Use of the gyrB gene for the identification of Pandoraea species

The recently described genus Pandoraea consists of five named species and four unnamed genomospecies, several of which have been identified in clinical specimens including respiratory secretions from persons with cystic fibrosis. We investigated whether it is possible to distinguish species of the genus Pandoraea by means of restriction fragment length polymorphism (RFLP) analysis and direct sequencing of the gyrB gene. Sixty-seven Pandoraea isolates were included. Species-specific RFLP patterns were obtained following digestion of the PCR-amplified gyrB gene with MspI. Specificity of RFLP groupings was confirmed by direct sequencing of several representative isolates. Our results indicate that RFLP analysis and sequencing of the gyrB gene are useful for the identification of Pandoraea species. We also found that further taxonomic studies within the beta-Proteobacteria using the gyrB gene would benefit from the development of additional primers allowing more efficient amplification of the gyrB gene. Our data also indicate that the taxonomic status of Pandoraea genomospecies 2 should be reinvestigated.     

嗜麦芽窄食单胞菌血流感染的临床回顾性分析

目的 探讨嗜麦芽窄食单胞菌血流感染的临床特点、治疗方案和预后。方法 对浙江大学医学院附属第一医院2012年1月至2015年8月诊断为嗜麦芽窄食单胞菌血流感染的住院患者的临床资料进行回顾性分析。结果 54例嗜麦芽窄食单胞菌血流感染患者平均年龄50.5岁,检出前平均住院16天。17例（31%）合并有其他细菌感染,43例（80%）合并其他系统感染,其中合并肺部感染最常见（32例,占59.3%）。嗜麦芽窄食单胞菌血流感染多发生于恶性实体肿瘤、血液病患者（各占29.6%）,最常见于肝胆外科（25.9%）等科室。54例患者好转32例（59%）,死亡22例（41%）;好转组APACHEII评分（14.13±4.54）、死亡组APACHEII评分（27.59±8.17）,两组有统计学差异（P<0.05）。合并其他部位感染、高APACHEII评分组死亡率更高（P<0.05）,而使用敏感抗生素组死亡率更低（P<0.05）。结论嗜麦芽窄食单胞菌血流感染多见于免疫力低下的危重患者,多发生在院内外科手术操作的科室,并容易合并其他部位感染。规范使用敏感抗菌药物,积极治疗合并感染,可有助于改善嗜麦芽窄食单胞菌血流感染患者预后。     

Identification of Stenotrophomonas maltophilia strains isolated from environmental and clinical samples: a rapid and efficient procedure

Aims: Aim of the study is to identify accurately Stenotrophomonas maltophilia isolates recovered from environmental and clinical samples. Methods and Results: Recovery of Sten. maltophilia‐like isolates from soil samples using the vancomycin, imipenem, amphotericin B (VIA) selective agar medium enabled distinction of various morphotype colonies. A set of soil and clinical isolates was tested for species identification using different methods. 16S rDNA analyses showed the dark green with a blue halo morphotype to be typical Sten. maltophilia strains. The API‐20NE, Vitek‐2 and Biolog phenotypic analyses typically used for the identification of clinical isolates did not perform well on these soil isolates. The species‐specific PCR screening targeting Sten. maltophilia 23S rDNA and the multiplex smeD/ggpS PCR, differentiating Sten. maltophilia from Stenotrophomonas rhizophila, were tested for improvement of these identification schemes. The latter multiplex PCR identified all isolates tested in this study, whatever be their origin. Conclusions: Isolation on VIA medium and confirmation of Sten. maltophilia species membership by smeD PCR is proposed to identify environmental and clinical isolates of Sten. maltophilia. Significance and Impact of the Study: The proposed approach enables isolation and identification of Sten. maltophilia from different environments in an easy and rapid way. This approach will be useful to accurately manage studies on the abundance and distribution of Sten. maltophilia in hospital and nonhospital environments.     

广东少数民族Morquio综合征StuI位点的RFLP分析

研究广东少数民族群体GALNS基因StuI位点的遗传多态性以及该位点等位基因片段传递的规律,为今后的连锁分析打下基础。采用PCR-RFLP方法,对72例无血缘关系的健康广东少数民族个体的144条染色体和3个家系9位成员的18条染色体进行检测,然后用χ2检验进行统计学处理。等位基因片段D1的频率为0.70, D2为0.30,杂合率为29%,D1、D2的传递规律与理论上预计的完全符合。广东少数民族群体中StuI位点具有多态性,其基因频率（D1和D2）与国外高加索群体的有显著差别,与日本群体及中国南方汉族群体的则无显著差别;而杂合率与高加索群体及日本群体的均有显著差异,但与中国南方汉族群体的则无显著差异。 Abstract:To investigate the genetic polymorphism of the StuI site in the GALNS gene from a national minority population in Guangdong and to study the mode of transmission of alleles,PCR-RFLP was used to analyze 144 chromosomes from 72 Guangdong unrelated healthy national minority individuals,and the genotypes of members in three families.To compare the frequencies and heterzygosity between Guangdong national minority people and Caucasians,Japanese and Chinese Han people by using χ2 test.The frequency of allele D1(295bp) was 0.70,allele D2(138 plus 157 bp)0.30,the heterozygosity was 29%.The genotypes of each member of all families detected were completely agreement with the theorical assessment.The site of StuI in the GALNS gene from national minority population in Guangdong has polymorphism.There is significant difference between Guangdong national minority population and Caucasians in Western countries,but no significant difference was found between Guangdong national minority population and Japanese and Chinese Han population.In addition,there is significant difference between Guangdong national minority population and Caucasians and Japanese in the heterzygosity,but no significant difference between Guangdong national minority population and Chinese Han population.The transmission of alleles was completely in agreement with the Mendelian genetic law.     

Molecular phytogeny of conifers using RFLP analysis of PCR-amplified specific chloroplast genes

We investigated the molecular phylogeny of conifers using restriction endonuclease fragment length polymorphism of six polymerase chain reaction-amplified chloroplast genes — frxC, rbcL, psbA, psbD, trnK, and 16S. We detected 227 total site changes among species, representing 23, 26, 38, 48, 67, and 25 site changes in frxC, psbA, psbD, rbcL, trnK and 16S, respectively. The mean nucleotide substitution was 10.75% (SD 0.573) among species in five families. Forty maximally parsimonious trees were obtained using the Wagner parsimony method, and a 50% majority-rule consensus tree was obtained from them. Data analysis produced similar basic patterns when both the Wagner parsimony and the neighbor-joining methods were applied, and the main lineages were clearly separated. Taxaceae and Cephalotaxaceae species were used as the out-groups when applying Wagner parsimony methods. With the Wagner method, the consistency index was 0.510, the retention index was 0.879, and tree length was 435 steps. Our results indicated that Cupressaceae and Taxodiaceae are closely related families and that Sciadopitys verticillata is the basal lineage of Cupressaceae and Taxodiaceae. The neighbor-joining tree is similar to the 50% majority-rule consensus of the 40 Wagner parsimony trees except for the position of Keteleeria daversifolia, the Picea and Cedrus group, and the divergence within Cupressaceae.     

1株高产蛋白酶嗜麦芽寡养单胞菌的分离鉴定及其酶学活性的研究

双齿围沙蚕消化道中分离1株高产蛋白酶菌株D2(CGMCC保藏号:1868),经形态学、生理学、16S rRNA基因序列测定及系统发育分析确定为嗜麦芽寡养单胞菌。Lowry法检测显示该菌株产酶能力为1104 U/mL,最佳产酶条件为pH 8.0、25℃培养48 h;酪蛋白酶图谱法和凝胶成像分析证实其蛋白酶分子量约为42 ku,在培养上清液中纯度大于97%;该酶对粗酶品比活性为301 U/mg,酶活性的最适pH值为9,是一种碱性蛋白酶;最适温度为60℃;在55℃以下及pH 6~10的环境中具有较好的稳定性。嗜麦芽寡养单胞菌D2株有望成为一种新的蛋白酶生产资源。     

RFLP of 16S-ITSrDNA Region to Differentiate Lactobacilli at Species Level

The 16S-ITS (internal transcribed spacer) region of the rrn operon was amplified by polymerase chain reaction (PCR). The amplification products were analysed by restriction fragment length polymorphism (RFLP) using a set of restriction enzymes, AluI, HaeIII, and TaqI. Restriction pattern analyses revealed that TaqI restriction enzyme could clearly differentiate the nine reference strains of Lactobacillus used in the study. This revised version was published online in July 2006 with corrections to the Cover Date.     

肿瘤患者并发嗜麦芽窄食单胞菌感染

目的 了解医院内肿瘤患者感染嗜麦芽窄食单胞菌情况及其耐药性,以利于临床合理选用抗生素。方法 用法国生物梅里埃公司API 20 NE细菌鉴定试验条及ATB试验条进行菌种鉴定及药敏试验。结果 58株嗜麦芽窄食单胞菌中有42株（72.4%）分离自肿瘤患者呼吸道标本（痰34份,气管切口分泌物8份）;药敏试验显示其耐药率对环丙沙星最低（10.3%）,其次为头孢他定（13.8%）,再次为哌拉西林-他唑巴坦（41.4%）,对亚胺培南天然耐药。结论 嗜麦芽窄食单胞菌主要引起呼吸道感染,尤其是院内的肿瘤患者呼吸道特别易受感染：其多重耐药状况十分严重,对其感染的治疗,应在抗生素敏感试验的指导下进行.     

Comparative genomic analysis of isolates belonging to the six species of the genus Thermus using pulsed-field gel electrophoresis and ribotyping

Fifty isolates belonging to the six validly described species of the genus Thermus (T. aquaticus, T. filiformis, T. thermophilus, T. scotoductus, T. brockianus, and T. oshimai) isolated from hot springs of different geographical areas were compared using macrorestriction analysis of genomic DNA and ribotyping. With the exception of presumed clones, the macrorestriction patterns of isolates obtained with EcoRI or NdeI were distinct. However, isolates belonging to the same species exhibited similar profiles particularly when they were isolated from the same hot spring. The estimated genomic size of strains of the Thermus spp. varied between approximately 1.8 and 2.5 Mbp. Ribotyping with BamHI and HindIII produced 30 and 35 distinct ribotypes, respectively. In spite of the variability of the hybridization patterns produced, the ribotypes obtained for isolates belonging to the same species also shared, in general, several fragments of identical size, and these fragments were similar when isolates originated from the same spring. Received: 7 October 1996 / Accepted: 10 March 1997     

Species identification of Chilean Artemia populations based on mitochondrial DNA RFLP analysis

Aim Two species of the brine shrimp, namely Artemia franciscana Kellogg and A. persimilis Piccinelli and Prosdocimi, inhabit Chile. Most studies so far have shown that A. franciscana is the most widely distributed species in Chile, with A. persimilis present only in Chilean Patagonia. In general, there is good agreement between morphological and genetic comparisons of Chilean populations with respect to species discrimination. However, a number of results indicate an overlap with some populations tending to diverge from A. franciscana and/or resembling A. persimilis. Prior to the mid 90's the use of DNA markers in Artemia was rather limited, despite their successful application in numerous other species. In this study, we investigate whether the conclusions drawn from traditional comparative tools are congruent with the pattern of genetic divergence depicted by DNA analysis at the mitochondrial level. Location Eight sites in Chile and two reference samples of A. franciscana and A. persimilis from San Francisco Bay (USA) and Buenos Aires (Argentina), respectively. Methods Restriction fragment length polymorphism (RFLP) analysis of a 535 bp segment of the mitochondrial 16S rRNA gene with nine restriction enzymes in 240 individuals. Results No haplotype was shared between the two species. Five restriction enzymes produced species‐specific patterns, enabling the unambiguous assignment of populations to species. Very high (100%) bootstrap values supported the clustering of haplotypes in two groups corresponding to the two species. The two species were clearly differentiated with average sequence divergence of 12.3%. High genetic differentiation was also found among con‐specific populations of A. franciscana with an F_ST estimate of 91%. Main conclusions The mitochondrial DNA (mtDNA) results of this study show a broadly similar pattern to those of previous allozyme and nuclear DNA analyses, with the two New World species appearing as highly divergent. The presence of A. persimilis in southern Chile (Chilean Patagonia) was confirmed. Hence, a species previously regarded as geographically restricted mainly to Argentina, appears to have expanded its range. Populations of A. franciscana appear highly structured with a level of inter‐population genetic differentiation much higher for mtDNA than previously reported with allozymes. Clustering of these populations does not follow a clear geographic pattern. The identification of population‐specific genetic markers for A. persimilis and A. franciscana will help to tackle further aspects of the speciation patterns of these species.     

都柏林念珠菌的PCR-RFLP鉴定

目的建立一种准确、可靠的鉴定都柏林念珠菌基因型的方法。方法临床念珠菌分离自临床生殖器念珠菌病患者,45℃温度试验时几乎不生长,且其他表型实验结果也符合都柏林念珠菌特征。对41例临床念珠菌和1例白念珠菌标准株、1例都柏林念珠菌标准株rDNA内部转录间隔区的基因进行聚合酶链反应（PCR）扩增,HpyF10Ⅵ酶切后观察PAGE图谱。结果聚合酶链反应-限制性片段长度多态性（PCR-RFLP）后,39例临床株鉴定为白念珠菌。2例临床菌株带型特殊,测序后行BLAST比对分析,1例鉴定为白念珠菌,另1例尚不能肯定为都柏林念珠菌,还需要进一步以其他分子生物学方法鉴定。结论PCR-RFLP方法酶切后两种念珠菌带型区分明显,可以鉴别大部分临床菌株。基因测序是该方法有意义的补充。     

Identification of bovine K-casein genotypes at the DNA level

By using a bovine kappa-Cn cDNA as probe and the PstI endonuclease we demonstrate that the DNA restriction patterns of kappa-Cn AA and kappa-Cn BB cows are different. Besides two invariant fragments (about 6.8kb and 1.1kb) the former shows two fragments of about 4.3 kb and 0.3 kb and the latter one fragment of about 4.6 kb. kappa-Cn AB cows show intermediate pattern. Therefore, it is possible to determine the bovine kappa-Cn genotypes even in absence of gene product.     

Detection and Identification of Aster Yellows (16SrI) Phytoplasma in Peach Trees in Jordan by RFLP Analysis of PCR-Amplified Products (16S rDNAs)

Aster yellows phytoplasma were detected, for the first time, in peach trees in Al‐Jubiha and Homret Al‐Sahen area. Leaves of infected trees showed yellow or reddish, irregular water‐soaked blotches. Discoloured areas become dry and brittle and the dead tissues dropped out. Under severe infections, leaves fall down and fruits dropped prematurely. Phytoplasmas were detected from all symptomatic peach trees by polymerase chain reaction (PCR) using universal phytoplasmas primers P1/P7 followed by R16F2/R2. No amplification products were obtained from templates of asymptomatic peaches. PCR products (1.2 kb) used for restriction fragment length polymorphism analysis (RFLP) after digestion with endonuclease AluI, HpaII, KpnI and RsaI produced the same restriction profiles for all samples, and they were identical with those of American aster yellows (16SrI) phytoplasma strain. This paper is the first report on aster yellows phytoplasma affecting peach trees in Jordan.     

Tools for T-RFLP data analysis using Excel

Background

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a DNA-fingerprinting method that can be used for comparisons of the microbial community composition in a large number of samples. There is no consensus on how T-RFLP data should be treated and analyzed before comparisons between samples are made, and several different approaches have been proposed in the literature. The analysis of T-RFLP data can be cumbersome and time-consuming, and for large datasets manual data analysis is not feasible. The currently available tools for automated T-RFLP analysis, although valuable, offer little flexibility, and few, if any, options regarding what methods to use. To enable comparisons and combinations of different data treatment methods an analysis template and an extensive collection of macros for T-RFLP data analysis using Microsoft Excel were developed.

Results

The Tools for T-RFLP data analysis template provides procedures for the analysis of large T-RFLP datasets including application of a noise baseline threshold and setting of the analysis range, normalization and alignment of replicate profiles, generation of consensus profiles, normalization and alignment of consensus profiles and final analysis of the samples including calculation of association coefficients and diversity index. The procedures are designed so that in all analysis steps, from the initial preparation of the data to the final comparison of the samples, there are various different options available. The parameters regarding analysis range, noise baseline, T-RF alignment and generation of consensus profiles are all given by the user and several different methods are available for normalization of the T-RF profiles. In each step, the user can also choose to base the calculations on either peak height data or peak area data.

Conclusions

The Tools for T-RFLP data analysis template enables an objective and flexible analysis of large T-RFLP datasets in a widely used spreadsheet application.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0361-7) contains supplementary material, which is available to authorized users.     

Comparing Techniques for the Identification of the MTHFR A1298C Polymorphism

The restriction fragment length polymorphism (RFLP) technique with the MboII enzyme is used by a number of researchers as a methodology for the identification of the genetic polymorphism MTHFR A1298C. However, the reliability of this enzyme for genotyping this polymorphism has been questioned, since the silent polymorphism T1317C, located close to the polymorphic region A1298C on gene MTHFR, also has a recognition site for MboII. Thus, the fragments formed by the digestion of MboII present similar sizes, making it difficult to differentiate the allele MTHFR 1298A in the presence of the allele MTHFR 1317C. Hence, we investigated the A1298C polymorphism in a Brazilian population of renal transplant patients, using the RFLP technique with digestion by Mbo II and using sequencing, in order to examine the concordance between the two techniques. Our results showed an 8.6% difference in genotyping between RFLP and sequencing, but the statistical concordance test presented a kappa coefficient equal to 0.81 (CI 95% 0.74–88), which indicates a virtually perfect concordance, according to the criterion of Landis and Koch. Therefore, we concluded that the RFLP technique is concordant with automated sequencing in the detection of polymorphism A1298C under our laboratory conditions.     

Identification of the double genome donor in spontaneous triploid tomato plants by RFLP analysis

Independent spontaneous triploid tomato plants (Lycopersicon esculentum Mill.) were collected among diploid hybrids growing in commercial greenhouses. Ploidy levels were verified by counting chromosomes, and the donor of the double genome dose was determined by restriction fragment length polymorphism (RFLP) analysis. The TG101 probe, which is tightly linked to the Tm-2 ^a locus, revealed different restriction patterns between TMV-resistant and TMV-susceptible parent lines. The parent donor which provided two genomes to the triploid was identified by comparing the relative intensity of alleles in the triploid with that in the diploid. The results indicate that both parents can serve as a double genome donor.     

Restriction fragment length polymorphism (RFLP) analysis of bovine nuclear protein genes

Summary We have recently cloned both the bovine protamine (Krawetz et al. 1987, DNA 6: 47–57) and high mobility group (HMG-1) cDNAs (Pentecost and Dixon 1984, Bioscience Reports 4: 49–57). They have been used as probes for Restriction Fragment Length Polymorphism analysis of male-female pairs of different species and breeds, within the genus Bos. Utilizing this approach we have studied inheritance, chromosomal location and gene copy number of the bovine protamine and HMG-1 genes. This revealed that these nuclear protein genes are highly conserved suggesting that selective pressure has maintained their gene structures during evolution. A polymorphic Taq 1 restriction fragment was identified that was shown to be a heritable marker. These genes are not sex-linked and are present in a single copy for protamine and at least two copies for the HMG-1.     

嗜麦芽窄食单胞菌感染的临床特征、高危因素及预后分析

目的了解嗜麦芽窄食单胞菌感染的临床特点、危险因素、预后及耐药现状,为有效预防和治疗该病原菌感染提供依据。方法收集2013年11月至2014年4月浙江大学医学院附属第一医院收治的129例细菌培养为嗜麦芽窄食单胞菌患者的临床资料进行回顾性统计分析。结果 129例细菌培养确诊嗜麦芽窄食单胞菌感染患者平均年龄（65.1±17.0）岁,包括下呼吸道感染和非呼吸道感染患者分别为100例和29例,下呼吸道感染患者存在原发肺部疾病的患病率、ICU入住率、气管切开比例、广谱抗生素的使用率、患病年龄等均高于非呼吸道感染患者（P〈0.05）。而非呼吸道感染患者的外科手术、无菌腔内置管比例及免疫抑制剂使用率高于下呼吸道感染患者（P〈0.05）。嗜麦芽窄食单胞菌感染后选择敏感抗生素治疗的患者的死亡率明显低于未选择敏感抗生素的患者（15.0%/30.4%,P〈0.05）。结论原发肺部疾病、入住ICU、气管切开、广谱抗生素使用、年龄大是下呼吸道感染嗜麦芽窄食单胞菌的高危因素,外科手术、无菌腔内置管、免疫抑制剂使用是非呼吸道感染嗜麦芽窄食单胞菌的高危因素。使用敏感抗生素可以降低嗜麦芽窄食单胞菌感染患者的死亡率。     

医院感染嗜麦芽窄食单胞菌的分子分型研究

摘要 目的：对医院感染分离的嗜麦芽窄食单胞菌进行耐药谱和分子分型研究分析,为制订合理的感染控制方案提供依据。方法：对临床分离的42株嗜麦芽窄食单胞菌用质谱仪进行鉴定并分析其耐药表型,根据其对头孢他啶、复方新诺明、左氧氟沙星、米诺环素及头孢吡肟5种抗生素的药敏结果的不同分组,药敏结果相同的菌株分为一型,药敏结果不同的分为另一型;采用PFGE对其做分子分型分析。结果：42株嗜麦芽窄食单胞菌分为6个不同的耐药表型;对42个株菌株做PFGE电泳,共得到A-D 4个基因型。结论：嗜麦芽窄食单胞菌耐药谱特征和PFGE 分型检测结果对抗生素选择性治疗有很好的指导作用。