生物学分型方法的研究

联合运用HSV-1和HSV-2型特异性核酸探针对28株HSV临床分离株做了鉴定分型,并与应用HSV型特异性单克隆抗体的三种免疫学方法的检测结果进行了比较。核酸探针与单克隆抗体之间的符合率为100%,但分型率不同,核酸探针为100%,单克隆抗体为64—82%。研究结果表明,本实验所建立的HSV-McAb-APAAP桥联免疫酶染技术具有简便、敏感、实用的特点。     

Development of a new MLST scheme for differentiation of Fusarium solani Species Complex (FSSC) isolates

Fungi belonging to the Fusarium solani Species Complex (FSSC) are well known plant pathogens. In addition to being the causative agent of some superficial infections, FSSC has recently emerged as a group of common opportunistic moulds, mainly in patients with haematological malignancies. Molecular typing methods are essential in order to better understand the epidemiology of such opportunistic agents with the final goal of preventing contamination. A three-locus typing scheme has thus been developed for FSSC; based on polymorphisms in the domains of the ITS, EF-1 alpha, and RPB2 genes. This method is now considered to be a useful reference for phylogenetic and taxonomic studies. In other significant clinical fungi (e.g., Candida sp., Cryptococcus neoformans, and Aspergillus fumigatus), genes coding for metabolic enzymes have been widely used and proven to be very informative for diagnosis and epidemiology. The contribution of these genes has never been evaluated for Fusarium sp. and more specifically for F. solani Species Complex.Here, we have evaluated the contribution of 25 genes for diagnosis and epidemiological purposes. We then report a new five-locus MLST scheme useful for diagnosis and typing of clinical FSSC isolates. The method has been validated on 51 epidemiologically unrelated strains of FSSC and presents a high power of discrimination calculated at 0.991.     

Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction followed by probe hybridization on a nylon membrane, which is re-usable. Probes are 5'' amine modified to allow fixation to the membrane. Primers are 5'' biotin modified which allows detection of hybridized PCR products using streptavidin-peroxidase and a chemiluminescent substrate via photosensitive film. With low setup and consumable costs, this technique is inexpensive (approximately US$2 per sample), high throughput (multiple membranes can be processed simultaneously) and has a short turnaround time (approximately 10 hours).The technique can be utilized in a number of ways. Multiple probes can be designed to detect sequence variation within a single amplified product, or multiple products can be amplified simultaneously, with one (or more) probes used for subsequent detection. A combination of both approaches can also be used within a single assay. The ability to include multiple probes for a single target sequence makes the assay highly specific.Published applications of mPCR/RLB include detection of antibiotic resistance genes^1,2, typing of methicillin-resistant Staphylococcus aureus^3-5 and Salmonella sp⁶, molecular serotyping of Streptococcus pneumoniae^7,8, Streptococcus agalactiae⁹ and enteroviruses^10,11, identification of Mycobacterium sp¹², detection of genital^13-15 and respiratory tract¹⁶ and other¹⁷ pathogens and detection and identification of mollicutes¹⁸. However, the versatility of the technique means the applications are virtually limitless and not restricted to molecular analysis of micro-organisms.The five steps in mPCR/RLB are a) Primer and Probe design, b) DNA extraction and PCR amplification c) Preparation of the membrane, d) Hybridization and detection, and e) Regeneration of the Membrane.     

Trypanosoma cruzi: sequence analysis of the variable region of kinetoplast minicircles

The comparisons of 170 sequences of kinetoplast DNA minicircle hypervariable region obtained from 19 stocks of Trypanosoma cruzi and 2 stocks of Trypanosoma cruzi marenkellei showed that only 56% exhibited a significant homology one with other sequences. These sequences could be grouped into homology classes showing no significant sequence similarity with any other homology group. The 44% remaining sequences thus corresponded to unique sequences in our data set. In the DTU I ("Discrete Typing Units") 51% of the sequences were unique. In contrast, in the DTU IId, 87.5% of sequences were distributed into three classes. The results obtained for T. cruzi marinkellei, showed that all sequences were unique, without any similarity between them and T. cruzi sequences. Analysis of palindromes in all sequence sets show high frequency of the EcoRI site. Analysis of repetitive sequences suggested a common ancestral origin of the kDNA. The editing mechanism that occurs in kinetoplastidae is discussed.     

Genetic diversity and population structure of food-borne Staphylococcus carnosus strains

The species Staphylococcus carnosus is a non-pathogenic representative of the coagulase negative staphylococci. Specific strains are applied as meat starter cultures. The species consists of two subspecies, S. carnosus ssp. carnosus and S. carnosus ssp. utilis. In order to place S. carnosus strains, characterized in former studies, in a genetic background that allows a typing of candidates for starter cultures, a Multilocus Sequence Typing (MLST) scheme was developed. Internal fragments of the genes tpiA, xprT, dat, gmk, glpK, narG, cstA, encoding triosephosphate isomerase, xanthine phosphoribosyltransferase, d-amino acid aminotransferase, guanylate kinase, glycerol kinase, the α-chain of the respiratory nitrate reductase, and a carbon starvation protein where chosen. Genes were selected based on their equal distribution in the genome, taxonomic value in subspecies differentiation and metabolic function. This MLST was applied to 44 S. carnosus strains, most of them previously analyzed for their suitability as starter cultures.The number of alleles varied between zero (tpiA) and five (cstA) and allowed the definition of nine sequence types (ST). ST1 was most abundant (18 strains), followed by ST2 (8) and ST4 (6). The nine STs confirmed a close relationship of all strains despite their isolation source and year, but lacked correlation with physiological activities relevant for starter cultures. The low amount of STs in the strain set lets us suggest that recombination between strains is rare. Thus, it is hypothesized that evolutionary events seem to be due to single point mutations rather than intrachromosomal recombination, and that the species possesses a clonal population structure.