Characterisation of extended-spectrum beta-lactamases of the SHV family using a combination of PCR-single strand conformational polymorphism (PCR-SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP)

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) has been developed to extend the identification of SHV beta-lactamases previously characterised by PCR-single strand conformational polymorphism (PCR-SSCP) analysis alone. Eight bacteria, each producing a different SHV beta-lactamase, were used in this study. These bacteria harbour bla(SHV-1), bla(SHV-2a), bla(SHV-3), bla(SHV-4), bla(SHV-5) (two strains), bla(SHV-11) and bla(SHV-12). All isolates were characterised by PCR-SSCP and PCR-RFLP with DdeI and NheI digestion. By a combination of these techniques, the genes encoding these beta-lactamases could be differentiated from each other. In addition, the PCR-RFLP technique theoretically can be applied to distinguish the genes encoding SHV-7, SHV-9, SHV-10, SHV-15, SHV-17 and SHV-24 from those encoding other SHV variants. We report a simple PCR-RFLP technique that can be used in epidemiological studies to enable the rapid characterisation of known SHV beta-lactamases in a combination with the previously published PCR-SSCP analysis.     

Differentiation of Coxiella burnetii isolates by sequence determination and PCR-restriction fragment length polymorphism analysis of isocitrate dehydrogenase gene

The isocitrate dehydrogenase (icd) gene of Coxiella burnetii was cloned and sequenced to differentiate between isolates with various geographic origins and phenotypic properties. Based on the gene sequences all 19 isolates studied could be divided into three groups. Group 1 contained isolates originating from acute cases of Q fever, ticks and cows. Groups 2 and 3 included isolates from chronic Q fever patients and a prototype strain from an aborted goat. Although the icd gene profiles were different among isolates of the latter two groups, there were two base differences common for both groups which could be used as markers to distinguish them from group 1 isolates. Based on one of the markers a simple method using PCR-restriction fragment length polymorphism analysis was developed for rapid differentiation of C. burnetii isolates as well as for direct detection and differentiation of the bacterium in human serum samples. Taken together, the study results suggest that the icd-based differentiation method may be useful in clinical investigation of Coxiella infections.     

Association between the −159C/T CD14 gene polymorphism and tuberculosis in a Korean population

The aim of the present study was to confirm the association between the CD14 −159C/T polymorphism and tuberculosis in the Korean population and to elucidate the functional basis for this putative association. CD14 −159C/T genotypes were determined by PCR – restriction fragment length polymorphism analysis in 274 tuberculosis patients and 422 healthy controls. Recombinant CD14 promoter–luciferase reporter constructs, including the −159T or −159C allele, were transfected into K562 and BEAS-2B cells, and luciferase activities were measured and compared. Levels of serum sCD14 and interferon-γ secreted by peripheral blood mononuclear cells (PBMCs) were measured using enzyme-linked immunosorbent assay.The frequency of −159TT genotypes was higher in tuberculosis patients than in healthy controls. The promoter activity of the −159T allele was higher than that of the −159C allele. Serum sCD14 levels were higher among tuberculosis patients with −159TT genotypes than among those with −159CC genotypes and interferon-γ release by PBMCs was decreased in subjects with −159TT genotypes. In conclusion, the −159TT CD14 genotypes were associated with tuberculosis development in Koreans. This association might be a result of the higher promoter activity of the −159T allele, the higher level of sCD14, and the decreased interferon-γ secretion in subjects with −159TT genotypes.     

Comparison of 23S polymerase chain reaction-restriction fragment length polymorphism and amplified fragment length polymorphism techniques as typing systems for thermophilic campylobacters

In this study, we evaluated the combination of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and amplified fragment length polymorphism (AFLP) molecular typing techniques for the analysis of thermophilic campylobacter species isolated from clinical and poultry samples. 23S PCR-RFLP analysis performed to fingerprint 69 strains exhibited an excellent level of typability. Eleven different types were defined at 100% linkage level following numerical analysis of band patterns. Differentiation of Campylobacter jejuni and Campylobacter coli at species level was achieved although no significant relationship could be observed between the profiles and the origin of the strains. Simplified AFLP analysis of the isolates disclosed the presence of 66 different banding patterns. The resulting dendrogram showed a high diversity among the strains studied. All the isolates were grouped within eight main types with a 69% homology degree among them. Differentiation at subspecies level was possible but no significant relationship could be observed between the AFLP profiles and the origin of the strains. When used in combination, 23S PCR-RFLP and single-enzyme AFLP methods can be applied to determine taxonomic and epidemiological relationships among thermophilic campylobacters.     

Species level identification of conifer associated Ceratocystis sapstain fungi by PCR-RFLP on a beta-tubulin gene fragment

The genus Ceratocystis includes several morphologically similar species commonly found as agents of sapstain in coniferous trees. In this paper we describe a simple and reliable polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique that aids in the identification and differentiation of these fungi. PCR was used to amplify a 1.3-kb fragment of the beta-tubulin gene from C. coerulescens, C. pinicola, C. douglassi, C. resinifera, C. rufipenni, C. polonica and C. adiposa. The PCR amplicon from representative isolates was sequenced. This information was utilized to select restriction enzymes that generated species-specific RFLP patterns. This approach was tested on our collection of over 200 Ceratocystis isolates and identified the fungi with a high level of confidence, reducing the time needed to identify these species by classical methods.     

Detection of high-level tetracycline resistance in clinical isolates of Helicobacter pylori using PCR-RFLP

Tetracycline is one of four antibiotics commonly used for the treatment of Helicobacter pylori infection, but its effectiveness is decreasing as the incidence of tetracycline resistance is increasing. In five Brazilian tetracycline-resistant (Tet(R)) H. pylori isolates, high-level tetracycline resistance is mediated by the triple-base-pair substitution AGA(926-928)-->TTC in both 16S rRNA genes, as was previously observed in two independent high-level Tet(R) H. pylori strains. A polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) assay was developed for the detection of the AGA(926-928)-->TTC substitution, and confirmed the presence of the aforementioned triple-base-pair substitution in all five Brazilian Tet(R) isolates. This PCR-RFLP-based approach distinguishes the high-level Tet(R) isolates from low-level Tet(R) and Tet(S) H. pylori strains and thus allows the direct detection of Tet(R) H. pylori isolates.     

Characterisation of the lpdA gene from Neisseria meningitidis by polymerase chain reaction, restriction fragment length polymorphism and sequencing

P64k protein from Neisseria meningitidis is well recognised in sera from individuals convalescent from meningococcal disease or vaccinated with the Cuban antimeningococcal vaccine VA-MENGOC-BC. The presence of the protein in more than 80 meningococcal strains has also been verified. It is immunogenic in animal models and the antibodies elicited show bactericidal activity against meningococci. To further investigate at the molecular level whether lpdA, the gene coding for P64k protein, is conserved among different N. meningitidis strains, a total of 20 strains isolated from different geographic areas were differentiated on the basis of restriction fragment length polymorphism (RFLP) patterns after polymerase chain reaction (PCR) amplification of the lpdA gene and restriction endonuclease digestion with HpaII. Although a total of five different PCR-RFLP patterns were present, nucleotide sequence determination showed that identity levels were as high as 93-99% among the N. meningitidis strains analysed.     

Comparison of two PCR methods for rapid identification of Leptospira genospecies interrogans

Based on (i) an analysis of Leptospira 16S rDNA sequences determined by us and of those from databases and (ii) a previously published finding that restriction fragment length polymorphisms (RFLPs) within the Leptospira 16S and 23S rDNA were detected by nine restriction enzymes and these RFLPs allowed categorisation of Leptospira into eight genospecies, we predicted that one particular Dde I restriction site polymorphism within 16S rDNA could be independently used for identifications of Leptospira strains belonging to the genospecies interrogans . Two PCR-based methods, namely allele-specific amplification (ASA) and PCR-RFLP, were tested for the rapid detection of the Dde I restriction site polymorphism. One or two representative strains from each of nine genospecies were tested by ASA, whereas 73 strains from nine genospecies and two field isolates were tested by PCR-RFLP. Our experiments showed that the ASA method was not as specific as intended, but the PCR-RFLP method was useful for rapid identifications of the genospecies interrogans . We have not only confirmed a previous finding and extended the number of samples particularly from the genospecies biflexa , weilii , and inadai , but also simplified a previous PCR-RFLP protocol.     

Variants of the CD40 ligand gene are not associated with increased susceptibility to tuberculosis in West Africa

Evidence for linkage between tuberculosis and human chromosomal region Xq26 has previously been described. The costimulatory molecule CD40 ligand, encoded by TNFSF5 and located at Xq26.3, is a promising positional candidate. Interactions between CD40 ligand and CD40 are involved in the development of humoral- and cell-mediated immunity, as well as the activation of macrophages, which are the primary host and effector cells for Mycobacterium tuberculosis. We hypothesised that common variation within TNFSF5 might affect susceptibility to tuberculosis disease and, thus, might be responsible for the observed linkage to Xq26. Sequencing 32 chromosomes from a Gambian population identified nine common polymorphisms within the coding, 3 and 5 regulatory sequences of the gene. Six single nucleotide polymorphisms (SNPs) and a 3 microsatellite were genotyped in 121 tuberculosis patients and their available parents. No association with tuberculosis was detected for these variants using a transmission disequilibrium test, although one SNP at –726 showed some evidence of association in males. This finding, however, did not replicate in a separate case control study of over 1,200 West African individuals. We conclude that common genetic variation in TNFSF5 is not likely to affect tuberculosis susceptibility in West Africa and the linkage observed in this region is not due to variation in TNFSF5.Sadly, Professor Steve Bennett passed away in March 2003     

Genomic species typing of acinetobacters by polymerase chain reaction amplification of the recA gene

Abstract The recA gene has been used as a target in screening for the presence of acinetobacters on the genospecies level and differentiation of relevant acinetobacter species from one another by PCR. Primers deduced from known recA gene sequences of Acinetobacter calcoaceticus and Neisseria gonorrhoeae allowed the amplification of DNAs from all Acinetobacter genospecies. The size of the amplified DNA fragment from all genospecies tested was approximately 435–500 bp relative to DNA size markers. The amplified products were examined further by restriction fragment length polymorphism (RFLP) analysis. Restriction analysis with only two enzymes, Mbo I and Hin fI, enabled us to identify all known genospecies. Since this method uses conserved recA gene sequences for primers, it is expected to be applicable for the identification of most bacterial species.     

Restriction analysis of PCR amplified nrDNA regions revealed intraspecific variation within populations of Fusarium culmorum

Seventy-five isolates of Fusarium culmorum with diverse geographical origin and host were analyzed using restriction digestion of polymerase chain reaction amplified nuclear ribosomal DNA intergenic spacer (IGS) and 28S gene regions. The 28S gene was conserved and has produced identical restriction patterns, however, the IGS region was substantially variable. The isolates were divided into 29 unique IGS haplotypes. There was limited resolution between clustering of isolates and their origin and/or host. The variability was distributed largely equally at both macro- and micro-geographical scale. The phylogeographic distribution pattern suggests a seed-borne dispersal of F. culmorum.     

贵州荔波封闭人群MTHFR基因多态性研究分析

目的 对贵州汉族、布依族亚甲基四氢叶酸还原酶（Methylenetetrahydrofolate Reductase,MTHFR）基因多态性进行研究,为贵州少数民族基因多态性数据库的建立提供相关数据。方法 应用聚合酶链式反应及限制性片段长度多态性检测贵州荔波汉族90例、布依族119例MTHFR基因两个单核苷酸（677及1298位）多态位点的基因频率及基因型频率。结果 汉族、布依族MTHFR 677位T等位基因的分布频率分别是22、8％,16．1％,x^2=1．561,P〉0．1;MTHFR 1298位C等位基因的分布频率分别是28．9％,39、1％,x^2=2．075,P〉0．1;677CT／1298AC双杂合子的分布频率分别是16．66％,22．7％。结论 MTHFRC 677T和A1298C多态性在中国南方和北方人群存在群体差异;贵州汉族与布依族此两位点无显著性差异。贵州荔波布依族MTHFR 1298位有较高的C等位基因频率。     

A Lactobacillus helveticus plasmid detects restriction fragment length polymorphism in different bacterial species

A small cryptic Lactobacillus helveticus plasmid, pLBL4, was able to reveal restriction fragment length polymorphism in different bacterial species including Lactobacillus species, Bacillus species, and Escherichia coli when used as a DNA probe. The observed polymorphism was a result of the combined hybridization of several microsatellite sequences. The 6-bp sequence (TTGTTT) was repeated 12 times, seven of which were concentrated within the region between 1791 and 1997 bp of the plasmid sequence. The polymorphic patterns generated with pLBL4 differed from those obtained with M13 DNA in the larger number of bands observed. The results presented here open the possibility of using pLBL4 as a new broad-spectrum polymorphic DNA probe for fingerprint analysis.     

亚甲基四氢叶酸还原酶基因C677T、G1793A 单核苷酸多态性 与散发性乳腺癌易感性的关系

目的:研究亚甲基四氢叶酸还原酶(MTHFR)基因C677T、G1793A位点单核苷酸多态性与散发性乳腺癌易感性关系。方法:采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法,对200例乳腺癌患者及200例正常对照者MTHFR基因C677T、G1793A位点单核苷酸多态性进行分析,logistic回归分析不同基因型与乳腺癌风险的关系。结果:乳腺癌组MTHFR 677TT基因型频率为25.00%显著高于正常对照组的10.50%(X2=14.401,P=0.001),CT基因型频率为44.50%低于正常对照组的54.50%,CC基因型频率在乳腺癌组和正常对照组中无差别;MTHFR 1793GA基因型频率为18.50%显著高于正常对照者的8.50%(X2=8.563,P=0.003)。乳腺癌患者MTHFR 677T和1793A等位基因频率分别为47.25%、9.25%,显著高于对照组中的37.75%、4.25%。MTHFR 677TT基因型携带者罹患乳腺癌的风险是677CC基因型携带者的2.732倍(95%CI=1.418～5.051,P=0.001),MTHFR1793GA基因型携带者罹患乳腺癌的风险是1793GG基因型携带者的2.444倍(95%CI=1.325～4.505,P=0.003)。另外,乳腺癌组中MTHFR C677T基因多态性与肿瘤大小相关(x2=7.431,P=0.024,MTHFR G1793A基因多态性与淋巴结转移情况(x2=8.939,P=0.011)、癌组织学分级(x2=9.983,P=0.007)相关。结论:MTHFR C677T、G1793A基因多态性与散发性乳腺癌的易感性相关。     

HLA-DRB1基因多态性与新疆哈萨克族人群结核病易感性的研究

目的:研究HLA-DRB1基因多态性与新疆哈萨克族人群结核病(TB)的相关性。方法:采用病例-对照的研究方法,应用聚合酶链反应-序列特异性引物(PCR-SSP)技术对231例新疆哈萨克族肺结核患者和230例新疆哈萨克族健康对照者的13个HLA-DRB1等位基因进行分型,比较其等位基因频率(GF)并计算其比值比(OR)。结果:与新疆哈萨克族人群对照组相比,新疆哈萨克族人群结核病例组中HLA-DRB1*04显著增高(11.72%比6.75%,p0.05,OR=1.889),HLA-DRB1*10也增高(2.86%比1.09%),但统计学上无显著性差异(Pc0.05)。结论:HLA-DRB1*04可能是新疆哈萨克族人群结核病的易感基因。     

亚甲基四氢叶酸还原酶基因C677T、G1793A单核苷酸多态性与散发性乳腺癌易感性的关系

目的：研究亚甲基四氢叶酸还原酶（MTHFR）基因C677T、G1793A位点单核苷酸多态性与散发性乳腺癌易感性关系。方法：采用聚合酶链反应-限制性片段长度多态性（PCR-RFLP）方法,对200例乳腺癌患者及200例正常对照者MTHFR基因C677T、G1793A位点单核苷酸多态性进行分析,logistic回归分析不同基因型与乳腺癌风险的关系。结果：乳腺癌组MTHFR 677TT基因型频率为25.00%显著高于正常对照组的10.50%（X2=14.401,P=0.001）,CT基因型频率为44.50%低于正常对照组的54.50%,CC基因型频率在乳腺癌组和正常对照组中无差别;MTHFR 1793GA基因型频率为18.50%显著高于正常对照者的8.50%（X2=8.563,P=0.003）。乳腺癌患者MTHFR 677T和1793A等位基因频率分别为47.25%、9.25%,显著高于对照组中的37.75%、4.25%。MTHFR 677TT基因型携带者罹患乳腺癌的风险是677CC基因型携带者的2.732倍（95%CI=1.418～5.051,P=0.001）,MTHFR1793GA基因型携带者罹患乳腺癌的风险是1793GG基因型携带者的2.444倍（95%CI=1.325～4.505,P=0.003）。另外,乳腺癌组中MTHFR C677T基因多态性与肿瘤大小相关（x2=7.431,P=0.024,MTHFR G1793A基因多态性与淋巴结转移情况（x2=8.939,P=0.011）、癌组织学分级（x2=9.983,P=0.007）相关。结论：MTHFR C677T、G1793A基因多态性与散发性乳腺癌的易感性相关。     

Coagulase gene polymorphism of Staphylococcus aureus isolated from goat mastitis in Brazilian dairy herds

AIMS: To investigate the coagulase gene polymorphism of Staphylococcus aureus isolated from mastitic goat's milk. METHODS AND RESULTS: A typing procedure based on coagulase gene polymorphism was used to discriminate S. aureus isolated from goat mastitis. Thirty-six strains collected from goats belonging to herds from northern Ceara State and Serrana region of Rio de Janeiro State were analysed. Based on restriction fragment length polymorphism of 3' end coagulase gene, the goat strains were grouped into 11 types. In northern Ceara herds, the predominant type was found in 60% of the strains, while in the Serrana region herds the two most common accounting for 62.5% of the strains. CONCLUSIONS: The analysis of the coa gene proved to be useful for typing S. aureus from goat mastitis. Although the results showed that goats from the studied regions were infected by S. aureus strains harbouring more than one coagulase genotype, only one or two types predominated. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of the prevalent strains within a herd or region is a necessity. The important virulence factors could be identified in such strains and this information can then be used as a specific base to develop S. aureus mastitis control measures.