Peter Schierack

Brandenburg University of Technology Cottbus - Senftenberg, Kottbus, Brandenburg, Germany

Are you Peter Schierack?

Claim your profile

Publications (68)187.32 Total impact

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: There is an ever-increasing number of applications, which use quantitative PCR (qPCR) or digital PCR (dPCR) to elicit fundamentals of biological processes. Moreover, quantitative isothermal amplification (qIA) methods have become more prominent in life sciences and point-of-care- diagnostics. Additionally, the analysis of melting data is essential during many experiments. Several software packages have been developed for the analysis of such datasets. In most cases, the software is either distributed as closed source software or as monolithic block with little freedom to perform highly customized analysis procedures. We argue, among others, that R is an excellent foundation for reproducible and transparent data analysis in a highly customizable cross-platform environment. However, for novices it is often challenging to master R or learn capabilities of the vast number of packages available. In the paper, we describe exemplary workflows for the analysis of qPCR, qIA or dPCR experiments including the analysis of melting curve data. Our analysis relies entirely on R packages available from public repositories. Additionally, we provide information related to standardized and reproducible research.
    The R Journal 06/2015; 7(1):127-150. · 0.90 Impact Factor
  • Source
    [Show description] [Hide description]
    DESCRIPTION: LoopTag Real-Time PCR Probe System for Sensitive Pathogen Detection
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: (qPCR) and quantitative isothermal amplification (qIA) are standard methods for nucleic acid quantification. Numerous real-time read-out technologies have been developed. Despite the continuous interest in amplification-based techniques, there are only few tools for pre-processing of amplification data. However, a transparent tool for precise control of raw data is indispensable in several scenarios, for example, during the development of new instruments. Results: chipPCR is an R package for the pre-processing and quality analysis of raw data of amplification curves. The package takes advantage of R's S4 object model and offers an extensible environment. chipPCR contains tools for raw data exploration: normalization, baselining, imputation of missing values, a powerful wrapper for amplification curve smoothing and a function to detect the start and end of an amplification curve. The capabilities of the software are enhanced by the implementation of algorithms unavailable in R, such as a 5-point stencil for derivative interpolation. Simulation tools, statistical tests, plots for data quality management, amplification efficiency/quantification cycle calculation, and data sets from qPCR and qIA experiments are part of the package. Core functionalities are integrated in GUIs (web-based and standalone shiny applications), thus streamlining analysis and report generation. Availability: http://cran.r-project.org/web/packages/chipPCR newline Source code: https://github.com/michbur/chipPCR Supplementary: Supplementary data are available at Bioinformatics online.
    Bioinformatics 04/2015; DOI:10.1093/bioinformatics/btv205 · 4.62 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Background: For the serological diagnosis of systemic autoimmune rheumatic diseases, a two-tier approach starting with sensitive antinuclear antibody (ANA) detection by indirect immunofluorescence (IIF) on HEp-2 cells followed by characterization of positive findings with different immunoassays is recommended. To overcome drawbacks of this approach, we developed a novel technique allowing the combination of screening and simultaneous confirmatory testing. For the first time, this creates the basis for second generation ANA testing. Methods: ANA and autoantibodies (autoAb) to dsDNA, CENP-B, SS-A/Ro52, SS-A/Ro60, SS-B/La, RNP-Sm, Sm, and Scl-70 were determined by IIF and enzyme-linked immunosorbent assay (ELISA), respectively, and compared to simultaneous analysis thereof by second generation ANA analysis in patients with systemic lupus erythematosus (n=174), systemic sclerosis (n=103), Sjögren’s syndrome (n=46), rheumatoid arthritis (n=36), mixed and undetermined connective tissue diseases (n=13), myositis (n=21), infectious disease (n=21), autoimmune liver disease (n=93), inflammatory bowel disease (n=78), paraproteinemia (n=11), and blood donors (n=101). Results: There was very good agreement of second generation ANA testing with classical one by IIF and ELISA regarding testing for ANA and autoAb to dsDNA, CENP-B, SS-B, RNP-Sm, Scl-70, SS-A/Ro52, and SS-A/Ro60 (Cohen’s kappa [κ]>0.8). The agreement for anti-Sm autoAb was good (κ=0.77). The differences of both approaches were not significant for autoAb to SS-B/La, RNP-Sm, Scl-70, SS-A/Ro60, and SS-A/Ro52 (McNemar’s test, p>0.05, respectively). Conclusions: Second generation ANA testing can replace the two-tier analysis by combining IIF screening with multiplex confirmative testing. This addresses shortcomings of classical ANA analysis like false-negative ANA findings and lack of laboratory efficiency and standardization.
    Clinical Chemistry and Laboratory Medicine 04/2015; · 2.96 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Background: For the serological diagnosis of systemic autoimmune rheumatic diseases, a two-tier approach starting with sensitive antinuclear antibody (ANA) detection by indirect immunofluorescence (IIF) on HEp-2 cells followed by characterization of positive findings with different immunoassays is recommended. To overcome drawbacks of this approach, we developed a novel technique allowing the combination of screening and simultaneous confirmatory testing. For the first time, this creates the basis for second generation ANA testing. Methods: ANA and autoantibodies (autoAb) to dsDNA, CENP-B, SS-A/Ro52, SS-A/Ro60, SS-B/La, RNP-Sm, Sm, and Scl-70 were determined by IIF and enzyme-linked immunosorbent assay (ELISA), respectively, and compared to simultaneous analysis thereof by second generation ANA analysis in patients with systemic lupus erythematosus (n=174), systemic sclerosis (n=103), Sjögren’s syndrome (n=46), rheumatoid arthritis (n=36), mixed and undetermined connective tissue diseases (n=13), myositis (n=21), infectious disease (n=21), autoimmune liver disease (n=93), inflammatory bowel disease (n=78), paraproteinemia (n=11), and blood donors (n=101). Results: There was very good agreement of second generation ANA testing with classical one by IIF and ELISA regarding testing for ANA and autoAb to dsDNA, CENP-B, SS-B, RNP-Sm, Scl-70, SS-A/Ro52, and SS-A/Ro60 (Cohen’s kappa [κ]>0.8). The agreement for anti-Sm autoAb was good (κ=0.77). The differences of both approaches were not significant for autoAb to SS-B/La, RNP-Sm, Scl-70, SS-A/Ro60, and SS-A/Ro52 (McNemar’s test, p>0.05, respectively). Conclusions: Second generation ANA testing can replace the two-tier analysis by combining IIF screening with multiplex confirmative testing. This addresses shortcomings of classical ANA analysis like false-negative ANA findings and lack of laboratory efficiency and standardization.
    Clinical Chemistry and Laboratory Medicine 04/2015; DOI:10.1515/cclm-2015-0083 · 2.96 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The efficacy of many chemotherapeutic agents relies on the preferential destruction of rapidly dividing cancer cells by inducing various kinds of DNA damage. The most deleterious type of DNA lesions are DNA double-strand breaks (DSB), which can be detected by immunofluorescence staining of phosphorylated histone protein H2AX (γH2AX). Furthermore, γH2AX has been suggested as clinical pharmacodynamic biomarker in chemotherapeutic cancer treatment. A great challenge in treating neoplastic diseases is the varying response behavior among cancer patients. Thus, intrinsic or drug-induced overexpression of efflux pumps often leads to multiple drug resistance (MDR) and treatment failure. In particular, inter-individual differences in expression levels of efflux pumps, such as the permeability glycoprotein (P-gp), were shown to correlate with cancer progression. Several efficient cytostatic drugs, including the DSB-inducing agent etoposide (ETP) are known P-gp substrates. In this respect, modulation of MDR by P-gp inhibitors, like the immunosuppressives cyclosporine A (CsA) and rapamycin (Rapa) have been described. Here, we investigated the application of γH2AX focus assay to monitor the impact of CsA and Rapa on ETP-induced cytotoxicity in human peripheral blood mononuclear cells. Evaluation of γH2AX foci was performed by the automated fluorescence microscopy and interpretation system AKLIDES. Compared to ETP treatment alone, our results revealed a significant rise in γH2AX focus number and percentage of DSB-positive cells after cells have been treated with ETP in the presence of either CsA or Rapa. In contrast, DSB levels of cells incubated with CsA or Rapa alone were comparable to focus number of untreated cells. Our results successfully demonstrated how automated γH2AX analysis can be used as fast and reliable approach to monitor drug resistance and the impact of MDR modulators during treatment with DSB-inducing cytostatics. © 2015 International Society for Advancement of Cytometry. © 2015 International Society for Advancement of Cytometry.
    Cytometry Part A 04/2015; DOI:10.1002/cyto.a.22667 · 3.07 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Escherichia coli bacteria are the most common causes of diarrhea and septicemia in calves. Moreover, calves form a major reservoir for transmission of pathogenic E. coli to humans. Systematic reviews and meta-analyses of publications on Escherichia coli as calf pathogens and the role of calves as reservoir have not been done so far. We reviewed studies between 1951 and 2013 reporting the presence of virulence associated factors (VAFs) in calf E. coli and extracted the following information: year(s) and country of sampling, animal number, health status, isolate number, VAF prevalence, serotypes, diagnostic methods and biological assays. The prevalence of VAFs or E. coli pathotypes was compared between healthy and diarrheic animals and was analysed for time courses. Together, 106 papers with 25982 E. coli isolates from 27 countries tested for VAFs were included. F5, F17 and F41 fimbriae and heat-stable enterotoxin (ST) - VAFs of enterotoxigenic E. coli (ETEC) were significantly associated with calf diarrhea. On the contrary, ETEC VAF F4 fimbriae and heat-labile enterotoxin as well as enteropathogenic (EPEC), Shiga toxin-producing (STEC), and enterohaemorrhagic E. coli (EHEC) were not associated with diarrhea. The prevalence increased overtime for ST-positive isolates, but decreased for F5- and STEC-positive isolates. Our study provides useful information about the history of scientific investigations performed in this domain so far, and helps to define etiological agents of calf disease, and to evaluate calves as reservoir hosts for human pathogenic E. coli.
    Frontiers in Cellular and Infection Microbiology 03/2015; 5. DOI:10.3389/fcimb.2015.00023 · 2.62 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Mallard ducks have demonstrated to be a likely reservoir for zoonotic E. coli strains; thus, it is possible that these ducks could also act as a reservoir for other Enterobacteriaceae members. The present study was initiated to evaluate the species distribution of Enterobacteriaceae other than E. coli in 175 fresh faecal samples collected from a population of mallard ducks. Sixty-four samples displayed detectable colonies of Enterobacteriaceae (excluding E. coli), which resulted in 75 pulsed-field gel electrophoresis (PFGE) types. Seventy-five single representatives of each PFGE type were subjected to identification with API 32NE and MALDI TOF MS systems due to the practical difficulties in species differentiation of Enterobacteriaceae. Those isolated were found to be from nine genera: Buttiauxella (15 %), Citrobacter (5 %), Enterobacter (32 %), Hafnia (1 %), Leclercia (1 %), Pantoea (7 %), Raoultella (21 %), Rahnella (7 %) and Serratia (11 %). Evaluation of antimicrobial resistance phenotypes using the disc method and detection of resistance genes using the microarray method revealed that these microbes possess resistance to β-lactams, aminoglycosides, macrolides, quinolones, rifamycine, sulphonamides, streptogramins and diaminopyrimidines. In conclusion, mallard ducks harbour a variety of non-pathogenic and pathogenic Enterobacteriaceae species like Enterobacter cloacae and Enterobacter amnigenus in their intestine and could act as a reservoir of resistant Enterobacteriaceae.
    Environmental Monitoring and Assessment 03/2015; 187(3):4346. DOI:10.1007/s10661-015-4346-4 · 1.68 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: We report the population structure and dynamics of one E. coli population of wild mallard ducks in their natural environment over four winter seasons, following the characterization of 100 isolates each consecutive season. Macrorestriction analysis was used to define isolates variously as multi- or one-year PFGE types. Isolates were characterized genotypically based on virulence-associated genes (VAGs), phylogenetic markers, and phenotypically based on hemolytic activity, antimicrobial resistance, adhesion to epithelial cells, microcin production, motility and carbohydrate metabolism. Only 12 out of 220 PFGE types were detectable over more than one winter, and classified as multi-year PFGE types. There was a dramatic change of PFGE types within two winter seasons. Nevertheless, the genetic pool (VAGs) and antimicrobial resistance pattern remained remarkably stable. The high diversity and dynamics of this E. coli population were also demonstrated by the occurrence of PFGE subtypes and differences between isolates of one PFGE type (based on VAGs, antimicrobial resistance, and adhesion rates). Multi- and one-year PFGE types differed in antimicrobial resistance, VAGs and adhesion. Other parameters were not prominent colonization factors. In conclusion, the high diversity, dynamics and stable genetic pool of an E. coli population seems to enable their successful colonization of host animal population overtime. This article is protected by copyright. All rights reserved.
    Environmental Microbiology 02/2015; DOI:10.1111/1462-2920.12807 · 6.24 Impact Factor
  • Laboratoriums Medizin 01/2015; 38(6). DOI:10.1515/labmed-2015-0036 · 0.30 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The aim of this study has been to elucidate the effect of the probiotic Enterococcus faecium NCIMB 10415 on epithelial integrity in intestinal epithelial cells and whether pre- and coincubation with this strain can reproducibly prevent damage induced by enterotoxigenic (ETEC) and enteropathogenic Escherichia coli (EPEC). Porcine (IPEC-J2) and human (Caco-2) intestinal epithelial cells were incubated with bacterial strains and epithelial integrity was assessed by measuring transepithelial electrical resistance (TEER) and mannitol flux rates. E. faecium alone increased TEER of Caco-2 cells without affecting mannitol fluxes whereas the E. coli strains decreased TEER and concomitantly increased mannitol flux rates in both cell lines. Preincubation with E. faecium had no effect on the TEER decrease induced by E. coli in preliminary experiments. However, in a second set of experiments using a slightly different protocol, E. faecium ameliorated the TEER decrease induced by ETEC at 4 h in IPEC-J2 and at 2, 4, and 6 h in Caco-2 cells. We conclude that E. faecium positively affected epithelial integrity in monoinfected Caco-2 cells and could ameliorate the damage on TEER induced by an ETEC strain. Reproducibility of the results is, however, limited when experiments are performed with living bacteria over longer periods.
    01/2015; 2015:235184. DOI:10.1155/2015/235184
  • [Show abstract] [Hide abstract]
    ABSTRACT: Background: Quantification cycle (Cq) and amplification efficiency (AE) are parameters mathematically extracted from raw data to characterize quantitative PCR (qPCR) reactions and quantify the copy number in a sample. Little attention has been paid to the effects of preprocessing and the use of smoothing or filtering approaches to compensate for noisy data. Existing algorithms largely are taken for granted, and it is unclear which of the various methods is most informative. We investigated the effect of smoothing and filtering algorithms on amplification curve data. Methods: We obtained published high-replicate qPCR datasets from standard block thermocyclers and other cycler platforms and statistically evaluated the impact of smoothing on Cq and AE. Results: Our results indicate that selected smoothing algorithms affect estimates of Cq and AE considerably. The commonly used moving average filter performed worst in all qPCR scenarios. The Savitzky–Golay smoother, cubic splines, and Whittaker smoother resulted overall in the least bias in our setting and exhibited low sensitivity to differences in qPCR AE, whereas other smoothers, such as running mean, introduced an AE-dependent bias. Conclusions: The selection of a smoothing algorithm is an important step in developing data analysis pipelines for real-time PCR experiments. We offer guidelines for selection of an appropriate smoothing algorithm in diagnostic qPCR applications. The findings of our study were implemented in the R packages chipPCR and qpcR as a basis for the implementation of an analytical strategy.
    Clinical Chemistry 12/2014; DOI:10.1373/clinchem.2014.230656 · 7.77 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Abstract Background: Autoantibodies to exocrine-pancreatic glycoprotein 2 (anti-GP2) are Crohn's disease (CD) markers. However, CD-specific antibodies have also been found in celiac-disease (CeD) patients, in which type 1 diabetes-specific autoantibodies against endocrine pancreatic targets can be present. We investigated whether anti-GP2 are also present in CeD, a disease like CD which is also characterised by intestinal mucosal inflammation with barrier impairment. Methods: Antibodies against GP2, tissue transglutaminase (tTG), deamidated gliadin (dGD), glutamic decarboxylase (GAD), and islet antigen-2 (IA2) were tested in sera from 73 CD patients, 90 blood donors (BD), and 79 (58 de novo) CeD patients (2 consecutive sera were available from 40 patients). Results: IgA and/or IgG anti-GP2 were found in 15/79 (19.0%) CeD patients on at least one occasion, in 25/73 (34.2%) CD patients, and in 4/90 (4.4%) BD (CeD vs. CD, p=0.042; BD vs. CeD and CD, p<0.001, respectively). Amongst the 58 de novo CeD patients, anti-GP2 IgA and/or IgG were present in 11 (19.0%). Anti-GP2 IgA was significantly less prevalent in CeD compared with CD (p=0.004). Anti-GP2 IgA and IgG in CD patients demonstrated a significantly higher median level compared to patients with CeD (p<0.001, p=0.008, respectively). IgA anti-GP2 levels correlated significantly with IgA anti-tTG and anti-dGD levels in CeD Spearman's coefficient of rank correlation (ρ)=0.42, confidence interval (CI): 0.26-0.56, p<0.001; ρ=0.54, CI 0.39-0.65, p<0.001, respectively. Conclusions: The presence of anti-GP2 in CeD patients supports the notion that loss of tolerance to GP2 can probably be a manifestation of an autoinflammatory process in this intestinal disorder.
    Clinical Chemistry and Laboratory Medicine 11/2014; DOI:10.1515/cclm-2014-0238 · 2.96 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Due to increased release or production of Shiga toxin by Enterohemorrhagic Escherichia coli (EHEC) after exposure to antimicrobial agents, the role of antimicrobial agents in EHEC mediated infections remains controversial. Probiotics are therefore rapidly gaining interest as an alternate therapeutic option. The well-known probiotic strain Escherichia coli Nissle 1917 (EcN) was tested in vitro to determine its probiotic effects on growth, Shiga toxin (Stx) gene expression, Stx amount and associated cytotoxicity on the most important EHEC strains of serotype O104:H4 and O157:H7. Following co-culture of EcN:EHEC in broth for 4 and 24 h, the probiotic effects on EHEC growth, toxin gene expression, Stx amount and cytotoxicity were determined using quantitative real time-PCR, Stx-ELISA and Vero cytotoxicity assays.Probiotic EcN strongly reduced EHEC numbers (cfu) of O104:H4 up to (68%) and O157:H7 to (72.2%) (P < 0.05) in LB broth medium whereas the non-probiotic E. coli strain MG1655 had no effect on EHEC growth. The level of stx expression was significantly down-regulated, particularly for the stx2a gene. The stx down-regulation in EcN co-culture was not due to reduced numbers of EHEC. A significant inhibition in Stx amounts and cytotoxicity were also observed in sterile supernatants of EcN:EHEC co-cultures.These findings indicate that probiotic EcN displays strong inhibitory effects on growth, Shiga toxin gene expression, amount and cytotoxicity of EHEC strains. Thus, EcN may be considered as a putative therapeutic candidate, in particular against EHEC O104:H4 and O157:H7.
    International Journal of Medical Microbiology 10/2014; 305(1). DOI:10.1016/j.ijmm.2014.10.003 · 3.42 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The international consensus for the classification of antiphospholipid syndrome (APS) requires clinical and laboratory criteria to be considered at an equal level for diagnosing APS. Thus, detection of antiphospholipid antibodies (aPL) being a hallmark of APS has been the object of intensive investigation over the past 40 years. However, appropriate detection of aPL still remains a laboratory challenge due to their heterogeneity comprising autoantibodies reactive to different phospholipid-binding plasma proteins, such as beta-2 glycoprotein I (β2GPI) and prothrombin. The relevance of aPL interacting with phospholipids other than cardiolipin (CL, diphosphatidylglycerol), such as phosphatidylserine (PS), remains elusive with regard to the diagnosis of APS. Recently, the concept of aPL profiling has been introduced to assess the risk of thrombotic complications in patients with APS. New assay techniques, apart from enzyme-linked immunosorbent assays (ELISAs) recommended by the international consensus for the classification of APS, have been proposed for multiplexing of aPL testing. Line immunoassays (LIAs) employing a novel hydrophobic solid phase for the simultaneous detection of different aPL seem to be an intriguing alternative. We evaluated a novel multiplex LIA employing a hydrophobic membrane coated with different phospholipid (PL)-binding proteins or PLs. The performance characteristics of this new multiplexing assay technique demonstrated its usefulness for aPL profiling.
    Lupus 10/2014; 23(12):1262-4. DOI:10.1177/0961203314534305 · 2.48 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Anti-neutrophil cytoplasmic antibodies (ANCA) are the serological hallmark of small vessel vasculitis, so called ANCA-associated vasculitis. The international consensus requires testing by indirect immunofluorescence (IIF) on human ethanol-fixed neutrophils (ethN) as screening followed by confirmation with enzyme-linked immunosorbent assays (ELISAs). This study evaluates the combination of cell- and microbead-based digital IIF analysis of ANCA in one reaction environment by the novel multiplexing CytoBead technology for simultaneous screening and confirmatory ANCA testing. Sera of 592 individuals including 118 patients with ANCA-associated vasculitis, 133 with rheumatoid arthritis, 49 with infectious diseases, 77 with inflammatory bowel syndrome, 20 with autoimmune liver diseases, 70 with primary sclerosing cholangitis and 125 blood donors were tested for cytoplasmic ANCA (C-ANCA) and perinuclear ANCA (P-ANCA) by classical IIF and ANCA to proteinase 3 (PR3) and myeloperoxidase (MPO) by ELISA. These findings were compared to respective ANCA results determined by automated multiplex CytoBead technology using ethN and antigen-coated microbeads for microbead immunoassays. There was a good agreement for PR3- and MPO-ANCA and a very good one for P-ANCA and C-ANCA by classical and multiplex analysis (Cohen's kappa [κ] = 0.775, 0.720, 0.876, 0.820, respectively). The differences between classical testing and CytoBead analysis were not significant for PR3-ANCA, P-ANCA, and C-ANCA (p<0.05, respectively). The prevalence of confirmed positive ANCA findings by classical testing (IIF and ELISA) compared with multiplex CytoBead analysis (IIF and microbead immunoassay positive) resulted in a very good agreement (κ = 0.831) with no significant difference of both methods (p = 0.735). Automated endpoint-ANCA titer detection in one dilution demonstrated a very good agreement with classical analysis requiring dilution of samples (κ = 0.985). Multiplexing by CytoBead technology can be employed for simultaneous screening and quantitative confirmation of ANCA. This novel technique provides fast and cost-effective ANCA analysis by automated digital IIF for the first time.
    PLoS ONE 09/2014; 9(9):e107743. DOI:10.1371/journal.pone.0107743 · 3.53 Impact Factor
  • Source
  • [Show abstract] [Hide abstract]
    ABSTRACT: Microbead-based technologies represent elegant and versatile approaches for highly parallelized quantitative multiparameter assays. They also form the basis of various techniques for detection and quantification of nucleic acids and proteins. Nucleic acid-based methods include hybridization assays, solid-phase PCR, sequencing, and trapping assays. Microbead assays have been improved in the past decades and are now important tools in routine and point-of-care diagnostics as well as in life science. Its advances include low costs, low workload, high speed and high-throughput automation. The potential of microbead-based assays therefore is apparent, and commercial applications can be found in the detection and discrimination of single nucleotide polymorphism, of pathogens, and in trapping assays. This review provides an overview on microbead-based platforms for biosensing with a main focus on nucleic acid detection (including amplification strategies and on selected probe systems using fluorescent labeling). Specific sections cover chemical properties of microbeads, the coupling of targets onto solid surfaces, microbead probe systems (mainly oligonucleotide probes), microbead detection schemes (with subsections on suspension arrays, microfluidic devices, and immobilized microbeads), quantification of nucleic acids, PCR in solution and the detection of amplicons, and methods for solid-phase amplification. We discuss selected trends such as microbead-coupled amplification, heterogeneous and homogenous DNA hybridization assays, real-time assays, melting curve analysis, and digital microbead assays. We finally discuss the relevance and trends of the methods in terms of high-level multiplexed analysis and their potential in diagnosis and personalized medicine. Contains 211 references.
    Microchimica Acta 08/2014; 181(11-12):1-18. DOI:10.1007/s00604-014-1243-4 · 3.72 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Dear Editor, With interest we read the paper by Juste et al1 proposing the amount of zymogen-granule membrane glycoprotein 2 (GP2) on the surface of intestinal bacteria as a Crohn's disease (CD) marker. Indeed, a decreased GP2 level was found on microbes in patients with CD as compared to those of healthy controls. GP2 is a homologue to the urinary Tamm–Horsefall protein demonstrating an antimicrobial function by binding type 1-fimbriated uropathogenic Escherichia coli (UPEC). Likewise, GP2 seems to interact with intestinal bacteria as a specific receptor of bacterial type-1 fimbriae (FimH) on intestinal microfold cells that are partaking in immune responses against such microbes.2 GP2 is overexpressed in the inflamed intestine of patients with CD and has an immunomodulating role in innate and acquired immune responses.3 ,4 Interestingly, GP2 was identified as autoantigen of pancreatic antibodies in CD.4 Altogether, these findings indicate two major GP2 sources (pancreatic/intestinal) and support a role for GP2 in the interaction between the immune system and intestinal microbiota.3 Thus, loss of tolerance to GP2 could play a role in CD's pathophysiology supposed to be exacerbated by preceding intestinal infections. In general, the findings by Juste et al1 may be explained by a lower pancreatic GP2 secretion, an impaired GP2 binding to bacteria, or by a higher prevalence of bacteria with poor or no GP2 binding in patients with CD.
    Gut 07/2014; 64(3). DOI:10.1136/gutjnl-2014-307854 · 13.32 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The edition of this Special Issue was commenced in 2013 at the occasion of the 60th anniversary of the elucidation of DNA structure. This milestone has completely changed biological and medical sciences and, more recently, has triggered the development of sophisticated instrumentation. The discovery of the polymerase chain reaction (PCR) has further promoted this trend and now allows the analysis of a DNA sequences even at a level of a single molecule. Similarly, various miniaturized chip-based approaches have been introduced in the past 10 years. The transition from a laboratory scale to a microscale implies many advantages. These include, in particular, reduced sample volumes, reduced costs, shorter assay times, faster heating/cooling rates, higher throughput, and the integration of processing module cascades. The chip approach enabled, in particular, the development of sophisticated PCR assays. These include single-well and multi-well continuous flow PCR devices, stationary chamber-based PCR chips, and planar microbead-based chips. Most of these systems have common components such as miniaturized reaction chambers, heating/cooling units, and an analyzer/detector unit. Some of the devices evolved from the end-point quantification to the multiplex real-time monitoring of amplification reactions and high-resolution analysis of melting curves. These technologies contributed to breakthroughs that were envisioned years before. In particular, PCR in microfluidics and isothermal amplification have radically changed medical diagnostics and life sciences in general. Any development in field is based on the efforts of experts from highly different fields, examples being molecular biology, surface chemistry, microfluidics, and of course engineering. Systems are becoming smaller and more widely applicable, for example to diagnostics, environmental monitoring, and life sciences. This Special Issue gives a representative selection of the progress made in this field and in technologies related to affinity biosensors in a wider sense. The Issue includes reviews and original papers. Many of them are based on presentations given at the 7th Senftenberg Innovationsforum on Multiparameter Diagnostics in April 2013 which was organized by Brandenburg University of Technology Cottbus - Senftenberg (Germany). We have mainly included manuscripts on new analytical techniques, on end-user applications, new instrumentation, sensors and materials, with a main focus on the analysis of nucleic acids. The first part (4 reviews) gives an overview on current technologies such as extraction, amplification and detection of DNA in microfluidic chip-based and microbead-based assays. This includes recent developments in solid-phase enzymatic assays and the application in multiparametric diagnostics. The second part (Chip-based technologies for amplification, detection and analyses of nucleic acids) mainly focuses on end-point and real-time PCR techniques. Articles cover topics ranging from fundamental research to commercial applications. Part 3 (Application of chip-based technologies for amplification, detection and analyses) covers aspects of reference gene selection, quantification of gene expression, PCR in microfluidics, microarray technology in PCR, conventional and isothermal amplification methods as well as fluorescent detection. The final part (Chip-based technologies for affinity sensing) focuses on novel techniques for use in affinity sensing which have a wide application potential including analysis of nucleic acids. We are confident that this Special Issue represents a timely overview on this field and will fuel novel ideas for microanalytical systems. Last, but not least, we would like to thank the authors of this Special Issue for their excellent articles. We also are grateful to the reviewers for their numerous constructive comments on these manuscripts. We truly hope that readers will enjoy reading the result of this collective effort. V. M. Mirsky, S. Rödiger, P. Schierack, D. Roggenbuck (Editors) Brandenburg University of Technology, Cottbus – Senftenberg (Germany)
    Microchimica Acta 04/2014; 181(13-14). DOI:10.1007/s00604-014-1246-1 · 3.72 Impact Factor

Publication Stats

800 Citations
187.32 Total Impact Points

Institutions

  • 2014–2015
    • Brandenburg University of Technology Cottbus - Senftenberg
      • Faculty of Natural Sciences (Faculty 6)
      Kottbus, Brandenburg, Germany
  • 2005–2015
    • Freie Universität Berlin
      • Institute of Microbiology and Epizootics
      Berlín, Berlin, Germany
  • 2011–2013
    • Fachhochschule der Wirtschaft
      Paderborn, North Rhine-Westphalia, Germany
  • 2009
    • Bundesamt für Verbraucherschutz
      Brunswyck, Lower Saxony, Germany
  • 2006
    • Bundesinstitut für Risikobewertung
      Berlín, Berlin, Germany