PCR Amplification Techniques Research Articles

Immunohistochemical analysis of thyroid follicular neoplasms and BRAF mutation correlation.

The accurate diagnosis of benign and malign thyroid tumors is very important for the clinical management of patients. The distinction of thyroid papillary carcinoma follicular variant and follicular adenoma can be difficult. To investigate the alternative methods like immunohistochemistry and exon 15 in the BRAF gene 1799 T/A mutation analyses for distinguishing thyroid tumors. We applied immunohistochemical markers; CK19, HMWCK, Galectin-3, HBME-1 and Fibronectin and mutant allele-specific PCR amplification technique was used to determine 1799 T/A mutation within the BRAF gene. Formalin-fixed parafin embedded tissues from 45 surgically total resected thyroids, included 26 thyroid papillary carcinoma follicular variant (FV-TPC), 8 Follicular Adenoma (FA), 6 Minimal invasive follicular carcinoma (MIFC) and 5 Follicular Carcinoma (FC). Statistical Analyses Used: Pearson Chi-Square and Kruskal Wallis tests were performed. There was a positive correlation between FV-TPC and HMWCK, CK 19, HBME1, Galectin 3, fibronectin (P < 0.05), but there was no correlation with FV-TPC and BRAF gene mutation (P > 0.05). HBME-1 and CK 19 stained strong and diffuse positive in FV-TPCs but weak and focal in FAs. Our study suggests that morphologic features combined with immunohistochemical panel of HMWCK, CK19, HBME-1, Galectin-3 and fibronectin can help to distinguish benign and malign thyroid neoplasms and FV-TPC from follicular adenomas. BRAF gene 1799 T/A mutation has been non-specific but its detection can be a useful tool combined with immunohistochemistry for diagnosing FV-TPC.

Cotton GASL genes encoding putative gibberellin-regulated proteins are involved in response to GA signaling in fiber development

GAST (GA-stimulated transcript)-like genes have been reported as targets of GA regulation in some plant species. In this study, we isolated seven GAST-like cDNAs from cotton (Gossypium hirsutum) cDNA libraries (designated as GhGASL1-GhGASL7). Meanwhile, the genomic DNA clones corresponding to the seven GhGASL genes were isolated by using PCR amplification technique. Analysis of gene structure revealed that four genes (GhGASL1/3/5/6) contain two exons and one intron, while the rest have four exons and three introns. All of the deduced GhGASL proteins contain a putative signal peptide in the N-terminus and a conservative cysteine-rich C-terminal domain. Quantitative RT-PCR analysis indicated that the seven GhGASL genes are differentially expressed in cotton tissues. Among them, GhGASL1/4/7 were predominantly expressed in cotyledons, while the transcripts of GhGASL2/5 were preferentially accumulated at hypocotyls. GhGASL3 mRNA was largely accumulated in fibers, while GhGASL6 transcripts were mainly detected in ovules. Furthermore, GhGASL2/3/5 displayed a relatively high expression levels during early fiber elongation stages, and were regulated by GA. These data suggested that GhGASL genes may be involved in fiber elongation and in response to GA signaling during fiber development.

Cloning, characterization and expression analysis of a 1-aminocyclopropane-1-carboxylate synthase gene from pear

Shi, H., Zhang, Y. and Chen, L. 2013. Cloning, characterization and expression analysis of a 1-aminocyclopropane-1-carboxylate synthase gene from pear. Can. J. Plant Sci. 93: 465–471. In this study, a cDNA clone encoding putative 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) that catalyzes the conversion of S-adenosyl-L-methionine to ACC in ethylene biosynthetic pathway was isolated from a cDNA library produced using mRNA from pear (Pyrus pyrifolia). The cDNA clone, designated PpACS2, comprised an open reading frame of 1, 341 bp encoding a protein of 446 amino acids that shares high similarity with the known plant ACSs. Using PCR amplification technique, a genomic clone (GenBank accession number: KC146402) corresponding to PpACS2 was isolated and shown to contain two introns. The PpACS2 gene product shared 97% identity with an ACC synthase from pear (Pyrus communis). Phylogenetic analyses clearly placed the gene product in the ACC synthase cluster of plant ACS superfamily tree. Quantitative RT-PCR analysis indicated that the PpACS2 gene was preferentially expressed in young pear leaves and shoots. The transcript of PpACS2 gene was accumulated at relatively high levels in anthers, but no signal was detected in the petals and mesocarp of pear. These results suggest that the PpACS2 may participate in the regulation of ethylene production in pear leaves, shoots, and anthers.

Two pear auxin-repressed protein genes, PpARP1 and PpARP2, are predominantly expressed in fruit and involved in response to salicylic acid signaling

Two genes encoding putative auxin-repressed proteins, designated PpARP1 and PpARP2, were isolated from pear (Pyrus pyrifolia). The deduced PpARP1 and PpARP2 proteins contain the typical auxin_repressed Dormancy/auxin associated protein domains. Phylogenetic analyses clearly demonstrated that PpARP1 has high homology with pear ARPs except PpARP2 which belongs to another clade. Using PCR amplification technique, the genomic clones corresponding to PpARP1 and PpARP2 were isolated and shown to contain two introns with typical GT/AG boundaries defining the splice junctions. PpARP1 and PpARP2 genes were predominantly expressed in fruit. PpARP1 transcripts were also detected in shoots, petals, and anthers, but a relatively low expression signal was detected in young leaves. PpARP2 transcripts were also detected in other tissues. In particular, expression of PpARP1 and PpARP2 was developmentally regulated in fruit. Further study demonstrated that PpARP1 and PpARP2 expression in pear fruit was remarkably inhibited by salicylic acid and induced by IAA. These data suggested that PpARP1 and PpARP2 might be involved in the response to salicylic acid signaling during fruit development of pear. The study of the pear PpARP genes also demonstrated a link between salicylic acid and auxin signaling.

Are histopathological findings of diagnostic value in native valve endocarditis?

Optimal management of infective endocarditis (IE) depends on the early detection of IE-causing pathogens and on appropriate antimicrobial and surgical therapy. The current guidelines of the European Society of Cardiology (ESC) recommend histopathological examination as the gold standard for diagnosing IE Habib et al. (Eur Heart J 30:2369-2413, 2005). We hypothesize that histopathological findings do not provide additional information relevant to clinical decision-making. We retrospectively reviewed a cohort of patients who had undergone surgery for native valve endocarditis (NVE) at the University Hospital Regensburg between September 1994 and February 2005. All episodes of intraoperatively confirmed endocarditis during this period were included in the study. Data were retrieved from surgical records, microbiological and histopathological reports, and medical files of the treating as well as admitting hospital. Pathogens were correlated with the site of manifestation of the affected heart valve and with clinical and histopathological findings. A total of 163 episodes of NVE were recorded and entered into our study for analysis. The valves affected were the aortic valve (45 %), the mitral valve (28 %), the aortic and mitral valve (22 %), and other valves (5 %). IE-causing pathogens were Staphylococcus aureus (22 %), viridans streptococci (18 %), enterococci (10 %), streptococci other than Streptococcus viridans (9 %), coagulase-negative staphylococci (5 %), miscellaneous pathogens (4 %), and culture-negative endocarditis (33 %). Infection with S. aureus was associated with high rates of sepsis, septic foci, and embolic events, while patients with enterococcal IE showed the highest rate of abscesses. Mortality rate in all subgroups was low without significant differences. However, histopathological findings correlated poorly with the pathogen involved and showed only few significant associations that were without clinical relevance. The clinical presentation of IE depends on the pathogen involved. Among the episodes of NVE examined, the histopathological examination of resected heart valves did not show any pathogen-specific morphological patterns and therefore did not provide any additional information of clinical value. Based on our findings, we recommend complementary cultures of the resected materials (valve tissue, thrombotic material, pacer wire) and implementation of molecular diagnostic methods (e.g., broad-range PCR amplification techniques) instead of histopathological analyses of resected valve tissue.

Development of single-nucleotide polymorphism-based phylum-specific PCR amplification technique: application to the community analysis using ciliates as a reference organism.

Despite recent advance in mass sequencing technologies such as pyrosequencing, assessment of culture-independent microbial eukaryote community structures using universal primers remains very difficult due to the tremendous richness and complexity of organisms in these communities. Use of a specific PCR marker targeting a particular group would provide enhanced sensitivity and more in-depth evaluation of microbial eukaryote communities compared to what can be achieved with universal primers. We discovered that many phylum- or group-specific single-nucleotide polymorphisms (SNPs) exist in small subunit ribosomal RNA (SSU rRNA) genes from diverse eukaryote groups. By applying this discovery to a known simple allele-discriminating (SAP) PCR method, we developed a technique that enables the identification of organisms belonging to a specific higher taxonomic group (or phylum) among diverse types of eukaryotes. We performed an assay using two complementary methods, pyrosequencing and clone library screening. In doing this, specificities for the group (ciliates) targeted in this study in bulked environmental samples were 94.6% for the clone library and 99.2% for pyrosequencing, respectively. In particular, our novel technique showed high selectivity for rare species, a feature that may be more important than the ability to identify quantitatively predominant species in community structure analyses. Additionally, our data revealed that a target-specific library (or ciliate-specific one for the present study) can better explain the ecological features of a sampling locality than a universal library.

Pear ACO genes encoding putative 1-aminocyclopropane-1-carboxylate oxidase homologs are functionally expressed during fruit ripening and involved in response to salicylic acid

1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the final reaction of the ethylene biosynthetic pathway, converting ACC into ethylene. Past studies have shown a possible link between ACC oxidase and salicylic acid during fruit ripening in pear, but the relationship has received no more than modest study at the gene expression level. In this study, two cDNA clones encoding putative ACC oxidase, PpACO1 and PpACO2, were isolated from a cDNA library constructed by our own laboratory and produced using mRNA from mesocarp of pear (Pyrus pyrifolia Nakai. cv.Whangkeumbae). One cDNA clone, designated PpACO1 (GenBank accession No. JN807390), comprised an open reading frame of 945 bp encoding a protein of 314 amino acids. The other cDNA, designated PpACO2 (GenBank accession No. JN807392), encodes a protein with 322 amino acids that shares high similarity with the known plant ACOs. Using PCR amplification techniques, two genomic clones corresponding to PpACO1 and PpACO2 were isolated and shown to contain independently three introns with typical GT/AG boundaries defining the splice junctions. The PpACO1 gene product shared 99 % identity with an ACC oxidase from pear (Pyrus × bretschneideri Rehd.cv.Yali), and phylogenetic analyses clearly placed the gene product in the ACC oxidase cluster of the pear 2-oxoglutarate-dependent dioxygenase superfamily tree. Quantitative RT-PCR analysis indicated that the two PpACO genes are differentially expressed in pear tissues. PpACO1 and PpACO2 were predominantly expressed in fruit. The transcripts of PpACO1 were accumulated at relatively low levels in early fruit, but strongly high levels in fruit ripening and senescence stages, while the transcripts of PpACO2 were accumulated at higher levels in early fruit and much lower levels with further fruit cell development than the transcripts of PpACO1. In addition, PpACO1 gene was down-regulated in fruit by salicylic acid (SA). Nevertheless, PpACO2 gene was dramatically up-regulated in fruit by SA. These results suggested that the PpACOs may participate in regulation of fruit ripening and in response to SA in pear.

Molecular Characterization of Pear 1-Aminocyclopropane-1-Carboxylate Synthase Gene Preferentially Expressed in Leaves

1-Aminocyclopropane-1-carboxylate (ACC) synthase catalyzes the conversion of S-adenosyl-L-methionine to ACC in the ethylene biosynthetic pathway. Lots of important ACC synthase genes have been isolated and characterized from the plant kingdom. In this study, a cDNA clone encoding putative ACC synthase was isolated from a cDNA library produced using mRNA from pear (Pyrus pyrifolia). The cDNA clone, designated PpACS1 (GenBank accession No. JQ284383), comprised an open reading frame of 1, 341 bp encoding a protein of 446 amino acids that shares high similarity with the known plant ACSs. Using PCR amplification techniques, a genomic clone corresponding to PpACS1 was isolated and shown to contain two introns with typical GT/AG boundaries defining the splice junctions. The PpACS1 gene product shared 97% identity with an ACC synthase from pear (Pyrus communis), and phylogenetic analyses clearly placed the gene product in the ACC synthase cluster of the plant ACS superfamily tree. RT-PCR analysis indicated that the PpACS1 gene was preferentially expressed in pear leaves. The transcript of PpACS1 gene was accumulated at relatively high levels in anthers. The expression signal was detected in shoot at relatively low levels, but none signal was detected in developing fruit of pear. These results suggested that the PpACS1 may participate in the regulation of ethylene production in pear leaves and anthers.

Characterization of vancomycin resistant Enterococcus faecium from clinical and chicken sources

ObjectiveThe study was undertaken to investigate the genomic and phenotypic relationship among E.faecium strains isolated from chicken and clinical sources. MethodsEnterococci were isolated and identified by conventional biochemical methods and the antibiotic susceptibility was determined by disk diffusion methods. Phenotypes and genotypes of vancomycin resistance were detected by E tests and PCR amplification techniques respectively. Genotyping of the vancomycin resistant E.faecium from two sources were done by RAPD typing. ResultsThe Vancomycin resistant E.faecium identified was selected for this comparative study. Among the VREF from two sources minor biochemical difference with regards to raffinose fermentation and haemolytic properties was observed. The RAPD tests using random primers also showed polymorphism. ConclusionThe results of the study showed that the strains from two different sources were not identical.

Bacterial endophyte communities of two wheatgrass varieties following propagation in different growing media

Bacterial endophyte communities of two wheatgrass varieties currently being used in the revegetation of military training ranges were studied. Culturable and direct 16S rDNA PCR amplification techniques were used to describe bacterial communities present in Siberian and slender wheatgrass seeds, leaf tissues, and root tissues following propagation in either sand or a peat-based growing mix. Our hypothesis was that the resulting plant endophytic communities would be distinct, showing not only the presence of endophytes originating from the seed but also the characteristics of growth in the two different growing media. Both culture and culture-independent assays showed the likely translocation of Actinobacteria, Firmicutes, and Gammaproteobacteria from seed to mature plant tissues as well as subsequent colonization by exogenous organisms. Statistical analysis of 16S terminal restriction fragment profiles identified growing media as having a greater significant effect on the formation of the endpoint endophytic communities than either plant tissue or wheatgrass variety. In silico digests of the ribosomal database produced putative identifications indicating an increase in overall species diversity and increased relative abundances of Firmicutes and Cyanobacteria following propagation in sand and Betaproteobacteria following propagation in the peat-based growing mix. Results indicated a substantial translocation of endophytes from seed to mature plant tissues for both growing media and that growing medium was a dominant determinant of the final taxonomy of the endpoint plant endophytic communities.

Rapid differentiation of phenotypically and genotypically similar Synechococcus elongatus strains by PCR fingerprinting.

PCR amplification techniques viz., repetitive DNA element PCR (REP-PCR), short tandemly repeated repetitive PCR (STRR-PCR) and arbitrarily primed PCR (RAPD-PCR) were used for the taxonomic discrimination among the strains of the unicellular cyanobacterium Synechococcus elongatus collected across the coastal regions of the Indian subcontinent. These strains showed similar phenotypic and genotypic characteristics. Data obtained from genomic fingerprinting were used to perform cluster analysis and demonstrated ability to differentiate strains at intra-specific level. Polymorphisms of different PCR amplification products can serve as strain-specific molecular fingerprints. In comparison with the STRR and RAPD, the REP primer set generates fingerprints of lower complexity, but still the phenogram clearly differentiated the strains. In conclusion, described PCR fingerprinting methods can be considered as promising tools for the differentiation at the strain level of cyanobacteria from the same species.

Morphological and genetic diversity of symbiotic cyanobacteria from cycads

The morphological and genetic diversity of cyanobacteria associated with cycads was examined using PCR amplification techniques and 16S rRNA gene sequence analysis. Eighteen symbiotic cyanobacteria were isolated from different cycad species. One of the symbiotic isolates was a species of Calothrix, a genus not previously reported to form symbioses with Cycadaceae family, and the remainder were Nostoc spp. Axenic cyanobacterial strains were compared by DNA amplification using PCR with either short arbitrary primers or primers specific for the repetitive sequences. Based on fingerprint patterns and phenograms, it was revealed that cyanobacterial symbionts exhibit important genetic diversity among host plants, both within and between cycad populations. A phylogenetic analysis based on 16S rRNA gene sequence analysis revealed that most of the symbiotic cyanobacterial isolates fell into well-separated clades.

A comparative retrospective study of RT-PCR-based liquid hybridization assay for early, definitive diagnosis of dengue

Dengue is an important flaviviral infection in tropical and subtropical regions. Early diagnosis of dengue infection helps in monitoring the disease, determining when hospital admission is necessary and reducing case fatalities. The objective of this study was to carry out a retrospective comparison of an RT-PCR-based liquid hybridization (RT-PCR-LH) assay with PCR amplification, virus isolation and serological techniques for laboratory diagnosis of dengue infection. Amplified products of non-structural 3 gene were hybridized with a mixture of four dengue type-specific DNA probes in liquid phase. The assay was validated in a comparative retrospective study using acute serum samples collected from 119 fever patients with or without dengue, confirmed by haemagglutination inhibition (HAI) assay, the gold standard assay for diagnosis of dengue infection. The RT-PCR-LH assay was highly specific for dengue and, as an early laboratory diagnostic method, had 100% sensitivity in detecting dengue patients confirmed by HAI assay. A high analytical sensitivity of two fluorescent focus units of dengue virus/reaction was achieved. This RT-PCR-LH assay using a single serum specimen offers distinct advantages of specificity and sensitivity over other diagnostic techniques for early definitive laboratory diagnosis of dengue infection when serological methods are of little value.

ACC oxidase genes expressed in the wood-forming tissues of loblolly pine ( Pinus taeda L.) include a pair of nearly identical paralogs (NIPs)

1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the final reaction of the ethylene biosynthetic pathway, converting the unusual cyclic amino acid, ACC, into ethylene. Past studies have shown a possible link between ethylene and compression wood formation in conifers, but the relationship has received no more than modest study at the gene expression level. In this study, a cDNA clone encoding a putative ACC oxidase, PtACO1, was isolated from a cDNA library produced using mRNA from lignifying xylem of loblolly pine ( Pinus taeda) trunk wood. The cDNA clone comprised an open reading frame of 1461 bp encoding a protein of 333 amino acids. Using PCR amplification techniques, a genomic clone corresponding to PtACO1 was isolated and shown to contain three introns with typical GT/AG boundaries defining the splice junctions. The PtACO1 gene product shared 70% identity with an ACC oxidase from European white birch ( Betula pendula), and phylogenetic analyses clearly placed the gene product in the ACC oxidase cluster of the Arabidopsis thaliana 2-oxoglutarate-dependent dioxygenase superfamily tree. The PtACO1 sequence was used to identify additional ACC oxidase clones from loblolly pine root cDNA libraries characterized as part of an expressed sequence tag (EST) discovery project. The PtACO1 sequence was also used to recover additional paralogous sequences from genomic DNA, one of which ( PtACO2) turned out to be > 98% identical to PtACO1 in the nucleotide coding sequence, leading to its classification as a “nearly identical paralog” (NIP). Quantitative PCR analyses showed that the expression level of PtACO1-like transcripts varied in different tissues, as well as in response to hormonal treatments and bending. Possible roles for PtACO1 in compression wood formation in loblolly pine and the discovery of its NIP are discussed in light of these results.

Isothermal Single Nucleotide Polymorphism Genotyping and Direct PCR from Whole Blood Using a Novel Whole-Blood Lysis Buffer

Cell lysis and subsequent release of genomic DNA is an ongoing dilemma for molecular biological techniques. In most cases, technologies such as PCR and other amplification techniques require DNA extraction and purification steps. The Smart Amplification Process Version 2 (SmartAmp2) is an isothermal and integrated amplification technology that eliminates the need for time-consuming sample preparation for the rapid detection of nucleic acids, including single nucleotide polymorphisms (SNPs), mutations, and other targets. In addition, DNA amplification directly from whole blood is beneficial and lessens the risk of cross-contamination. Traditional SmartAmp2 assays entail two steps and require an alkali pretreatment step at 98 degrees C prior to the 60 degrees C run. To make SmartAmp2 truly isothermal and to simplify DNA amplification, we hereby introduce the SmartAmp Isothermal Lysis Buffer (SIL-B), a newly developed chaotropic lysis buffer that enables the simultaneous recovery and denaturation of genomic material directly from whole blood at a uniform 60 degrees C. The improved method for isolating nucleic acids from whole blood is a critical milestone in making SmartAmp2 truly isothermal from start to finish at one temperature, increasing its potential to be routinely used in field point-of-care testing. Furthermore, pretreatment with SIL-B enables the PCR amplification of genomic material directly from whole blood.

Detection of herpetic viruses in gingival crevicular fluid of patients suffering from periodontal diseases: prevalence and effect of treatment

Although the role of bacteria in the etiology of periodontitis is well established, it has been suggested that herpetic viruses could contribute to the initiation and progression of this disease. The aim of this study was to determine the prevalence of human cytomegalovirus (HCMV), Epstein-Barr virus (EBV) and herpes simplex virus (HSV) in gingival crevicular fluid (GCF) samples obtained from periodontally healthy, gingivitis and periodontitis patients. In addition, the effect of periodontal treatment (scaling and root planing) on the persistence of herpetic viruses was evaluated in a sub-group of patients suffering from chronic periodontitis. The presence of viruses in GCF samples was assessed by a nested PCR amplification technique. The persistence of viruses in periodontal sites was evaluated following a scaling and root planing therapy. A statistically significant higher prevalence of HCMV was observed in periodontitis patients as compared to healthy control subjects (35 vs. 8%, respectively; P = 0.0377). A trend for a higher prevalence of HSV was also noted in the periodontitis group, in comparison with healthy control subjects. In addition, a higher prevalence of HCMV was associated with deep periodontal pockets in subjects suffering from periodontitis. In the sub-group of periodontitis patients, periodontal therapy resulted in the elimination (HCMV and EBV) or reduction (HSV) of the herpetic viruses. This study showed that the prevalence of HCMV and HSV viruses in GCF is higher in patients suffering from periodontitis compared to periodontally healthy subjects, and that the prevalence of HCMV is higher in deep periodontal pockets. It also brought evidences that periodontal therapy may be associated with virus elimination in diseased sites.

Health-related Physical Fitness Of Portuguese Twins. A Quantitative Genetic Analysis

PURPOSE: The purposes of this study were: (1) to evaluate familial aggregation in heterogeneity levels of physical fitness, and (2) to quantify the magnitude of genetic and environmental effects in different components of the Fitnessgram health-related physical fitness. METHODS: The sample comprises 231 twin pairs from different regions of Portugal of both zygosity (93 Monozygotic, and 138 Dizygotic) and was divided in two groups according to their age: group 1 (G1), from 5 to 11 years; group 2 (G2) from 12 to 19 years. Zygosity was assessed with 16 random DNA microsattelies using PCR amplification techniques. Physical fitness was evaluated with the Fitnessgram test battery, which included curl-ups (CU), push-ups (PU), trunk-lift (TL), one-mile run-walk (1M), and body mass index (BMI). Data analysis was done in SPSS 15, and SYSTAT 11. ANOVA models were used to estimate intraclass correlations (t) to compute heritability estimates (h2). RESULTS: Intra pair differences showed remarkable familial aggregation, because intraclass correlations were systematically greater in MZ twins as compared to DZ twins, showing a smaller intra-pair difference in MZ twins. In all fitness components, all t values in MZ in both groups varied from 0.72 to 0.90, whereas in DZ varied from 0.43 to 0.55. h2 estimates ranged from 0.75 (CU), 0.53 (PU), 0.63 (TL), 0.77 (1M) to 0.84 (BMI) in G1, and from 0.76 (CU), 0.59 (PU), 0.68 (TL), 0.65 (1M) to 0.81 (BMI) G2 showing very similar values in the two age groups. CONCLUSIONS: These results support the idea of familial aggregation in health-related physical fitness among twin pairs, and the importance of genetic factors in explaining interindividual differences. Genetic factors consistently explain from 50 to 80 % of the total variation. This calls for a more precise understanding of physical fitness levels of children and adolescents, the need to implement differential physical activity and sports programs to youth to meet the variation of their potential for change, as well as a careful interpretation of their response to such programs at the population level.

Dual Roles of the Central Domain of Colicin D tRNase in TonB-mediated Import and in Immunity

Colicin D import into Escherichia coli requires an interaction via its TonB box with the energy transducer TonB. Colicin D cytotoxicity is inhibited by specific tonB mutations, but it is restored by suppressor mutations in the TonB box. Here we report that there is a second site of interaction between TonB and colicin D, which is dependent upon a 45-amino acid region, within the uncharacterized central domain of colicin D. In addition, the 8th amino acids of colicin D (a glycine) and colicin B (a valine), adjacent to their TonB boxes, are also required for TonB recognition, suggesting that high affinity complex formation involves multiple interactions between these colicins and TonB. The central domain also contributes to the formation of the immunity complex, as well as being essential for uptake and thus killing. Colicin D is normally secreted in association with the immunity protein, and this complex involves the following two interactions: a major interaction with the C-terminal tRNase domain and a second interaction involving the central domain of colicin D and, most probably, the alpha4 helix of ImmD, which is on the opposite side of ImmD compared with the major interface. In contrast, formation of the immunity complex with the processed cytotoxic domain, the form expected to be found in the cytoplasm after colicin D uptake, requires only the major interaction. Klebicin D has, like colicin D, a ribonuclease activity toward tRNAArg and a central domain, which can form a complex with ImmD but which does not function in TonB-mediated transport.

Identification and detection of Pseudomonas plecoglossicida isolates with PCR primers targeting the gyrB region

Pseudomonas plecoglossicida is the agent of bacterial haemorrhagic ascites (BHA) in freshwater fish farming in Japan. To develop a rapid identification and detection method for P. plecoglossicida, a PCR amplification technique targeting the chromosomal DNA region coding the B subunit of the DNA gyrase (gyrB) was used. The nucleotide sequences of gyrB were determined in nine isolates of P. plecoglossicida and two other Pseudomonas species. On the basis of these determined sequences and the gyrB sequences of other Pseudomonas species or fish pathogenic bacteria deposited in international nucleotide sequence databases (GenBank/EMBL/DDBJ), PCR primers PL-G1F, PL-G1R, PL-G2F and PL-G2R were designed for specific amplification of the partial gyrB of P. plecoglossicida. The specificity of these primers in amplifying the gyrB of P. plecoglossicida was verified using selected strains of related bacterial species. The nested PCR technique was used to detect P. plecoglossicida from kidney and intestine of ayu. Primer pair PL-G1F and PL-G1R was used for the external PCR, and primer pair PL-G2F and PL-G2R for the internal PCR. Of 10 ayu juveniles, expected size PCR products were observed from intestine and kidney samples in one and two specimens, respectively. The PCR technique with primers based on the gyrB sequence is thus useful for the diagnosis of BHA.

Identification of CTX-M-Type Extended-Spectrum-β-Lactamase Genes Using Real-Time PCR and Pyrosequencing

CTX-M extended-spectrum beta-lactamases (ESBLs) are increasingly prevalent worldwide among Escherichia coli bacteria, mostly in community-acquired urinary tract infections. Finding a fast and reliable technique for identification of CTX-M enzymes is becoming a challenge for the microbiology laboratory. A fast real-time PCR amplification technique, using degenerated primers specific for all the bla(CTX-M) alleles, coupled to real-time pyrosequencing was developed. The five CTX-M groups were unambiguously identified by pyrosequencing a 13-bp DNA region. Further sequencing of an additional 16-bp region allowed further division into subgroups. Phylogenetic trees constructed with the entire bla(CTX-M) genes and with both pyrosequenced regions (29 bp) gave similar results, suggesting that this technique, termed the real-time detection and sequencing method, has a powerful discriminatory ability. This high-throughput technique has been evaluated by screening 48 ESBL-producing E. coli isolates recovered from the Bicêtre hospital (France) in 2004. Forty-four of these strains were CTX-M positive by real-time PCR detection and direct pyrosequencing of the PCR products, which identified CTX-M-15 as the main CTX-M-type beta-lactamase. Pulsed-field gel electrophoresis analysis of these strains revealed that several clones, of which one CTX-M-15-positive clone was predominant (60%), were identified both in nosocomial and in community-acquired isolates. The combination of real-time PCR with pyrosequencing represents a powerful tool for epidemiological studies of CTX-M producers. This assay has the potential to be used in a diagnostic laboratory since up to 96 bacterial isolates may be screened in less than 3 h.