Development of a real-time PCR assay for the specific detection and identification of Streptococcus pseudopneumoniae using the recA gene

AbstractWe sequenced the evolutionarily conserved genes 16S rRNA, atpD, tuf, and recA from Streptococcus pseudopneumoniae, Streptococcus pneumoniae, Streptococcus mitis, and Streptococcus oralis. Phylogenetic analysis revealed that recA provided good resolution between these species, including discrimination of the novel species S. pseudopneumoniae. By contrast, the more conserved 16S rRNA, tuf and atpD are not sufficiently discriminatory. Therefore, recA sequences were used to develop a real-time PCR assay with a locked nucleic acid-mediated TaqMan probe for the specific detection and identification of S. pseudopneumoniae. The PCR assay showed excellent specificity and a detection limit of <10 genome copies for the detection and identification of S. pseudopneumoniae strains, which makes it a promising tool for molecular identification and epidemiological studies. In conclusion, this article describes for the first time a PCR assay for the specific identification of S. pseudopneumoniae

DNA Detection Using Recombination Proteins

DNA amplification is essential to most nucleic acid testing strategies, but established techniques require sophisticated equipment or complex experimental procedures, and their uptake outside specialised laboratories has been limited. Our novel approach, recombinase polymerase amplification (RPA), couples isothermal recombinase-driven primer targeting of template material with strand-displacement DNA synthesis. It achieves exponential amplification with no need for pretreatment of sample DNA. Reactions are sensitive, specific, and rapid and operate at constant low temperature. We have also developed a probe-based detection system. Key aspects of the combined RPA amplification/detection process are illustrated by a test for the pathogen methicillin-resistant Staphylococcus aureus. The technology proves to be sensitive to fewer than ten copies of genomic DNA. Furthermore, products can be detected in a simple sandwich assay, thereby establishing an instrument-free DNA testing system. This unique combination of properties is a significant advance in the development of portable and widely accessible nucleic acid–based tests

Evaluation of single and double-locus real-time PCR assays for methicillin-resistant Staphylococcus aureus (MRSA) surveillance

<p>Abstract</p> <p>Background</p> <p>Methicillin-resistant <it>Staphylococcus aureus </it>(MRSA) is a human pathogen, representing an infection control challenge. Conventional MRSA screening takes up to three days, therefore development of rapid detection is essential. Real time-PCR (rt-PCR) is the fastest method fulfilling this task. All currently published or commercially available rt-PCR MRSA assays relay on single or double-locus detection. Double-locus assays are based on simultaneous detection of <it>mecA </it>gene and a <it>S. aureus</it>-specific gene. Such assays cannot be applied on clinical samples, which often contain both coagulase-negative staphylococci (CoNS) and <it>S. aureus</it>, either of which can carry <it>mecA</it>. Single-locus assays are based on detection of the staphylococcal cassette chromosome <it>mec </it>(SCC<it>mec</it>) element and the <it>S. aureus</it>-specific <it>orfX </it>gene, assuming that it is equivalent to <it>mecA </it>detection.</p> <p>Findings</p> <p>Parallel evaluation of several published single and double-locus rt-PCR MRSA assays of 150 pure culture strains, followed by analysis of 460 swab-derived clinical samples which included standard identification, susceptibility testing, followed by PCR detection of staphylococcal suspected isolates and in-PCR mixed bacterial populations analysis indicated the following findings.</p> <p>Pure cultures analysis indicated that one of the single-locus assay had very high prevalence of false positives (Positive predictive value = 77.8%) and was excluded from further analysis. Analysis of 460 swab-derived samples indicated that the second single-locus assay misidentified 16 out of 219 MRSA's and 13 out of 90 methicillin-sensitive <it>S</it>. <it>aureus</it>'s (MSSA) were misidentified as MRSA's. The double-locus detection assay misidentified 55 out of 90 MSSA's. 46 MSSA containing samples were misidentified as MRSA and 9 as other than <it>S. aureus </it>ending with low positive predicted value (<85%) and very low specificity (<62%).</p> <p>Conclusion</p> <p>The results indicate that high prevalence of false-positive and false-negative reactions occurs in such assays.</p

External quality assessment of the molecular diagnostics and genotyping of meticillin-resistant Staphylococcus aureus

Two multicentre external quality assessments (EQA) for the molecular detection and genotyping of meticillin-resistant Staphylococcus aureus (MRSA) were arranged. Firstly, 11 samples containing various amounts of inactivated MRSA strains, meticillin-susceptible S. aureus (MSSA), meticillin-resistant coagulase-negative staphylococci (MRCoNS) or Escherichia coli were distributed to 82 laboratories. Samples containing 102 or 103 MRSA cells were correctly scored in only 16 and 46% of the datasets returned, respectively. Two of the used MSSA strains contained an SCCmec cassette lacking the mecA gene. There was a marked difference in the percentage of correct results for these two MSSA strains (37 and 39%) compared to the MSSA strain lacking the SCCmec cassette (88%). Secondly, a panel for MRSA genotyping, consisting of ten samples (two identical, three genetically related and five unique strains) was distributed to 19 laboratories. Seventy-three percent of the datasets recorded all samples correctly. Most pulsed-field gel electrophoresis (PFGE) protocols proved to be suboptimal, resulting in inferior resolution in the higher or lower fragment regions. The performance of molecular diagnostics for MRSA shows no significant changes since our first EQA in 2006. The first molecular typing results are encouraging. Both assessments indicate that programme expansion is required and that major performance discrepancies continue to exist

Detection of novel chromosome-SCCmec variants in Methicillin Resistant Staphylococcus aureus and their inclusion in PCR based screening

Findings. To facilitate automation, a novel DNA extraction method for MRSA was adopted. The MRSA specific chromosome-SCCmec PCR was adapted, additional primers were added, and the performance was validated. From various laboratories in The Netherlands we received a total of 86 MRSA clinical isolates, that were negative in commercially available tests. We identified 14 MRSA strains with new variant chromosome-SCCmec junctions by sequence analysis. These MRSA strains appeared to carry SCCmec sequences with a high degree of homology to SCC regions of S. epidermidis and S. haemolyticus. All were included for detection in chromosome-SCCmec based PCR. Background: Efficient management of Methicillin Resistant Staphylococcus aureus (MRSA) in the hospital is needed to prevent dissemination. It is important that MRSA can be rapidly identified, and effective infection control measures can be initiated. Equally important is a rapid MRSA negative report, especially for patients in isolation. For negative screening we implemented fully automated high through-put molecular screening for MRSA. Conclusions: Fourteen variant chromosome-SCCmec junctions in MRSA, that are not detected in commercially available MRSA detection kits were added to our PCR to detect all currently known variant SCC-mec types of MRSA

Rapid Detection of Chlamydia trachomatis and Typing of the Lymphogranuloma venereum associated L-Serovars by TaqMan PCR

<p>Abstract</p> <p>Background</p> <p>Infection due to <it>Chlamydia trachomatis </it>is the most common sexually transmitted bacterial disease of global health significance, and especially the L-serovars causing lymphogranuloma venereum are increasingly being found in Europe in men who have sex with men.</p> <p>Results</p> <p>The design and evaluation of a rapid, multiplex, real-time PCR targeting the major outer membrane protein (<it>omp-1</it>) -gene and a L-serovar-specific region of the polymorphic protein H (<it>pmp-H</it>) -gene for the detection of <it>Chlamydia trachomatis </it>is reported here. The PCR takes place as a single reaction with an internal control. For L1-, L2- and L3-serovar differentiation a second set of real-time PCRs was evaluated based on the amplification of serovar-specific <it>omp-1</it>-regions. The detection limit of each real-time PCR, multiplexed or not, was 50 genome copies per reaction with an efficiency ranging from 90,5–95,2%.</p> <p>In a retrospective analysis of 50 ocular, rectal and urogenital specimens formerly tested to be positive for <it>C. trachomatis </it>we identified six L2-serovars in rectal specimens of HIV-positive men, one in a double-infection with L3, and one L2 in a urethral specimen of an HIV-negative male.</p> <p>Conclusion</p> <p>This unique real-time PCR is specific and convenient for the rapid routine-diagnostic detection of lymphogranuloma venereum-associated L-serovars and enables the subsequent differentiation of L1, L2 and L3 for epidemiologic studies.</p

Development of a heptaplex PCR assay for identification of Staphylococcus aureus and CoNS with simultaneous detection of virulence and antibiotic resistance genes

Background Staphylococcal toxicity and antibiotic resistance (STAAR) have been menacing public health. Although vancomycin-resistant Staphylococcus aureus (VRSA) is currently not as widespread as methicillin-resistant S. aureus (MRSA), genome evolution of MRSA into VRSA, including strains engineered within the same patient under anti-staphylococcal therapy, may build up to future public health concern. To further complicate diagnosis, infection control and anti-microbial chemotherapy, non-sterile sites such as the nares and the skin could contain both S. aureus and coagulase-negative staphylococci (CoNS), either of which could harbour mecA the gene driving staphylococcal methicillin-resistance and required for MRSA-VRSA evolution. Results A new heptaplex PCR assay has been developed which simultaneously detects seven markers for: i) eubacteria (16S rRNA), ii) Staphylococcus genus (tuf), iii) Staphylococcus aureus (spa), iv) CoNS (cns), v) Panton-Valentine leukocidin (pvl), vi) methicillin resistance (mecA), and vii) vancomycin resistance (vanA). Following successful validation using 255 reference bacterial strains, applicability to analyse clinical samples was evaluated by direct amplification in spiked blood cultures (n = 89) which returned 100 % specificity, negative and positive predictive values. The new assay has LoD of 1.0x103 CFU/mL for the 16S rRNA marker and 1.0x104 CFU/mL for six other markers and completes cycling in less than one hour. Conclusion The speed, sensitivity (100 %), NPV (100 %) and PPV (100 %) suggest the new heptaplex PCR assay could be easily integrated into a routine diagnostic microbiology workflow. Detection of the cns marker allows for unique identification of CoNS in mono-microbial and in poly-microbial samples containing mixtures of CoNS and S. aureus without recourse to the conventional elimination approach which is ambiguous. In addition to the SA-CoNS differential diagnostic essence of the new assay, inclusion of vanA primers will allow microbiology laboratories to stay ahead of the emerging MRSA-VRSA evolution. To the best of our knowledge, the new heptaplex PCR assay is the most multiplexed among similar PCR-based assays for simultaneous detection of STAAR

Targeting poly(ADP-ribose) polymerase activity for cancer therapy

Poly(ADP-ribosyl)ation is a ubiquitous protein modification found in mammalian cells that modulates many cellular responses, including DNA repair. The poly(ADP-ribose) polymerase (PARP) family catalyze the formation and addition onto proteins of negatively charged ADP-ribose polymers synthesized from NAD+. The absence of PARP-1 and PARP-2, both of which are activated by DNA damage, results in hypersensitivity to ionizing radiation and alkylating agents. PARP inhibitors that compete with NAD+ at the enzyme’s activity site are effective chemo- and radiopotentiation agents and, in BRCA-deficient tumors, can be used as single-agent therapies acting through the principle of synthetic lethality. Through extensive drug-development programs, third-generation inhibitors have now entered clinical trials and are showing great promise. However, both PARP-1 and PARP-2 are not only involved in DNA repair but also in transcription regulation, chromatin modification, and cellular homeostasis. The impact on these processes of PARP inhibition on long-term therapeutic responses needs to be investigated

A Review of SHV Extended-Spectrum β-Lactamases: Neglected Yet Ubiquitous

Microbial Biotechnolog

Modulation de la structure de la chromatine par la poly(ADP-ribosyl)ation

Nous avons caractérisé les protéines chromosomales acceptrices depoly(ADP-ribose) dans différents niveaux d'organisation de la chromatine et nous avons étudié l'effet structural de leur poly(ADP-ribosyl)ation sur la structure chromatinienne. Premièrement, nous avons démontré à l'aide de techniques d'électrophorèse sur gel de polyacrylamide que le patron des histones poly(ADP-ribosyl)ées dans des préparations de chromatine native varie selon l'état d'automodification de la poly(ADP-ribose) polymérase. Les histones H1, H1°, H2B, H2A et A24 sont principalement modifiées dans des conditions où l'enzyme est faiblement modifiée ( ≤1 uM NAD+). Lorsque l'enzyme est fortement modifiée ( ≥ 10 uM NAD+), l'histone H1 est préférentiellement hyper(ADP-ribosyl)ée. Par la suite, nous avons démontré à l'aide de techniques de microscopie électronique et d'ultracentrifugation analytique qu'à des concentrations physiologiques de NAD+ (200 uM), la synthèse et l'hydrolyse du poly(ADP-ribose) par l'action séquentielle de la poly(ADP-ribose) polymérase et de la poly(ADP-ribose) glycohydrolase purifiées sur la chromatine native provoquent une décondensation-recondensation réversible de la fibre chromatinienne de 30 nm. Ce phénomène est attribué à la formation et à la dégradation des formes hyper(ADP-ribosyl)ées de l'histone H1. Deuxièmement, nous avons montré que le patron des histones modifiées varie également selon les différents niveaux d'organisation de la chromatine à des concentrations physiologiques de NAD+. L'histone H1 est l'histone acceptrice majeure dans la chromatine native tandis que les histones H2B, H2A et A24 sont principalement modifiées dans la chromatine dépourvue en histone H1 et dans des préparations de coeurs nucléosomaux purifiés. Nous avons par la suite observé par microscopie électronique que la poly(ADP-ribosyl)ation de coeurs nucléosomaux purifiés dans ces conditions provoque la dissociation de l'ADN qui entoure l'octamère d'histones. Nous avons également démontré que la poly(ADP-ribosyl)ation de la chromatine dépourvue en histone H1 empêche la génération de la fibre de 30 nm lors de l'addition de l'histone H1. Ce phénomène semble être attribué à la présence des histones modifiées dans l'octamère et/ou de la poly(ADP-ribose) polymérase modifiée puisqu'une forte interaction de celle-ci a été observée sur la chromatine dépourvue en histone H1 tandis qu'une interaction négligeable a été observée avec la chromatine native. Nous avons également observé une interaction importante du poly(ADP-ribose) avec des coeurs nucléosomaux purifiés. Troisièmement, par une approche immunologique, nous avons observé une plus grande accessibilité des histones de l'octamère à différents anticorps monoclonaux et polyclonaux anti histones dans la chromatine native et la chromatine dépourvue en histone H1 poly(ADP-ribosyl)ées. L'ensemble de ces résultats suggère une déstabilisation importante de la structure chromatinienne par la poly(ADP-ribosyl)ation. Nous proposons ainsi un modèle dans lequel la poly(ADP-ribosyl)ation serait impliquée dans la modulation de la structure chromatinienne lors de réparation d'ADN