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Sample records for detecting disease-associated genotype

  1. Utilising Family-Based Designs for Detecting Rare Variant Disease Associations

    PubMed Central

    Preston, Mark D; Dudbridge, Frank

    2014-01-01

    Summary Rare genetic variants are thought to be important components in the causality of many diseases but discovering these associations is challenging. We demonstrate how best to use family-based designs to improve the power to detect rare variant disease associations. We show that using genetic data from enriched families (those pedigrees with greater than one affected member) increases the power and sensitivity of existing case–control rare variant tests. However, we show that transmission- (or within-family-) based tests do not benefit from this enrichment. This means that, in studies where a limited amount of genotyping is available, choosing a single case from each of many pedigrees has greater power than selecting multiple cases from fewer pedigrees. Finally, we show how a pseudo-case–control design allows a greater range of statistical tests to be applied to family data. PMID:24571231

  2. Immunohistochemical characteristics of disease-associated PrP are not altered by host genotype or route of inoculation following infection of sheep with bovine spongiform encephalopathy.

    PubMed

    Martin, Stuart; González, Lorenzo; Chong, Angela; Houston, Fiona E; Hunter, Nora; Jeffrey, Martin

    2005-03-01

    It has previously been reported that disease-associated prion protein (PrP(d)) derived from natural scrapie and from sheep infected experimentally with bovine spongiform encephalopathy (BSE) differed in respect of their immunohistochemical and immunoblotting properties. For BSE, however, these initial observations were restricted to orally challenged sheep of the ARQ/ARQ PrP genotype. Here, extended examinations were performed on 28 sheep that developed neurological signs after BSE experimental infection by one of three routes. Intracerebrally infected ARQ/ARQ sheep showed more widespread and abundant accumulations of PrP(d) in tissues of the lymphoreticular system (LRS) than VRQ/VRQ animals, whereas no peripheral PrP(d) was detected in ARR/ARR sheep. The intensity and dissemination of PrP(d) accumulation in LRS tissues were less than those found previously in orally dosed sheep. AHQ/AHQ sheep challenged orally and ARQ/AHQ and ARQ/ARQ animals infected intravenously showed similar LRS-tissue PrP(d) distributions and levels to those of ARQ/ARQ sheep infected intracerebrally. The patterns of intra- and extracellular immunoreactivity to different PrP antibodies in brain and LRS tissues and the immunoblotting characteristics of PrP(res) from brain samples remained constant, irrespective of the route of inoculation and the PrP genotype, and were the same as described previously for ARQ/ARQ sheep dosed orally with BSE. These results suggest that the intracellular truncation of BSE PrP(d) and the proteinase K cleavage site of BSE PrP(res) are not altered by PrP genotype or by route of inoculation and that, therefore, screening tests based on these properties can be applied to identify potential sheep BSE cases occurring naturally.

  3. Metagenomic approaches to disclose disease-associated pathogens: detection of viral pathogens in honeybees.

    PubMed

    Granberg, Fredrik; Karlsson, Oskar E; Belák, Sándor

    2015-01-01

    Metagenomic approaches have become invaluable for culture-independent and sequence-independent detection and characterization of disease-associated pathogens. Here, the sequential steps from sampling to verification of results are described for a metagenomic-based approach to detect potential pathogens in honeybees. The pre-sequencing steps are given in detail, but due to the rapid development of sequencing technologies, all platform-specific procedures, as well as subsequent bioinformatics analysis, are more generally described. It should also be noted that this approach could, with minor modifications, be adapted for other organisms and sample matrices.

  4. Precise genotyping and recombination detection of Enterovirus

    PubMed Central

    2015-01-01

    Enteroviruses (EV) with different genotypes cause diverse infectious diseases in humans and mammals. A correct EV typing result is crucial for effective medical treatment and disease control; however, the emergence of novel viral strains has impaired the performance of available diagnostic tools. Here, we present a web-based tool, named EVIDENCE (EnteroVirus In DEep conception, http://symbiont.iis.sinica.edu.tw/evidence), for EV genotyping and recombination detection. We introduce the idea of using mixed-ranking scores to evaluate the fitness of prototypes based on relatedness and on the genome regions of interest. Using phylogenetic methods, the most possible genotype is determined based on the closest neighbor among the selected references. To detect possible recombination events, EVIDENCE calculates the sequence distance and phylogenetic relationship among sequences of all sliding windows scanning over the whole genome. Detected recombination events are plotted in an interactive figure for viewing of fine details. In addition, all EV sequences available in GenBank were collected and revised using the latest classification and nomenclature of EV in EVIDENCE. These sequences are built into the database and are retrieved in an indexed catalog, or can be searched for by keywords or by sequence similarity. EVIDENCE is the first web-based tool containing pipelines for genotyping and recombination detection, with updated, built-in, and complete reference sequences to improve sensitivity and specificity. The use of EVIDENCE can accelerate genotype identification, aiding clinical diagnosis and enhancing our understanding of EV evolution. PMID:26678286

  5. Detecting disease-associated genomic outcomes using constrained mixture of Bayesian hierarchical models for paired data

    PubMed Central

    Li, Yunfeng; Morrow, Jarrett; Raby, Benjamin; Tantisira, Kelan; Weiss, Scott T.; Huang, Wei

    2017-01-01

    Detecting disease-associated genomic outcomes is one of the key steps in precision medicine research. Cutting-edge high-throughput technologies enable researchers to unbiasedly test if genomic outcomes are associated with disease of interest. However, these technologies also include the challenges associated with the analysis of genome-wide data. Two big challenges are (1) how to reduce the effects of technical noise; and (2) how to handle the curse of dimensionality (i.e., number of variables are way larger than the number of samples). To tackle these challenges, we propose a constrained mixture of Bayesian hierarchical models (MBHM) for detecting disease-associated genomic outcomes for data obtained from paired/matched designs. Paired/matched designs can effectively reduce effects of confounding factors. MBHM does not involve multiple testing, hence does not have the problem of the curse of dimensionality. It also could borrow information across genes so that it can be used for whole genome data with small sample sizes. PMID:28358896

  6. Detection of the disease-associated form of the prion protein in biological samples.

    PubMed

    Nicholson, Eric M

    2015-01-01

    Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases that occur in a variety of mammals. In TSEs, a chromosomally encoded protein (PrPC) undergoes a conformational change to the disease-associated form (PrPd). PrPd is capable of inducing a change in additional molecules of PrPC to the PrPd conformation. TSEs are inevitably fatal and cross-species transmission is known to occur, and there is potential for transmission via blood transfusion and organ transplantation in humans. Thus, there is interest in high-quality diagnostics for both humans and animals. This review summarizes methods of TSE detection currently in use in diagnostic settings and discusses recent advances in PrPd detection that afford substantial enhancements in sensitivity over currently approved methods for use in clinical settings.

  7. Disease-associated prion protein detected in lymphoid tissues from pigs challenged with the agent of chronic wasting disease

    USDA-ARS?s Scientific Manuscript database

    Aims: Chronic wasting disease (CWD) is a naturally-occurring, fatal neurodegenerative disease of cervids. We previously demonstrated that disease-associated prion protein (PrPSc) can be detected in the brain and retina from pigs challenged intracranially or orally with the CWD agent. In that study,...

  8. Magnetic Resonance for Noninvasive Detection of Microcirculatory Disease Associated With Allograft Vasculopathy: Intracoronary Measurement Validation.

    PubMed

    Mirelis, Jesús G; García-Pavía, Pablo; Cavero, Miguel A; González-López, Esther; Echavarria-Pinto, Mauro; Pastrana, Miguel; Segovia, Javier; Oteo, Juan F; Alonso-Pulpón, Luis; Escaned, Javier

    2015-07-01

    comprehensive dobutamine stress magnetic resonance appears to be a reliable technique for noninvasive detection of microcirculatory coronary disease associated with cardiac allograft vasculopathy. Copyright © 2014 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.

  9. Detection of the disease associated form of the prion protein in biological samples

    USDA-ARS?s Scientific Manuscript database

    Transmissible spongiform encephalopathies (TSEs) or prion diseases are neurodegenerative diseases that occur in a variety of mammals. In these diseases, a chromosomally encoded protein (PrP**c) undergoes a conformational change to the disease associated form (PrP**d), and PrP**d is capable inducing ...

  10. SNP marker detection and genotyping in tilapia.

    PubMed

    Van Bers, N E M; Crooijmans, R P M A; Groenen, M A M; Dibbits, B W; Komen, J

    2012-09-01

    We have generated a unique resource consisting of nearly 175 000 short contig sequences and 3569 SNP markers from the widely cultured GIFT (Genetically Improved Farmed Tilapia) strain of Nile tilapia (Oreochromis niloticus). In total, 384 SNPs were selected to monitor the wider applicability of the SNPs by genotyping tilapia individuals from different strains and different geographical locations. In all strains and species tested (O. niloticus, O. aureus and O. mossambicus), the genotyping assay was working for a similar number of SNPs (288-305 SNPs). The actual number of polymorphic SNPs was, as expected, highest for individuals from the GIFT population (255 SNPs). In the individuals from an Egyptian strain and in individuals caught in the wild in the basin of the river Volta, 197 and 163 SNPs were polymorphic, respectively. A pairwise calculation of Nei's genetic distance allowed the discrimination of the individual strains and species based on the genotypes determined with the SNP set. We expect that this set will be widely applicable for use in tilapia aquaculture, e.g. for pedigree reconstruction. In addition, this set is currently used for assaying the genetic diversity of native Nile tilapia in areas where tilapia is, or will be, introduced in aquaculture projects. This allows the tracing of escapees from aquaculture and the monitoring of effects of introgression and hybridization.

  11. Niche harmony search algorithm for detecting complex disease associated high-order SNP combinations.

    PubMed

    Tuo, Shouheng; Zhang, Junying; Yuan, Xiguo; He, Zongzhen; Liu, Yajun; Liu, Zhaowen

    2017-09-14

    Genome-wide association study is especially challenging in detecting high-order disease-causing models due to model diversity, possible low or even no marginal effect of the model, and extraordinary search and computations. In this paper, we propose a niche harmony search algorithm where joint entropy is utilized as a heuristic factor to guide the search for low or no marginal effect model, and two computationally lightweight scores are selected to evaluate and adapt to diverse of disease models. In order to obtain all possible suspected pathogenic models, niche technique merges with HS, which serves as a taboo region to avoid HS trapping into local search. From the resultant set of candidate SNP-combinations, we use G-test statistic for testing true positives. Experiments were performed on twenty typical simulation datasets in which 12 models are with marginal effect and eight ones are with no marginal effect. Our results indicate that the proposed algorithm has very high detection power for searching suspected disease models in the first stage and it is superior to some typical existing approaches in both detection power and CPU runtime for all these datasets. Application to age-related macular degeneration (AMD) demonstrates our method is promising in detecting high-order disease-causing models.

  12. Detection of Mendelian consistent genotyping errors in pedigrees.

    PubMed

    Cheung, Charles Y K; Thompson, Elizabeth A; Wijsman, Ellen M

    2014-05-01

    Detection of genotyping errors is a necessary step to minimize false results in genetic analysis. This is especially important when the rate of genotyping errors is high, as has been reported for high-throughput sequence data. To detect genotyping errors in pedigrees, Mendelian inconsistent (MI) error checks exist, as do multi-point methods that flag Mendelian consistent (MC) errors for sparse multi-allelic markers. However, few methods exist for detecting MC genotyping errors, particularly for dense variants on large pedigrees. Here, we introduce an efficient method to detect MC errors even for very dense variants (e.g., SNPs and sequencing data) on pedigrees that may be large. Our method first samples inheritance vectors (IVs) using a moderately sparse but informative set of markers using a Markov chain Monte Carlo-based sampler. Using sampled IVs, we considered two test statistics to detect MC genotyping errors: the percentage of IVs inconsistent with observed genotypes (A1) or the posterior probability of error configurations (A2). Using simulations, we show that this method, even with the simpler A1 statistic, is effective for detecting MC genotyping errors in dense variants, with sensitivity almost as high as the theoretical best sensitivity possible. We also evaluate the effectiveness of this method as a function of parameters, when including the observed pattern for genotype, density of framework markers, error rate, allele frequencies, and number of sampled inheritance vectors. Our approach provides a line of defense against false findings based on the use of dense variants in pedigrees. © 2014 WILEY PERIODICALS, INC.

  13. Recommendations for Screening and Detection of Connective-Tissue Disease Associated Pulmonary Arterial Hypertension

    PubMed Central

    Khanna, Dinesh; Gladue, Heather; Channick, Richard; Chung, Lorinda; Distler, Oliver; Furst, Daniel E.; Hachulla, Eric; Humbert, Marc; Langleben, David; Mathai, Stephen C.; Saggar, Rajeev; Visovatti, Scott; Altorok, Nezam; Townsend, Whitney; FitzGerald, John; McLaughlin, Vallerie

    2013-01-01

    Objectives Pulmonary arterial hypertension (PAH) affects up to 15% of patients with connective tissue diseases (CTD). Previous recommendations developed as part of larger efforts in PAH did not provide detailed recommendations for patients with CTD-PAH. Therefore, we sought to develop recommendations for screening and early detection of CTD-PAH. Methods We performed a systematic review for the screening and diagnosis of PAH in CTD by searching the literature. Using the RAND/UCLA methodology, we developed case scenarios followed by 2 stages of voting—first international experts from a variety of specialties voted anonymously on the appropriateness of each case scenario and then the experts met in a face-to-face meeting to discuss and resolve discrepant votes to arrive at consensus recommendations. Results The key recommendations state that patients with systemic sclerosis (SSc) should be screened for PAH. In addition, mixed connective tissue diseases (MCTD) or other CTD’s with scleroderma features should also be screened for PAH (scleroderma-spectrum disorder). Initial screening evaluation in patients with SSc and scleroderma-spectrum disorders include pulmonary function test (PFT) including diffusion capacity carbon monoxide (DLCO), transthoracic echocardiogram (TTE), and NT- Pro BNP. In SSc and spectrum disorders, TTE and PFT should be performed on annual basis. The full screening panel (TTE, PFT, and NT-ProBNP) should be performed as soon as any new signs or symptoms are present. Conclusion We provide consensus-based, evidence-driven recommendations for screening and early detection of CTD-PAH. It is our hope that these recommendations will lead to earlier detection of CTD-PAH and ultimately improve patient outcomes. PMID:24022584

  14. Disulfide-modified antigen for detection of celiac disease-associated anti-tissue transglutaminase autoantibodies.

    PubMed

    Rosales-Rivera, Luis Carlos; Dulay, Samuel; Lozano-Sánchez, Pablo; Katakis, Ioanis; Acero-Sánchez, Josep Lluís; O'Sullivan, Ciara K

    2017-03-29

    A simple and rapid immunosensor for the determination of the celiac disease-related antibody, anti-tissue transglutaminase, was investigated. The antigenic protein tissue transglutaminase was chemically modified, introducing disulfide groups through different moieties of the molecule (amine, carboxylic, and hydroxyl groups), self-assembled on gold surfaces, and used for the detection of IgA and IgG autoantibodies. The modified proteins were evaluated using enzyme-linked immunosorbent assay and surface plasmon resonance, which showed that only introduction of the disulfide groups through amine moieties in the tissue transglutaminase preserved its antigenic properties. The disulfide-modified antigen was co-immobilized via chemisorption with a poly(ethylene glycol) alkanethiol on gold electrodes. The modified electrodes were then exposed to IgA anti-tissue transglutaminase antibodies and subsequently to horseradish peroxidase-labeled anti-idiotypic antibodies, achieving a detection limit of 260 ng ml(-1). Immunosensor performance in the presence of complex matrixes, including clinically relevant serum reference solutions and real patient samples, was evaluated. The introduction of disulfides in the antigenic protein enabled a simple and convenient one-step surface immobilization procedure involving only spontaneous gold-thiol covalent binding. Complete amperometric assay time was 30 min.

  15. Kullback-Leibler divergence for detection of rare haplotype common disease association.

    PubMed

    Lin, Shili

    2015-11-01

    Rare haplotypes may tag rare causal variants of common diseases; hence, detection of such rare haplotypes may also contribute to our understanding of complex disease etiology. Because rare haplotypes frequently result from common single-nucleotide polymorphisms (SNPs), focusing on rare haplotypes is much more economical compared with using rare single-nucleotide variants (SNVs) from sequencing, as SNPs are available and 'free' from already amassed genome-wide studies. Further, associated haplotypes may shed light on the underlying disease causal mechanism, a feat unmatched by SNV-based collapsing methods. In recent years, data mining approaches have been adapted to detect rare haplotype association. However, as they rely on an assumed underlying disease model and require the specification of a null haplotype, results can be erroneous if such assumptions are violated. In this paper, we present a haplotype association method based on Kullback-Leibler divergence (hapKL) for case-control samples. The idea is to compare haplotype frequencies for the cases versus the controls by computing symmetrical divergence measures. An important property of such measures is that both the frequencies and logarithms of the frequencies contribute in parallel, thus balancing the contributions from rare and common, and accommodating both deleterious and protective, haplotypes. A simulation study under various scenarios shows that hapKL has well-controlled type I error rates and good power compared with existing data mining methods. Application of hapKL to age-related macular degeneration (AMD) shows a strong association of the complement factor H (CFH) gene with AMD, identifying several individual rare haplotypes with strong signals.

  16. Probability of detecting disease-associated single nucleotide polymorphisms in case-control genome-wide association studies.

    PubMed

    Gail, Mitchell H; Pfeiffer, Ruth M; Wheeler, William; Pee, David

    2008-04-01

    Some case-control genome-wide association studies (CCGWASs) select promising single nucleotide polymorphisms (SNPs) by ranking corresponding p-values, rather than by applying the same p-value threshold to each SNP. For such a study, we define the detection probability (DP) for a specific disease-associated SNP as the probability that the SNP will be "T-selected," namely have one of the top T largest chi-square values (or smallest p-values) for trend tests of association. The corresponding proportion positive (PP) is the fraction of selected SNPs that are true disease-associated SNPs. We study DP and PP analytically and via simulations, both for fixed and for random effects models of genetic risk, that allow for heterogeneity in genetic risk. DP increases with genetic effect size and case-control sample size and decreases with the number of nondisease-associated SNPs, mainly through the ratio of T to N, the total number of SNPs. We show that DP increases very slowly with T, and the increment in DP per unit increase in T declines rapidly with T. DP is also diminished if the number of true disease SNPs exceeds T. For a genetic odds ratio per minor disease allele of 1.2 or less, even a CCGWAS with 1000 cases and 1000 controls requires T to be impractically large to achieve an acceptable DP, leading to PP values so low as to make the study futile and misleading. We further calculate the sample size of the initial CCGWAS that is required to minimize the total cost of a research program that also includes follow-up studies to examine the T-selected SNPs. A large initial CCGWAS is desirable if genetic effects are small or if the cost of a follow-up study is large.

  17. High-Throughput Variation Detection and Genotyping Using Microarrays

    PubMed Central

    Cutler, David J.; Zwick, Michael E.; Carrasquillo, Minerva M.; Yohn, Christopher T.; Tobin, Katherine P.; Kashuk, Carl; Mathews, Debra J.; Shah, Nila A.; Eichler, Evan E.; Warrington, Janet A.; Chakravarti, Aravinda

    2001-01-01

    The genetic dissection of complex traits may ultimately require a large number of SNPs to be genotyped in multiple individuals who exhibit phenotypic variation in a trait of interest. Microarray technology can enable rapid genotyping of variation specific to study samples. To facilitate their use, we have developed an automated statistical method (ABACUS) to analyze microarray hybridization data and applied this method to Affymetrix Variation Detection Arrays (VDAs). ABACUS provides a quality score to individual genotypes, allowing investigators to focus their attention on sites that give accurate information. We have applied ABACUS to an experiment encompassing 32 autosomal and eight X-linked genomic regions, each consisting of ∼50 kb of unique sequence spanning a 100-kb region, in 40 humans. At sufficiently high-quality scores, we are able to read ∼80% of all sites. To assess the accuracy of SNP detection, 108 of 108 SNPs have been experimentally confirmed; an additional 371 SNPs have been confirmed electronically. To access the accuracy of diploid genotypes at segregating autosomal sites, we confirmed 1515 of 1515 homozygous calls, and 420 of 423 (99.29%) heterozygotes. In replicate experiments, consisting of independent amplification of identical samples followed by hybridization to distinct microarrays of the same design, genotyping is highly repeatable. In an autosomal replicate experiment, 813,295 of 813,295 genotypes are called identically (including 351 heterozygotes); at an X-linked locus in males (haploid), 841,236 of 841,236 sites are called identically. PMID:11691856

  18. reGenotyper: Detecting mislabeled samples in genetic data

    PubMed Central

    Snoek, Basten L.; Elvin, Mark; Rodriguez, Miriam; Arends, Danny; Swertz, Morris A.; Kammenga, Jan E.; Li, Yang

    2017-01-01

    In high-throughput molecular profiling studies, genotype labels can be wrongly assigned at various experimental steps; the resulting mislabeled samples seriously reduce the power to detect the genetic basis of phenotypic variation. We have developed an approach to detect potential mislabeling, recover the “ideal” genotype and identify “best-matched” labels for mislabeled samples. On average, we identified 4% of samples as mislabeled in eight published datasets, highlighting the necessity of applying a “data cleaning” step before standard data analysis. PMID:28192439

  19. Universal SNP genotyping assay with fluorescence polarization detection.

    PubMed

    Hsu, T M; Chen, X; Duan, S; Miller, R D; Kwok, P Y

    2001-09-01

    The degree of fluorescence polarization (FP) of a fluorescent molecule is a reflection of its molecular weight (Mr). FP is therefore a useful detection methodfor homogeneous assays in which the starting reagents and products differ significantly in Mr. We have previously shown that FP is a good detection method for the single-base extension and the 5'-nuclease assays. In this report, we describe a universal, optimized single-base extension assay for genotyping single nucleotide polymorphisms (SNPs). This assay, which we named the template-directed dye-terminator incorporation assay with fluorescence polarization detection (FP-TDI), uses four spectrally distinct dye terminators to achieve universal assay conditions. Even without optimization, approximately 70% of all SNP markers tested yielded robust assays. The addition of an E. coli ssDNA-binding protein just before the FP reading significantly increased FP values of the products and brought the success rate of FP-TDI assays up to 90%. Increasing the amount of dye terminators and reducing the number of thermal cycles in the single-base extension step of the assay increased the separation of the FP values benveen the products corresponding to different genotypes and improved the success rate of the assay to 100%. In this study the genomic DNA samples of 90 individuals were typed for a total of 38 FP-TDI assays (using both the sense and antisense TDI primers for 19 SNP markers). With the previously described modifications, the FP-TDI assay gave unambiguous genotyping data for all the samples tested in the 38 FP-TDI assays. When the genotypes determined by the FP-TDI and 5'-nuclease assays were compared, they were in 100% concordance for all experiments (a total of 3420 genotypes). The four-dye-terminator master mixture described here can be used for assaying any SNP marker and greatly simplifies the SNP genotyping assay design.

  20. Detection and integration of genotyping errors in statistical genetics.

    PubMed

    Sobel, Eric; Papp, Jeanette C; Lange, Kenneth

    2002-02-01

    Detection of genotyping errors and integration of such errors in statistical analysis are relatively neglected topics, given their importance in gene mapping. A few inopportunely placed errors, if ignored, can tremendously affect evidence for linkage. The present study takes a fresh look at the calculation of pedigree likelihoods in the presence of genotyping error. To accommodate genotyping error, we present extensions to the Lander-Green-Kruglyak deterministic algorithm for small pedigrees and to the Markov-chain Monte Carlo stochastic algorithm for large pedigrees. These extensions can accommodate a variety of error models and refrain from simplifying assumptions, such as allowing, at most, one error per pedigree. In principle, almost any statistical genetic analysis can be performed taking errors into account, without actually correcting or deleting suspect genotypes. Three examples illustrate the possibilities. These examples make use of the full pedigree data, multiple linked markers, and a prior error model. The first example is the estimation of genotyping error rates from pedigree data. The second-and currently most useful-example is the computation of posterior mistyping probabilities. These probabilities cover both Mendelian-consistent and Mendelian-inconsistent errors. The third example is the selection of the true pedigree structure connecting a group of people from among several competing pedigree structures. Paternity testing and twin zygosity testing are typical applications.

  1. Detecting tissue-specific alternative splicing and disease-associated aberrant splicing of the PTCH gene with exon junction microarrays.

    PubMed

    Nagao, Kazuaki; Togawa, Naoyuki; Fujii, Katsunori; Uchikawa, Hideki; Kohno, Yoichi; Yamada, Masao; Miyashita, Toshiyuki

    2005-11-15

    Mutations in the human ortholog of Drosophila patched (PTCH) have been identified in patients with autosomal dominant nevoid basal cell carcinoma syndrome (NBCCS), characterized by minor developmental anomalies and an increased incidence of cancers such as medulloblastoma and basal cell carcinoma. We identified many isoforms of PTCH mRNA involving exons 1-5, exon 10 and a novel exon, 12b, generated by alternative splicing (AS), most of which have not been deposited in GenBank nor discussed earlier. To monitor splicing events of the PTCH gene, we designed oligonucleotide arrays on which exon probes and exon-exon junction probes as well as a couple of intron probes for the PTCH gene were placed in duplicate. Probe intensities were normalized on the basis of the total expression of PTCH and probe sensitivity. Tissue-specific regulation of AS identified with the microarrays closely correlated with the results obtained by RT-PCR. Of note, the novel exon, exon 12b, was specifically expressed in the brain and heart, especially in the cerebellum. Additionally, using these microarrays, we were able to detect disease-associated aberrant splicings of the PTCH gene in two patients with NBCCS. In both cases, cryptic splice donor sites located either in an exon or in an intron were activated because of the partial disruption of the consensus sequence for the authentic splice donor sites due to point mutations. Taken together, oligonucleotide microarrays containing exon junction probes are demonstrated to be a powerful tool to investigate tissue-specific regulation of AS and aberrant splicing taking place in genetic disorders.

  2. The Risk of Coronary Heart Disease Associated With Glycosylated Hemoglobin ≥ 6.5% is Pronounced in the Haptoglobin 2-2 Genotype

    PubMed Central

    Cahill, Leah E; Jensen, Majken K; Chiuve, Stephanie E; Shalom, Hadar; Pai, Jennifer K; Flint, Alan J; Mukamal, Kenneth J; Rexrode, Kathryn M; Levy, Andrew P; Rimm, Eric B

    2015-01-01

    BACKGROUND Research targeting glycosylated hemoglobin (HbA1c) to < 6.5% to prevent coronary heart disease (CHD) events has conflicting results. We previously observed the haptoglobin (Hp) Hp2-2 genotype is associated with a ~10-fold increased CHD risk among individuals with HbA1c ≥ 6.5%, and thus might be useful in identifying those at high risk of CHD who would benefit from maintaining HbA1c < 6.5%. OBJECTIVES We modeled whether HbA1c ≥ 6.5% in the Hp2-2 genotype is associated with CHD in a prospective case-control study nested within the Health Professionals Follow-Up Study (HPFS). METHODS HbA1c concentration and Hp genotype were determined for 695 incident cases of CHD from 1994–2010 and matched controls. Logistic regression models calculated relative risk (RR) and 95% confidence intervals (CI), for the first and second halves of follow-up, adjusting for confounding variables. A dataset from the Nurses’ Health Study (NHS) served as a replication cohort. RESULTS Prevalence of Hp2-2 genotype in HPFS was 39%. Compared to HbA1c < 6.5%, RR of CHD for HbA1c ≥ 6.5% for the Hp2-2 genotype over full follow-up was 3.07 (1.37–6.86)-- 3.88 (1.31–11.52) during the first half of follow-up and 2.16 (0.61–7.61) in the second half. The corresponding RRs (95% CI) for the Hp1-1 + Hp2-1 genotypes were 2.19 (1.14–4.24) (full follow-up), 1.60 (0.73–3.53) (first half), and 4.72 (1.26–17.65) (second half). CONCLUSIONS In 2 independent cohorts, risk of CHD associated with HbA1c ≥ 6.5% is pronounced in the Hp2-2 genotype, particularly in early cases. The Hp2-2 genotype may identify individuals at greatest CHD risk from hyperglycemia. PMID:26483103

  3. Detection of genotype recycling fraud in U.S. immigrants.

    PubMed

    Wenk, Robert E

    2011-01-01

    Relationship testing laboratories provide genetic evidence to support or refute claims of kinship between U.S. citizen petitioners and potential immigrant beneficiaries. One female beneficiary presented a male amelogenin type and alleles at 15 autosomal loci that were identical to an alleged brother's. Laboratory records showed that her alleged father had petitioned to have 15 children emigrate from Ghana. The petitioner's 15 paternity indices exceeded 10⁵, but the children shared only four short tandem repeat (STR) profiles, suggesting fraudulent reuse of genotypes in this alleged pedigree (AP). To determine the extent of this "genotype recycling," I examined the laboratory's 555 APs from Ghana and 532 control APs from Nigeria. Seventeen Ghanaian APs (3.1%) but no Nigerian APs showed genotype recycling. Of 90 tested people in the 17 APs, 56 shared identical STR profiles with others in their AP. Of these 56 people, 10 were petitioners with unexpectedly high parentage indices. Seven of 56 had amelogenin types that disagreed with their declared genders. Database searches for identical multilocus genotypes in allegedly different people would best detect this fraud.

  4. Detection of a novel genotype of Cryptosporidium in Antarctic pinnipeds.

    PubMed

    Rengifo-Herrera, Claudia; Ortega-Mora, Luis Miguel; Gómez-Bautista, Mercedes; García-Peña, Francisco Javier; García-Párraga, Daniel; Pedraza-Díaz, Susana

    2013-01-16

    A study was conducted to investigate the presence of Cryptosporidium and Giardia in Antarctic marine mammals. A total of 270 faecal samples from different species of pinnipeds from different locations in the South Shetland Islands and Antarctic Peninsula were analysed by immunofluorescence microscopy and PCR. Cryptosporidium was detected by PCR in three samples from Southern elephant seals (Mirounga leonina) and 2 Weddell seals (Leptonychotes weddellii). However, no oocysts were observed in any of the samples by immunofluorescence microscopy. Molecular characterisation of the isolates, using the 18S rDNA, the HSP70 and the COWP loci, revealed the presence of a Cryptosporidium sp., previously reported from an Antarctic Southern elephant seal, in the elephant seals and a novel genotype in Weddell seals. Giardia could not be detected in any of the samples analysed.

  5. High-resolution HLA-DRB1 genotyping in an Australian inclusion body myositis (s-IBM) cohort: an analysis of disease-associated alleles and diplotypes.

    PubMed

    Rojana-udomsart, Arada; James, Ian; Castley, Alison; Needham, Merrilee; Scott, Adrian; Day, Timothy; Kiers, Lynette; Corbett, Alastair; Sue, Carolyn; Witt, Campbell; Martinez, Patricia; Christiansen, Frank; Mastaglia, Frank

    2012-09-15

    We performed high-resolution (4-digit) HLA-DRB1 genotyping in an Australian cohort of 105s-IBM patients and 189 controls. Our findings showed that whilst the strongest association was with the HLA-DRB1*03:01 allele and the HLA-DRB1*03:01/*01:01 diplotype, HLA-DRB1*01:01 and HLA-DRB1*13:01 are also risk alleles. A number of other alleles, HLA-DRB1*04:01, *04:04, *07:01, *09:01, *11:01 and *15:01, as well as the HLA-DRB1*03:01/*04:01 and HLA-DRB1*03:01/*07:01 diplotypes were reduced in s-IBM cases and may be protective. The HLA-DRB1*03:01 and HLA-DRB1*13:01 alleles also appear to have an influence on the age at onset of the disease and severity of muscle weakness. Our findings indicate that the influence of HLA-DRB1 in s-IBM is complex and that epistatic interactions at the HLA-DRB1 locus contribute both to disease susceptibility and to the clinical phenotype. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. American Behcet's Disease Association

    MedlinePlus

    ... sponsors, Larry and Terri Gury. American Behcet's Disease Association PO BOX 80576 Rochester, MI 48308 Contact Us | Website Policy | webmaster@behcets.com American Behcet's Disease Association Copyright 2014

  7. Performance comparison of the versant HCV genotype 2.0 assay (LiPA) and the abbott realtime HCV genotype II assay for detecting hepatitis C virus genotype 6.

    PubMed

    Yang, Ruifeng; Cong, Xu; Du, Shaocai; Fei, Ran; Rao, Huiying; Wei, Lai

    2014-10-01

    The Versant HCV genotype 2.0 assay (line probe assay [LiPA] 2.0), based on reverse hybridization, and the Abbott Realtime HCV genotype II assay (Realtime II), based on genotype-specific real-time PCR, have been widely used to analyze hepatitis C virus (HCV) genotypes. However, their performances for detecting HCV genotype 6 infections have not been well studied. Here, we analyzed genotype 6 in 63 samples from the China HCV Genotyping Study that were originally identified as genotype 6 using the LiPA 2.0. The genotyping results were confirmed by nonstructural 5B (NS5B) or core sequence phylogenetic analysis. A total of 57 samples were confirmed to be genotype 6 (51 genotype 6a, 5 genotype 6n, and 1 genotype 6e). Four samples identified as a mixture of genotypes 6 and 4 by the LiPA 2.0 were confirmed to be genotype 3b. The remaining two samples classified as genotype 6 by the LiPA 2.0 were confirmed to be genotype 1b, which were intergenotypic recombinants and excluded from further comparison. In 57 genotype 6 samples detected using the Realtime II version 2.00 assay, 47 genotype 6a samples were identified as genotype 6, one 6e sample was misclassified as genotype 1, and four 6a and five 6n samples yielded indeterminate results. Nine nucleotide profiles in the 5' untranslated region affected the performances of both assays. Therefore, our analysis shows that both assays have limitations in identifying HCV genotype 6. The LiPA 2.0 cannot distinguish some 3b samples from genotype 6 samples. The Realtime II assay fails to identify some 6a and all non-6a subtypes, and it misclassifies genotype 6e as genotype 1. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  8. Performance Comparison of the Versant HCV Genotype 2.0 Assay (LiPA) and the Abbott Realtime HCV Genotype II Assay for Detecting Hepatitis C Virus Genotype 6

    PubMed Central

    Yang, Ruifeng; Cong, Xu; Du, Shaocai; Fei, Ran; Rao, Huiying

    2014-01-01

    The Versant HCV genotype 2.0 assay (line probe assay [LiPA] 2.0), based on reverse hybridization, and the Abbott Realtime HCV genotype II assay (Realtime II), based on genotype-specific real-time PCR, have been widely used to analyze hepatitis C virus (HCV) genotypes. However, their performances for detecting HCV genotype 6 infections have not been well studied. Here, we analyzed genotype 6 in 63 samples from the China HCV Genotyping Study that were originally identified as genotype 6 using the LiPA 2.0. The genotyping results were confirmed by nonstructural 5B (NS5B) or core sequence phylogenetic analysis. A total of 57 samples were confirmed to be genotype 6 (51 genotype 6a, 5 genotype 6n, and 1 genotype 6e). Four samples identified as a mixture of genotypes 6 and 4 by the LiPA 2.0 were confirmed to be genotype 3b. The remaining two samples classified as genotype 6 by the LiPA 2.0 were confirmed to be genotype 1b, which were intergenotypic recombinants and excluded from further comparison. In 57 genotype 6 samples detected using the Realtime II version 2.00 assay, 47 genotype 6a samples were identified as genotype 6, one 6e sample was misclassified as genotype 1, and four 6a and five 6n samples yielded indeterminate results. Nine nucleotide profiles in the 5′ untranslated region affected the performances of both assays. Therefore, our analysis shows that both assays have limitations in identifying HCV genotype 6. The LiPA 2.0 cannot distinguish some 3b samples from genotype 6 samples. The Realtime II assay fails to identify some 6a and all non-6a subtypes, and it misclassifies genotype 6e as genotype 1. PMID:25100817

  9. Wilson's Disease Association International

    MedlinePlus

    Administration Connect with Wilson Disease Association Send Email Physician Contacts List of Physicians and Institutions in Your Area View Contacts Support Contacts Individuals who can offer Support ...

  10. Detection and genotyping of enteroviruses in cerebrospinal fluid in patients in Victoria, Australia, 2007-2013.

    PubMed

    Papadakis, Georgina; Chibo, Doris; Druce, Julian; Catton, Michael; Birch, Chris

    2014-09-01

    Genotyping by VP1 fragment polymerase chain reaction (PCR) and nucleic acid sequencing to detect enterovirus (EV) genotypes was performed directly on 729 EV PCR positive cerebrospinal fluid (CSF) samples collected between 2007 and 2012 from Victorian hospital inpatients. The overall genotype identification rate from CSF-positive material was 43%. The four most common genotypes identified were Echovirus 6 (24%), Echovirus 30 (17%), Echovirus 25 (10%), and Coxsackievirus A9 (10%), together comprising 61% of all EVs typed. The seasonal distribution of all EVs identified followed the recognized pattern of mainly summer epidemics. Three of the four predominant genotypes were present in each of the 6 years in which the study was conducted, with 20 other EV genotypes also detected, often in only a single year. Genotyping of EVs directly in CSF is faster, simpler and more sensitive than traditional virus neutralization assays performed on EV positive samples.

  11. Rapid SNP Detection and Genotyping of Bacterial Pathogens by Pyrosequencing.

    PubMed

    Amoako, Kingsley K; Thomas, Matthew C; Janzen, Timothy W; Goji, Noriko

    2017-01-01

    Bacterial identification and typing are fixtures of microbiology laboratories and are vital aspects of our response mechanisms in the event of foodborne outbreaks and bioterrorist events. Whole genome sequencing (WGS) is leading the way in terms of expanding our ability to identify and characterize bacteria through the identification of subtle differences between genomes (e.g. single nucleotide polymorphisms (SNPs) and insertions/deletions). Modern high-throughput technologies such as pyrosequencing can facilitate the typing of bacteria by generating short-read sequence data of informative regions identified by WGS analyses, at a fraction of the cost of WGS. Thus, pyrosequencing systems remain a valuable asset in the laboratory today. Presented in this chapter are two methods developed in the Amoako laboratory that detail the identification and genotyping of bacterial pathogens. The first targets canonical single nucleotide polymorphisms (canSNPs) of evolutionary importance in Bacillus anthracis, the causative agent of Anthrax. The second assay detects Shiga-toxin (stx) genes, which are associated with virulence in Escherichia coli and Shigella spp., and differentiates the subtypes of stx-1 and stx-2 based on SNP loci. These rapid methods provide end users with important information regarding virulence traits as well as the evolutionary and biogeographic origin of isolates.

  12. Total Coronary Atherosclerotic Plaque Burden Assessment by CT Angiography for Detecting Obstructive Coronary Artery Disease Associated with Myocardial Perfusion Abnormalities

    PubMed Central

    Kishi, Satoru; Magalhães, Tiago A.; Cerci, Rodrigo J.; Matheson, Matthew B.; Vavere, Andrea; Tanami, Yutaka; Kitslaar, Pieter H.; George, Richard T.; Brinker, Jeffrey; Miller, Julie M.; Clouse, Melvin E.; Lemos, Pedro A.; Niinuma, Hiroyuki; Reiber, Johan H.C.; Rochitte, Carlos E.; Rybicki, Frank J.; Di Carli, Marcelo F.; Cox, Christopher; Lima, Joao A.C.; Arbab-Zadeh, Armin

    2016-01-01

    Background Total atherosclerotic plaque burden assessment by CT angiography (CTA) is a promising tool for diagnosis and prognosis of coronary artery disease (CAD) but its validation is restricted to small clinical studies. We tested the feasibility of semi-automatically derived coronary atheroma burden assessment for identifying patients with hemodynamically significant CAD in a large cohort of patients with heterogenous characteristics. Methods This study focused on the CTA component of the CORE320 study population. A semi-automated contour detection algorithm quantified total coronary atheroma volume defined as the difference between vessel and lumen volume. Percent atheroma volume (PAV = [total atheroma volume/total vessel volume]×100) was the primary metric for assessment (n=374). The area under the receiver operating characteristic curve (AUC) determined the diagnostic accuracy for identifying patients with hemodynamically significant CAD defined as ≥50% stenosis by quantitative coronary angiography and associated myocardial perfusion abnormality by SPECT. Results Of 374 patients, 139 (37%) had hemodynamically significant CAD. The AUC for PAV was 0.78 (95% confidence interval [CI] 0.73–0.83) compared to 0.84 [0.79–0.88] by standard expert CTA interpretation (p=0.02). Accuracy for both CTA (0.91 [0.87, 0.96]) and PAV (0.86 [0.81–0.91]) increased after excluding patients with history of CAD (p<0.01 for both). Bland-Altman analysis revealed good agreement between two observers ( bias of 280.2 mm3 [161.8, 398.7]). Conclusions A semi-automatically derived index of total coronary atheroma volume yields good accuracy for identifying patients with hemodynamically significant CAD, though marginally inferior to CTA expert reading. These results convey promise for rapid, reliable evaluation of clinically relevant CAD. PMID:26817414

  13. Kennedy's Disease Association

    MedlinePlus

    ... Disease Association A Public Benefit, Non-Profit Organization Register GTranslate GTranslate Javascript is required to use GTranslate multilingual website and translation delivery network Select Language English Afrikaans Albanian Arabic Armenian Azerbaijani Basque Belarusian ...

  14. Kennedy's Disease Association

    MedlinePlus

    Kennedy's Disease Association A Public Benefit, Non-Profit Organization Register GTranslate GTranslate Javascript is required to use ... worldwide. Males generally inherit the disease symptoms and females are the carriers. Help Us Find A Cure! ...

  15. Detection of disease-associated prion protein in the optic nerve and the adrenal gland of cattle with bovine spongiform encephalopathy by using highly sensitive immunolabeling procedures.

    PubMed

    Okada, Hiroyuki; Iwamaru, Yoshifumi; Fukuda, Shigeo; Yokoyama, Takashi; Mohri, Shirou

    2012-04-01

    A sensitive immunohistochemical procedure, the tyramide signal amplification (TSA) system, was applied to detect the localization of immunolabeled disease-associated prion protein (PrP(Sc)) in cattle affected with bovine spongiform encephalopathy (BSE). In this procedure, immunolabeling could be visualized in the optic nerve and the adrenal medulla. In the optic nerve, the dual immunofluorescent technique showed that the granular PrP(Sc) was occasionally detected in the astrocytes, microglia, and myelin sheath adjacent to the axon. Clustered PrP(Sc) was also scattered in association with microglial cells and astrocytes of the optic nerve. In the adrenal gland, PrP(Sc) immunolabeling was confined within the sympathetic nerve fibers and endings. The results suggest that (1) PrP(Sc) might centrifugally spread within and between glial cells and/or the non-axonal (also known as ad-axonal) region of nerve fibers, rather than the axonal and/or extracellular space pathway in the optic nerve, and (2) the sympathetic innervations might be important for the trafficking of BSE agent in the adrenal glands of cattle. This study also suggests that tyramide-based immunochemical analysis should be performed to detect immunolabeled PrP(Sc) in the extracerebral tissues of BSE-affected cattle.

  16. Detection of Disease-associated Prion Protein in the Optic Nerve and the Adrenal Gland of Cattle with Bovine Spongiform Encephalopathy by Using Highly Sensitive Immunolabeling Procedures

    PubMed Central

    Iwamaru, Yoshifumi; Fukuda, Shigeo; Yokoyama, Takashi; Mohri, Shirou

    2012-01-01

    A sensitive immunohistochemical procedure, the tyramide signal amplification (TSA) system, was applied to detect the localization of immunolabeled disease-associated prion protein (PrPSc) in cattle affected with bovine spongiform encephalopathy (BSE). In this procedure, immunolabeling could be visualized in the optic nerve and the adrenal medulla. In the optic nerve, the dual immunofluorescent technique showed that the granular PrPSc was occasionally detected in the astrocytes, microglia, and myelin sheath adjacent to the axon. Clustered PrPSc was also scattered in association with microglial cells and astrocytes of the optic nerve. In the adrenal gland, PrPSc immunolabeling was confined within the sympathetic nerve fibers and endings. The results suggest that (1) PrPSc might centrifugally spread within and between glial cells and/or the non-axonal (also known as ad-axonal) region of nerve fibers, rather than the axonal and/or extracellular space pathway in the optic nerve, and (2) the sympathetic innervations might be important for the trafficking of BSE agent in the adrenal glands of cattle. This study also suggests that tyramide-based immunochemical analysis should be performed to detect immunolabeled PrPSc in the extracerebral tissues of BSE-affected cattle. PMID:22260993

  17. Using Linkage Analysis to Detect Gene-Gene Interaction by Stratifying Family Data on Known Disease, or Disease-Associated, Alleles

    PubMed Central

    Corso, Barbara; Greenberg, David A.

    2014-01-01

    Detecting gene-gene interaction in complex diseases is a major challenge for common disease genetics. Most interaction detection approaches use disease-marker associations and such methods have low power and unknown reliability in real data. We developed and tested a powerful linkage-analysis-based gene-gene interaction detection strategy based on conditioning the family data on a known disease-causing allele or disease-associated marker allele. We computer-generated multipoint linkage data for a disease caused by two epistatically interacting loci (A and B). We examined several two-locus epistatic inheritance models: dominant-dominant, dominant-recessive, recessive-dominant, recessive-recessive. At one of the loci (A), there was a known disease-related allele. We stratified the family data on the presence of this allele, eliminating family members who were without it. This elimination step has the effect of raising the “penetrance” at the second locus (B). We then calculated the lod score at the second locus (B) and compared the pre- and post-stratification lod scores at B. A positive difference indicated interaction. We also examined if it was possible to detect interaction with locus B based on a disease-marker association (instead of an identified disease allele) at locus A. We also tested whether the presence of genetic heterogeneity would generate false positive evidence of interaction. The power to detect interaction for a known disease allele was 60–90%. The probability of false positives, based on heterogeneity, was low. Decreasing linkage disequilibrium between the disease and marker at locus A decreased the likelihood of detecting interaction. The allele frequency of the associated marker made little difference to the power. PMID:24690899

  18. Multicolor fluorescence detection for single nucleotide polymorphism genotyping using a filter-less fluorescence detector

    NASA Astrophysics Data System (ADS)

    Yamasaki, Keita; Nakazawa, Hirokazu; Misawa, Nobuo; Ishida, Makoto; Sawada, Kazuaki

    2013-06-01

    Single nucleotide polymorphism (SNP) analysis that is commonly performed using fluorescence is important in drug development and pathology research. In this study, to facilitate the analysis, multicolor fluorescence detection for SNP genotyping using a filter-less fluorescence detector (FFD) was investigated. FFDs do not require any optical filters for multicolor fluorescence detection. From the experimental results, FFD could identify 0 μM, 1 μM, and 10 μM solutions of Texas Red and fluorescein isothiocyanate. Moreover, a mixture of Texas Red and 6-FAM could be detected in the SNP genotyping simulation. Therefore, a small and low-cost SNP genotyping system is feasible.

  19. Mycobacterium genotypes in pulmonary tuberculosis infections and their detection by trained African giant pouched rats.

    PubMed

    Mgode, Georgies F; Cohen-Bacrie, Stéphan; Bedotto, Marielle; Weetjens, Bart J; Cox, Christophe; Jubitana, Maureen; Kuipers, Dian; Machang'u, Robert S; Kazwala, Rudovick; Mfinanga, Sayoki G; Kaufmann, Stefan H E; Drancourt, Michel

    2015-02-01

    Tuberculosis (TB) diagnosis in low-income countries is mainly done by microscopy. Hence, little is known about the diversity of Mycobacterium spp. in TB infections. Different genotypes or lineages of Mycobacterium tuberculosis vary in virulence and induce different inflammatory and immune responses. Trained Cricetomys rats show a potential for rapid diagnosis of TB. They detect over 28 % of smear-negative, culture-positive TB. However, it is unknown whether these rats can equally detect sputa from patients infected with different genotypes of M. tuberculosis. A 4-month prospective study on diversity of Mycobacterium spp. was conducted in Dar es Salaam, Tanzania. 252 sputa from 161 subjects were cultured on Lowenstein-Jensen medium and thereafter tested by rats. Mycobacterial isolates were subjected to molecular identification and multispacer sequence typing (MST) to determine species and genotypes. A total of 34 Mycobacterium spp. isolates consisting of 32 M. tuberculosis, 1 M. avium subsp. hominissuis and 1 M. intracellulare were obtained. MST analyses of 26 M. tuberculosis isolates yielded 10 distinct MST genotypes, including 3 new genotypes with two clusters of related patterns not grouped by geographic areas. Genotype MST-67, shared by one-third of M. tuberculosis isolates, was associated with the Mwananyamala clinic. This study shows that diverse M. tuberculosis genotypes (n = 10) occur in Dar es Salaam and trained rats detect 80 % of the genotypes. Sputa with two M. tuberculosis genotypes (20 %), M. avium hominissuis and M. intracellulare were not detected. Therefore, rats detect sputa with different M. tuberculosis genotypes and can be used to detect TB in resource-poor countries.

  20. Adhesion/growth-regulatory galectins in the human eye: localization profiles and tissue reactivities as a standard to detect disease-associated alterations.

    PubMed

    Schlötzer-Schrehardt, Ursula; André, Sabine; Janko, Christina; Kaltner, Herbert; Kopitz, Jürgen; Gabius, Hans-Joachim; Herrmann, Martin

    2012-08-01

    To characterize the localization profiles of seven adhesion/growth-regulatory galectins in the normal human eye using immunodetection of endogenous galectins and binding of labeled galectins to sections, useful as a basis to detect disease-associated alterations. Non-cross-reactive anti-galectin antibodies and biotinylated galectins were tested on acetone-fixed cryosections of normal human donor eyes. Controls included omission of first-step reagent, testing of an antibody against a galectin specific for rat (galectin-5), and blocking of galectin binding with lactose. Galectin presence was not restricted to one or few members of this family. Signal occurrence can even include all tested or most proteins (conjunctival or corneal epithelium), whereas choroid positivity is fully accounted for by galectin-9. Regional specificity and characteristic profiles for each protein, immuno- and galectin histochemically, were determined. Differences in tissue reactivity among the galectins were detected. That the galectins have characteristic localization/reactivity profiles supports the concept of a network with potential for non-overlapping functions. The reported data thus prompt to proceed to the respective analysis of specimens from ocular diseases.

  1. Detecting Identity by Descent and Estimating Genotype Error Rates in Sequence Data

    PubMed Central

    Browning, Brian L.; Browning, Sharon R.

    2013-01-01

    Existing methods for identity by descent (IBD) segment detection were designed for SNP array data, not sequence data. Sequence data have a much higher density of genetic variants and a different allele frequency distribution, and can have higher genotype error rates. Consequently, best practices for IBD detection in SNP array data do not necessarily carry over to sequence data. We present a method, IBDseq, for detecting IBD segments in sequence data and a method, SEQERR, for estimating genotype error rates at low-frequency variants by using detected IBD. The IBDseq method estimates probabilities of genotypes observed with error for each pair of individuals under IBD and non-IBD models. The ratio of estimated probabilities under the two models gives a LOD score for IBD. We evaluate several IBD detection methods that are fast enough for application to sequence data (IBDseq, Beagle Refined IBD, PLINK, and GERMLINE) under multiple parameter settings, and we show that IBDseq achieves high power and accuracy for IBD detection in sequence data. The SEQERR method estimates genotype error rates by comparing observed and expected rates of pairs of homozygote and heterozygote genotypes at low-frequency variants in IBD segments. We demonstrate the accuracy of SEQERR in simulated data, and we apply the method to estimate genotype error rates in sequence data from the UK10K and 1000 Genomes projects. PMID:24207118

  2. Sensitive discrimination and detection of prion disease-associated isoform with a dual-aptamer strategy by developing a sandwich structure of magnetic microparticles and quantum dots.

    PubMed

    Xiao, Sai Jin; Hu, Ping Ping; Wu, Xiao Dong; Zou, Yan Li; Chen, Li Qiang; Peng, Li; Ling, Jian; Zhen, Shu Jun; Zhan, Lei; Li, Yuan Fang; Huang, Cheng Zhi

    2010-12-01

    The major challenge of prion disease diagnosis at the presymptomatic stage is how to sensitively or selectively discriminate and detect the minute quantity of disease-associated prion protein isoform (PrP(Res)) in complex biological systems such as serum and brain homogenate. In this contribution, we developed a dual-aptamer strategy by taking the advantages of aptamers, the excellent separation ability of magnetic microparticles (MMPs), and the high fluorescence emission features of quantum dots (QDs). Two aptamers (Apt1 and Apt2), which can recognize their two corresponding distinct epitopes of prion proteins (PrP), were coupled to the surfaces of MMPs and QDs, respectively, to make MMPs-Apt1 and QDs-Apt2 ready at first, which then could be coassociated together through the specific recognitions of the two aptamers with their two corresponding distinct epitopes of PrP, forming a sandwich structure of MMPs-Apt1-PrP-Apt2-QDs and displaying the strong fluorescence of QDs. Owing to the different binding affinities of the two aptamers with PrP(Res) and cellular prion protein (PrP(C)), both of which have distinct denaturing detergent resistance, our dual-aptamer strategy could be applied to discriminate PrP(Res) and PrP(C) successfully in serum. Further identifications showed that the present dual-aptamer assay could be successfully applied to the detection of PrP in 0.01% brain homogenate, about 1000-fold lower than that of commonly applied antibody-mediated assays, which can detect PrP just in 10% brain homogenate, indicating that the present designed dual-aptamer assay is highly sensitive and adequate for clinical diagnosis without isolation of target protein prior to assay.

  3. Detection of genotype-specific Ehrlichia canis exposure in Brazilian dogs by TRP36 peptide ELISA.

    PubMed

    Aguiar, Daniel M; Zhang, Xiaofeng; Braga, Isis A; Taques, Isis I G G; McBride, Jere W

    2016-02-01

    We recently characterized a novel genotype of Ehrlichia canis based on the tandem repeat (TR) sequence of the TRP36 gene in Brazil. The TR amino acid sequence of the Brazilian (Br) genotype (ASVVPEAE) was divergent from the previously described US genotype (TEDSVSAPA) of E. canis. In this study, we developed an ELISA based on TRP36 TR synthetic peptides from both Br and US E. canis TRP36 genotypes to serologically detect and distinguish infections caused by these genotypes. Sera from 30 Brazilian dogs naturally infected with E. canis, sera from dogs experimentally infected E. canis (Jake and Cuiabá #1 strains) and E. chaffeensis (Arkansas strain) and 12 seronegative E. canis dogs were evaluated. Fifteen naturally infected Brazilian dogs had antibodies that reacted with the US TRP36 (n=9) or Br TRP36 (n=6) only, and 13 dogs had antibodies that reacted with both TPR36 peptides suggesting that these dogs were exposed to both genotypes. Most dogs (n=28) had antibodies that reacted with the highly conserved E. canis TRP19 peptide; however, two dogs had antibodies to E. canis TRP19, but did not have TRP36 antibodies, raising the possibility that another novel TRP36 genotype is circulating in Brazil. Our results demonstrate that synthetic peptides based on the TR region of E. canis TRP36 can be used to serologically distinguish infections or identify coinfections by different genotypes, and to determine the seroprevalence of various E. canis genotypes in Brazil.

  4. Universal Primers for Detection and Sequencing of Hepatitis B Virus Genomes across Genotypes A to G.

    PubMed

    Chook, Jack Bee; Teo, Woon Li; Ngeow, Yun Fong; Tee, Kok Keng; Ng, Kee Peng; Mohamed, Rosmawati

    2015-06-01

    Hepatitis B virus (HBV) has been divided into 10 genotypes, A to J, based on an 8% nucleotide sequence divergence between genotypes. The conventional practice of using a single set of primers to amplify a near-complete HBV genome is hampered by its low analytical sensitivity. The current practice of using overlapping conserved primer sets to amplify a complete HBV genome in a clinical sample is limited by the lack of pan-primers to detect all HBV genotypes. In this study, we designed six highly conserved, overlapping primer sets to cover the complete HBV genome. We based our design on the sequences of 5,154 HBV genomes of genotypes A to I downloaded from the GenBank nucleotide database. These primer sets were tested on 126 plasma samples from Malaysia, containing genotypes A to D and with viral loads ranging from 20 to >79,780,000 IU/ml. The overall success rates for PCR amplification and sequencing were >96% and >94%, respectively. Similarly, there was 100% amplification and sequencing success when the primer sets were tested on an HBV reference panel of genotypes A to G. Thus, we have established primer sets that gave a high analytical sensitivity for PCR-based detection of HBV and a high rate of sequencing success for HBV genomes in most of the viral genotypes, if not all, without prior known sequence data for the particular genotype/genome.

  5. Immunohistochemical detection of disease-associated prion protein in the peripheral nervous system in experimental H-type bovine spongiform encephalopathy.

    PubMed

    Okada, H; Iwamaru, Y; Yokoyama, T; Mohri, S

    2013-07-01

    H-type bovine spongiform encephalopathy (BSE) has been identified in aged cattle in Europe and North America. To determine the localization of disease-associated prion protein (PrP(Sc)) in the peripheral nerve tissues of cattle affected with H-type BSE, we employed highly sensitive immunohistochemical and immunofluorescence techniques with the tyramide signal amplification (TSA) system. PrP(Sc) deposition was detected in the inferior ganglia, sympathetic nerve trunk, vagus nerve, spinal nerves, cauda equina, and adrenal medulla, using this system. Notably, granular PrP(Sc) deposits were present mainly in the Schwann cells and fibroblast-like cells and occasionally along certain nerve fibers at the surface of the axons. In the adrenal gland, PrP(Sc) immunolabeling was observed within the sympathetic nerve fibers and nerve endings in the adrenal medulla. Although our results were limited to only 3 experimental cases, these results suggest that the TSA system, a highly sensitive immunohistochemical procedure, may help in elucidating the peripheral pathogenesis of H-type BSE.

  6. A complete molecular biology assay for hepatitis C virus detection, quantification and genotyping.

    PubMed

    Casanova, Yara Silva; Boeira, Thais da Rocha; Sisti, Elisa; Celmer, Álvaro; Fonseca, André Salvador Kazantzi; Ikuta, Nilo; Simon, Daniel; Lunge, Vagner Ricardo

    2014-01-01

    Molecular biology procedures to detect, genotype and quantify hepatitis C virus (HCV) RNA in clinical samples have been extensively described. Routine commercial methods for each specific purpose (detection, quantification and genotyping) are also available, all of which are typically based on polymerase chain reaction (PCR) targeting the HCV 5' untranslated region (5'UTR). This study was performed to develop and validate a complete serial laboratory assay that combines real-time nested reverse transcription-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) techniques for the complete molecular analysis of HCV (detection, genotyping and viral load) in clinical samples. Published HCV sequences were compared to select specific primers, probe and restriction enzyme sites. An original real-time nested RT-PCR-RFLP assay was then developed and validated to detect, genotype and quantify HCV in plasma samples. The real-time nested RT-PCR data were linear and reproducible for HCV analysis in clinical samples. High correlations (> 0.97) were observed between samples with different viral loads and the corresponding read cycle (Ct - Cycle threshold), and this part of the assay had a wide dynamic range of analysis. Additionally, HCV genotypes 1, 2 and 3 were successfully distinguished using the RFLP method. A complete serial molecular assay was developed and validated for HCV detection, quantification and genotyping.

  7. [Hepatitis B virus genotype E infection in Turkey: the detection of the first case].

    PubMed

    Sayan, Murat; Sanlıdağ, Tamer; Akçalı, Sinem; Arıkan, Ayşe

    2014-10-01

    Hepatitis B virus (HBV) infection is a global major health problem. Currently, 10 genotypes (A-J) of hepatitis B virus (HBV) are identified based on the nucleic acid sequence heterogeneity, and these genotypes have been shown to have distinct geographic distribution. Reports of the previous studies indicated that the genotype D is the predominant type among hepatitis B patients in different regions of Turkey. However, recent studies indicated that other HBV genotypes are also seen with an increasing rate. Although epidemiological and clinical information on genotype E infection is currently limited, it is known that genotype E infection is common in West and Central Africa. In this report, the first case of HBV genotype E infection in Turkey was presented. A 22-year-old Nigerian male employee who resided in Manisa for five years was admitted to Celal Bayar University Hospital Manisa, Turkey, for his routine check-up. Since HBsAg was found positive, other HBV markers were tested with a repeated serum sample. Laboratory findings were as follows; HBsAg (+), anti-HBs (-), HBeAg (-), anti-HBe (+), anti-HBc (+), anti-HCV (-), anti-HIV (-), ALT: 44 U/L and AST: 45 U/L. HBV-DNA level was detected as 700 IU/ml by real-time PCR (Artus HBV QS RGQ Qiagen, Germany). HBV-DNA isolated from the serum sample of the patient was amplified by PCR and polymerase gene segment of HBV was directly sequenced. UPGMA method was used for phylogenetic analysis and Inno-LIPA HBV genotyping method (Innogenetics, Belgium) was performed to determine multiple HBV genotype infection. On the basis of those methods the genotype of the virus was identified as genotype E. The partial sequences of the HBV polymerase gene were loaded to the international DNA data bank (GenBank) for contribution to the global HBV surveillance. This report emphasized that besides genotype D the other HBV genotypes could be found in Turkey. Since the patient was an inactive HBsAg carrier before his residence in Turkey, this

  8. Molecular detection of bovine Noroviruses in Argentinean dairy calves: Circulation of a tentative new genotype.

    PubMed

    Ferragut, Fátima; Vega, Celina G; Mauroy, Axel; Conceição-Neto, Nádia; Zeller, Mark; Heylen, Elisabeth; Uriarte, Enrique Louge; Bilbao, Gladys; Bok, Marina; Matthijnssens, Jelle; Thiry, Etienne; Badaracco, Alejandra; Parreño, Viviana

    2016-06-01

    Bovine noroviruses are enteric pathogens detected in fecal samples of both diarrheic and non-diarrheic calves from several countries worldwide. However, epidemiological information regarding bovine noroviruses is still lacking for many important cattle producing countries from South America. In this study, three bovine norovirus genogroup III sequences were determined by conventional RT-PCR and Sanger sequencing in feces from diarrheic dairy calves from Argentina (B4836, B4848, and B4881, all collected in 2012). Phylogenetic studies based on a partial coding region for the RNA-dependent RNA polymerase (RdRp, 503 nucleotides) of these three samples suggested that two of them (B4836 and B4881) belong to genotype 2 (GIII.2) while the third one (B4848) was more closely related to genotype 1 (GIII.1) strains. By deep sequencing, the capsid region from two of these strains could be determined. This confirmed the circulation of genotype 1 (B4848) together with the presence of another sequence (B4881) sharing its highest genetic relatedness with genotype 1, but sufficiently distant to constitute a new genotype. This latter strain was shown in silico to be a recombinant: phylogenetic divergence was detected between its RNA-dependent RNA polymerase coding sequence (genotype GIII.2) and its capsid protein coding sequence (genotype GIII.1 or a potential norovirus genotype). According to this data, this strain could be the second genotype GIII.2_GIII.1 bovine norovirus recombinant described in literature worldwide. Further analysis suggested that this strain could even be a potential norovirus GIII genotype, tentatively named GIII.4. The data provides important epidemiological and evolutionary information on bovine noroviruses circulating in South America. Copyright © 2016. Published by Elsevier B.V.

  9. FRET-based detection and genotyping of HPV-6 and HPV-11 causing recurrent respiratory papillomatosis.

    PubMed

    Combrinck, Catharina E; Seedat, Riaz Y; Burt, Felicity J

    2013-05-01

    Recurrent respiratory papillomatosis (RRP) is a potentially life-threatening disease caused by human papillomavirus (HPV), usually HPV types 6 and 11. The conventional method used for detection and typing the RRP isolates in our laboratory is the polymerase chain reaction (PCR) and DNA sequencing method. A real-time PCR assay based on fluorescence resonance energy transfer (FRET) probe technology was developed for the detection and rapid genotyping of HPV-6 and-11 isolates from biopsy material. The primers and probes were designed using multiple alignments of HPV-6 and HPV-11 partial E6 and E7 sequences that included prototypic and non-prototypic variants. Real-time PCR followed by probe-specific melting-curve analysis allowed differentiation of HPV-6 and HPV-11. HPV-6 and HPV-11 amplicons were used to determine detection limits and inter- and intra-assay variability. The detection limit of the assay was 12.8 DNA copies for HPV-6 and 22.5 DNA copies for HPV-11. A total of 60 isolates were genotyped using the FRET real-time PCR assay and a 100% concordance was obtained when results were compared with genotyping based on conventional DNA sequencing. The real-time PCR assay based on FRET technology was able to detect and rapidly genotype HPV from tissue biopsy obtained from patients with RRP. The assay reduces the time required for genotyping from three working days to less than a day.

  10. Detecting and Estimating Contamination of Human DNA Samples in Sequencing and Array-Based Genotype Data

    PubMed Central

    Jun, Goo; Flickinger, Matthew; Hetrick, Kurt N.; Romm, Jane M.; Doheny, Kimberly F.; Abecasis, Gonçalo R.; Boehnke, Michael; Kang, Hyun Min

    2012-01-01

    DNA sample contamination is a serious problem in DNA sequencing studies and may result in systematic genotype misclassification and false positive associations. Although methods exist to detect and filter out cross-species contamination, few methods to detect within-species sample contamination are available. In this paper, we describe methods to identify within-species DNA sample contamination based on (1) a combination of sequencing reads and array-based genotype data, (2) sequence reads alone, and (3) array-based genotype data alone. Analysis of sequencing reads allows contamination detection after sequence data is generated but prior to variant calling; analysis of array-based genotype data allows contamination detection prior to generation of costly sequence data. Through a combination of analysis of in silico and experimentally contaminated samples, we show that our methods can reliably detect and estimate levels of contamination as low as 1%. We evaluate the impact of DNA contamination on genotype accuracy and propose effective strategies to screen for and prevent DNA contamination in sequencing studies. PMID:23103226

  11. Giant Magnetoresistive Biochip for DNA Detection and HPV Genotyping

    PubMed Central

    Xu, Liang; Yu, Heng; Akhras, Michael S.; Han, Shu-Jen; Osterfeld, Sebastian; White, Robert L.; Pourmand, Nader; Wang, Shan X.

    2008-01-01

    A giant magnetoresistive (GMR) biochip based on spin valve sensor array and magnetic nanoparticle labels was developed for inexpensive, sensitive and reliable DNA detection. The DNA targets detected in this experiment were PCR products amplified from Human Papillomavirus (HPV) plasmids. The concentrations of the target DNA after PCR were around 10 nM in most cases, but concentrations of 10 pM were also detectable, which is demonstrated by experiments with artificial DNA samples. A mild but highly specific surface chemistry was used for probe oligonucleotide immobilization. Double modulation technique was used for signal detection in order to reduce the 1/f noise in the sensor. Twelve assays were performed with an accuracy of approximately 90%. Magnetic signals were consistent with particle coverage data measured with Scanning Electron Microscopy. More recent research on microfluidics showed the potential of reducing the assay time below one hour. This is the first demonstration of magnetic DNA detection using plasmid-derived samples. This study provides a direct proof that GMR sensors can be used for biomedical applications. PMID:18457945

  12. Detection of Rotavirus Genotypes in Korea 5 Years after the Introduction of Rotavirus Vaccines.

    PubMed

    Chung, Ju-Young; Kim, Min-Sung; Jung, Tae Woong; Kim, Seong Joon; Kang, Jin-Han; Han, Seung Beom; Kim, Sang Yong; Rhim, Jung Woo; Kim, Hwang-Min; Park, Jae Hong; Jo, Dae Sun; Ma, Sang Hyuk; Jeong, Hye-Sook; Cheon, Doo-Sung; Kim, Jong-Hyun

    2015-10-01

    Rotavirus (RV) is one of the most important viral etiologic agents of acute gastroenteritis (AGE) in children. Although effective RV vaccines (RVVs) are now used worldwide, novel genotypes and outbreaks resulting from rare genotype combinations have emerged. This study documented RV genotypes in a Korean population of children with AGE 5 yr after the introduction of RVV and assessed potential genotype differences based on vaccination status or vaccine type. Children less than 5-yr-old diagnosed with AGE between October 2012 and September 2013 admitted to 9 medical institutions from 8 provinces in Korea were prospectively enrolled. Stool samples were tested for RV by enzyme immunoassay and genotyped by multiplex reverse-transcription polymerase chain reaction. In 346 patients, 114 (32.9%) were RV-positive. Among them, 87 (76.3%) patients were infected with RV alone. Eighty-six of 114 RV-positive stool samples were successfully genotyped, and their combinations of genotypes were G1P[8] (36, 41.9%), G2P[4] (12, 14.0%), and G3P[8] (6, 7.0%). RV was detected in 27.8% of patients in the vaccinated group and 39.8% in the unvaccinated group (P=0.035). Vaccination history was available for 67 of 86 cases with successfully genotyped RV-positive stool samples; RotaTeq (20, 29.9%), Rotarix (7, 10.4%), unvaccinated (40, 59.7%). The incidence of RV AGE is lower in the RV-vaccinated group compared to the unvaccinated group with no evidence of substitution with unusual genotype combinations.

  13. Giant Magnetoresistive Biochips for Biomarker Detection and Genotyping: An Overview

    NASA Astrophysics Data System (ADS)

    Wang, Shan X.

    2008-06-01

    Giant magnetoresistive biochips based on spin valve sensor arrays and magnetic nanoparticle labels have been successfully applied to the detection of biological events in the form of both protein and DNA assays with great speed, sensitivity, selectivity and economy. The technology is highly scalable to deep multiplex detection of biomarkers in a complex disease, and amenable to integration of microfluidics and CMOS electronics for portable applications. The results suggest that a magneto-nano biochip holds great promises in biomedicine, particularly for point of care molecular diagnostics of cancer, infectious diseases, radiation injury, cardiac and other diseases.

  14. Genotypic and phenotypic detection of efflux pump in Rhodococcus equi.

    PubMed

    Gressler, Letícia Trevisan; de Vargas, Agueda Castagna; da Costa, Mateus Matiuzzi; Pötter, Luciana; da Silveira, Bibiana Petri; Sangioni, Luis Antônio; de Avila Botton, Sônia

    2014-01-01

    The req_39680 gene, associated to a putative efflux system, was detected in 60% (54/90) of R. equi isolates by PCR. The phenotypic expression of efflux mechanism was verified in 20% of the isolates using ethidium bromide. For the first time, the expression of efflux mechanism was demonstrated in R. equi.

  15. A sensitive method for detecting and genotyping Cryptosporidium parvum oocysts

    USDA-ARS?s Scientific Manuscript database

    Cryptosporidium parvum oocysts represent a considerable health risk to humans and animals because the parasite has a low infectious dose and usually exists in low numbers in environmental samples, which makes detection problematic. The purpose of this study was to evaluate Cryspovirus as a target f...

  16. Genotypic and phenotypic detection of efflux pump in Rhodococcus equi

    PubMed Central

    Gressler, Letícia Trevisan; de Vargas, Agueda Castagna; da Costa, Mateus Matiuzzi; Pötter, Luciana; da Silveira, Bibiana Petri; Sangioni, Luis Antônio; de Avila Botton, Sônia

    2014-01-01

    The req_39680 gene, associated to a putative efflux system, was detected in 60% (54/90) of R. equi isolates by PCR. The phenotypic expression of efflux mechanism was verified in 20% of the isolates using ethidium bromide. For the first time, the expression of efflux mechanism was demonstrated in R. equi. PMID:25242956

  17. Detection of Helicobacter pylori and the genotypes of resistance to clarithromycin and the heterogeneous genotype to this antibiotic in biopsies obtained from symptomatic children.

    PubMed

    Aguilera-Correa, John Jairo; Urruzuno, Pedro; Barrio, Josefa; Martinez, María José; Agudo, Sonia; Somodevilla, Angela; Llorca, Laura; Alarcón, Teresa

    2017-02-01

    The aim of this study was to use a commercially available kit (GenoType® HelicoDR; Hain Life Science, Germany) to detect Helicobacter pylori infection and clarithromycin resistance genotype in biopsies obtained from symptomatic children. 111 out of 136 (81.6%) biopsies were H. pylori positive by genotype: 47 (42.3%) showed wild-type genotype, 53 resistant genotype (47.7%) and 11 heterogeneous genotype (9.9%). Culture was negative in 27 out of the 111 genotyped biopsies. Mutation A2143G (87.5%), followed by A2142G (7.5%) and double mutant A2142C-A2143G (5%) were found. The 11 heterogeneous genotype biopsies showed wild-type plus A2143G in 9 and plus A2142G in 2. This kit is a rapid, culture-independent method for routine application in biopsies from the pediatric population that allows detection of clarithromycin resistance and heterogeneous genotypes. It is important to know the clinical impact of infection with this type of strains as well as the role in treatment success. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Electronic hybridization detection in microarray format and DNA genotyping

    PubMed Central

    Blin, Antoine; Cissé, Ismaïl; Bockelmann, Ulrich

    2014-01-01

    We describe an approach to substituting a fluorescence microarray with a surface made of an arrangement of electrolyte-gated field effect transistors. This was achieved using a dedicated blocking of non-specific interactions and comparing threshold voltage shifts of transistors exhibiting probe molecules of different base sequence. We apply the approach to detection of the 35delG mutation, which is related to non-syndromic deafness and is one of the most frequent mutations in humans. The process involves barcode sequences that are generated by Tas-PCR, a newly developed replication reaction using polymerase blocking. The barcodes are recognized by hybridization to surface attached probes and are directly detected by the semiconductor device. PMID:24569823

  19. Electronic hybridization detection in microarray format and DNA genotyping

    NASA Astrophysics Data System (ADS)

    Blin, Antoine; Cissé, Ismaïl; Bockelmann, Ulrich

    2014-02-01

    We describe an approach to substituting a fluorescence microarray with a surface made of an arrangement of electrolyte-gated field effect transistors. This was achieved using a dedicated blocking of non-specific interactions and comparing threshold voltage shifts of transistors exhibiting probe molecules of different base sequence. We apply the approach to detection of the 35delG mutation, which is related to non-syndromic deafness and is one of the most frequent mutations in humans. The process involves barcode sequences that are generated by Tas-PCR, a newly developed replication reaction using polymerase blocking. The barcodes are recognized by hybridization to surface attached probes and are directly detected by the semiconductor device.

  20. Influence of breed and genotype on the onset and distribution of infectivity and disease-associated prion protein in sheep following oral infection with the bovine spongiform encephalopathy agent.

    PubMed

    McGovern, G; Martin, S; Jeffrey, M; Bellworthy, S J; Spiropoulos, J; Green, R; Lockey, R; Vickery, C M; Thurston, L; Dexter, G; Hawkins, S A C; González, L

    2015-01-01

    The onset and distribution of infectivity and disease-specific prion protein (PrP(d)) accumulation was studied in Romney and Suffolk sheep of the ARQ/ARQ, ARQ/ARR and ARR/ARR prion protein gene (Prnp) genotypes (where A stands for alanine, R for arginine and Q for glutamine at codons 136, 154 and 171 of PrP), following experimental oral infection with cattle-derived bovine spongiform encephalopathy (BSE) agent. Groups of sheep were killed at regular intervals and a wide range of tissues taken for mouse bioassay or immunohistochemistry (IHC), or both. Bioassay results for infectivity were mostly coincident with those of PrP(d) detection by IHC both in terms of tissues and time post infection. Neither PrP(d) nor infectivity was detected in any tissues of BSE-dosed ARQ/ARR or ARR/ARR sheep or of undosed controls. Moreover, four ARQ/ARQ Suffolk sheep, which were methionine (M)/threonine heterozygous at codon 112 of the Prnp gene, did not show any biological or immunohistochemical evidence of infection, while those homozygous for methionine (MARQ/MARQ) did. In MARQ/MARQ sheep of both breeds, initial PrP(d) accumulation was identified in lymphoreticular system (LRS) tissues followed by the central nervous system (CNS) and enteric nervous system (ENS) and finally by the autonomic nervous system and peripheral nervous system and other organs. Detection of infectivity closely mimicked this sequence. No PrP(d) was observed in the ENS prior to its accumulation in the CNS, suggesting that ENS involvement occurred simultaneously to that of, or followed centrifugal spread from, the CNS. The distribution of PrP(d) within the ENS further suggested a progressive spread from the ileal plexus to other ENS segments via neuronal connections of the gut wall. Differences between the two breeds were noted in terms of involvement of LRS and ENS tissues, with Romney sheep showing a more delayed and less consistent PrP(d) accumulation than Suffolk sheep in such tissues. Whether this

  1. Detection of rare and possibly carcinogenic human papillomavirus genotypes as single infections in invasive cervical cancer.

    PubMed

    Geraets, Daan; Alemany, Laia; Guimera, Nuria; de Sanjose, Silvia; de Koning, Maurits; Molijn, Anco; Jenkins, David; Bosch, Xavier; Quint, Wim

    2012-12-01

    The contribution of carcinogenic human papillomavirus (HPV) types to the burden of cervical cancer has been well established. However, the role and contribution of phylogenetically related HPV genotypes and rare variants remains uncertain. In a recent global study of 8977 HPV-positive invasive cervical carcinomas (ICCs), the genotype remained unidentified in 3.7% by the HPV SPF10 PCR-DEIA-LiPA25 (version 1) algorithm. The 331 ICC specimens with unknown genotype were analysed by a novel sequence methodology, using multiple selected short regions in L1. This demonstrated HPV genotypes that have infrequently or never been detected in ICC, ie HPV26, 30, 61, 67, 68, 69, 73 and 82, and rare variants of HPV16, 18, 26, 30, 34, 39, 56, 67, 68, 69, 82 and 91. These are not identified individually by LiPA25 and only to some extent by other HPV genotyping assays. Most identified genotypes have a close phylogenetic relationship with established carcinogenic HPVs and have been classified as possibly carcinogenic by IARC. Except for HPV85, all genotypes in α-species 5, 6, 7, 9 and 11 were encountered as single infections in ICCs. These species of established and possibly carcinogenic HPV types form an evolutionary clade. We have shown that the possibly carcinogenic types were detected only in squamous cell carcinomas, which were often keratinizing and diagnosed at a relatively higher mean age (55.3 years) than those associated with established carcinogenic types (50.9 years). The individual frequency of the possibly carcinogenic types in ICCs is low, but together they are associated with 2.25% of the 8338 included ICCs with a single HPV type. This fraction is greater than seven of the established carcinogenic types individually. This study provides evidence that possibly carcinogenic HPV types occur as single infections in invasive cervical cancer, strengthening the circumstantial evidence of a carcinogenic role.

  2. Detection and Genotyping of Leuconostoc spp. in a Sausage Processing Plant.

    PubMed

    Padilla-Frausto, J J; Cepeda-Marquez, L G; Salgado, L M; Iturriaga, M H; Arvizu-Medrano, S M

    2015-12-01

    Some Leuconostoc spp. have the ability to produce slime and undesirable compounds in cooked sausage. The objectives of this research were to identify Leuconostoc sources in a Vienna-type sausage processing plant and to evaluate the genetic diversity of the isolated strains. Three hundred and two samples of sausage batter, sausages during processing, spoiled sausage, equipment surfaces, chilling brine, workers' gloves and aprons, and used casings were collected (March to November 2008 and February to April 2010) from a sausage processing plant. Lactic acid bacteria (LAB) were quantified, and Leuconostoc were detected using PCR. Strains were isolated and identified in Leuconostoc-positive samples. Leuconostoc strains were genotyped using randomly amplified polymorphic DNA and pulsed-field gel electrophoresis. LAB content of nonspoiled and spoiled sausage ranged from <0.8 to 4.4 log CFU/g and from 4.9 to 8.3 log CFU/g, respectively. LAB levels on equipment surfaces ranged from <1.3 to 4.8 log CFU/100 cm(2). Leuconostoc was detected in 35% of the samples, and 88 Leuconostoc spp. strains were isolated and genotyped. The main Leuconostoc spp. isolated were L. mesenteroides (37 genotypes), L. fallax (29 genotypes), and L. lactis (6 genotypes). Some strains of Leuconostoc isolated from equipment surfaces and sausages showed the same genotype. One L. lactis genotype included strains isolated from spoiled sausages analyzed in April 2008 and March to April 2010. Equipment and conveyor belts constitute Leuconostoc contamination sources. Leuconostoc persistence in the sausage processing environment and in the final product suggests the existence of microbial reservoirs, possibly on equipment surfaces.

  3. DEVELOPMENT AND EVALUATION OF A MICROARRAY APPROACH TO DETECT AND GENOTYPE NOROVIRUSES IN WATER

    EPA Science Inventory

    Noroviruses are the leading cause of nonbacterial gastroenteritis outbreaks in the United States, some of which are caused by the ingestion of contaminated water. These viruses are usually detected and genotyped using reverse transcription-polymerase chain reaction (RT-PCR) base...

  4. Linkage disequilibrium among commonly genotyped SNP and variants detected from bull sequence

    USDA-ARS?s Scientific Manuscript database

    Genomic prediction utilizing causal variants could increase selection accuracy above that achieved with SNP genotyped by commercial assays. A number of variants detected from sequencing influential sires are likely to be causal, but noticable improvements in prediction accuracy using imputed sequen...

  5. Development of reference materials to detect 15 different human papillomavirus genotypes.

    PubMed

    Rhee, Jee Eun; Kang, Young Soon; Seo, Hyun Hee; Choi, Ju-yeon; Kee, Mee-Kyung; Kim, Tae-Jin; Hong, Sung Ran; Kim, Sung Soon

    2014-06-10

    Accurate human papillomavirus (HPV) typing is essential for evaluating and monitoring HPV vaccines in cervical cancer screening and in epidemiological surveys. In our country, different HPV DNA detection and genotyping methodologies have been established for diagnosing and monitoring HPV-related disease in clinical practice and for research. However, there is a lack of reference materials to standardize the methods for HPV detection and genotyping. In this study, we constructed candidate reference materials comprising 15 targets (13 types of high-risk HPV, two types of low-risk HPV). We evaluated whether the candidate reference materials could be used as the reference for HPV detection and genotyping using quantitative real-time polymerase chain reaction. Standard curves for the wide linear range (10(1)-10(6)copies/μL) produced high correlation regression coefficient R(2) of 0.99. The reaction efficiencies were 96.3% to 101.2% for the standard curves, indicating highly efficient reactions. Specific genotypes were detected in single or multiple mixed samples. Our results suggest that these reference materials may provide useful standards for standardizing quality assurance for different HPV-typing assays and for proficiency testing in diagnostic laboratories.

  6. Comparison of human papillomavirus detection and genotyping with four different prime sets by PCR-sequencing.

    PubMed

    Cai, Yu Pin; Yang, Yi; Zhu, Bao Li; Li, Yuan; Xia, Xiao Yu; Zhang, Rui Fen; Xiang, Yang

    2013-01-01

    To assess and compare the Human Papillomavirus (HPV) detection efficiency and the potential clinical utility of PCR sequencing-based technology. Four HPV consensus primer sets (GP5+/6+, MGP, MY09/11, and PGMY09/11) were used in order to amplify a broad spectrum of HPV types for HPV infection in 325 cervical samples and the PCR products were sequenced afterwards for the HPV genotyping. The HPV-positive rate was 75.4%, of which 35.5% harbored more than one HPV genotype. A total of 36 different genotypes was found, with HPV 16 (24.1%) being the most prevalent, followed by HPV 58 (13.3%) and HPV 52 (9.6%). There were substantial to almost perfect agreements between different primer sets regarding HPV detection efficiency, with the kappa value varying from 0.751 to 0.925, MGP, and PGMY09/11 were the most effective in detecting multiple infections (P < 0.001). With each of the primer sets, a board range of HPV types could be identified, though there were several differences for a few genotypes. The substantial agreement between PCR-sequencing and HC2 for the detection of high-risk HPV (kappa=0.761) indicated that PCR-sequencing is also suitable for routine HPV screening. Copyright © 2013 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  7. DEVELOPMENT AND EVALUATION OF A MICROARRAY APPROACH TO DETECT AND GENOTYPE NOROVIRUSES IN WATER

    EPA Science Inventory

    Noroviruses are the leading cause of nonbacterial gastroenteritis outbreaks in the United States, some of which are caused by the ingestion of contaminated water. These viruses are usually detected and genotyped using reverse transcription-polymerase chain reaction (RT-PCR) base...

  8. Advances in ligase chain reaction and ligation-based amplifications for genotyping assays: Detection and applications.

    PubMed

    Gibriel, Abdullah A; Adel, Ola

    2017-07-01

    Genetic variants have been reported to cause several genetic diseases. Various genotyping assays have been developed for diagnostic and screening purposes but with certain limitations in sensitivity, specificity, cost effectiveness and/or time savings. Since the discovery of ligase chain reaction (LCR) in the late nineties, it became one of the most favored platforms for detecting these variants and also for genotyping low abundant contaminants. Recent and powerful modifications with the integration of various detection strategies such as electrochemical and magnetic biosensors, nanoparticles (NPs), quantum dots, quartz crystal and leaky surface acoustic surface biosensors, DNAzyme, rolling circle amplification (RCA), strand displacement amplification (SDA), surface enhanced raman scattering (SERS), chemiluminescence and fluorescence resonance energy transfer have been introduced to both LCR and ligation based amplifications to enable high-throughput and inexpensive multiplex genotyping with improved robustness, simplicity, sensitivity and specificity. In this article, classical and up to date modifications in LCR and ligation based amplifications are critically evaluated and compared with emphasis on points of strength and weakness, sensitivity, cost, running time, equipment needed, applications and multiplexing potential. Versatile genotyping applications such as genetic diseases detection, bacterial and viral pathogens detection are also detailed. Ligation based gold NPs biosensor, ligation based RCA and ligation mediated SDA assays enhanced detection limit tremendously with a discrimination power approaching 1.5aM, 2aM and 0.1fM respectively. MLPA (multiplexed ligation dependent probe amplification) and SNPlex assays have been commercialized for multiplex detection of at least 48 SNPs at a time. MOL-PCR (multiplex oligonucleotide ligation) has high-throughput capability with multiplex detection of 50 SNPs/well in a 96 well plate. Ligase detection reaction (LDR

  9. Rapid and Sensitive Detection of Lymphocystis Disease Virus Genotype VII by Loop-Mediated Isothermal Amplification.

    PubMed

    Valverde, Estefanía J; Cano, Irene; Castro, Dolores; Paley, Richard K; Borrego, Juan J

    2017-03-01

    Lymphocystis disease virus (LCDV) infections have been described in gilthead seabream (Sparus aurata L.) and Senegalese sole (Solea senegalensis, Kaup), two of the most important marine fish species in the Mediterranean aquaculture. In this study, a rapid, specific, and sensitive detection method for LCDV genotype VII based on loop-mediated isothermal amplification (LAMP) was developed. The LAMP assay, performed using an apparatus with real-time amplification monitoring, was able to specifically detect LCDV genotype VII from clinically positive samples in less than 12 min. In addition, the assay allowed the detection of LCDV in all asymptomatic carrier fish analysed, identified by qPCR, showing an analytical sensitivity of ten copies of viral DNA per reaction. The LCDV LAMP assay has proven to be a promising diagnostic method that can be used easily in fish farms to detect the presence and spread of this iridovirus.

  10. Development of T m -shift genotyping method for detection of cat-derived Giardia lamblia.

    PubMed

    Pan, Weida; Fu, Yeqi; Abdullahi, Auwalu Yusuf; Wang, Mingwei; Shi, Xianli; Yang, Fang; Yu, Xingang; Yan, Xinxin; Zhang, Pan; Hang, Jianxiong; Li, Guoqing

    2017-04-01

    To develop T m -shift genotyping method for detection of cat-derived Giardia lamblia, two sets of primers with two GC-rich tails of unequal length attached to their 5'-end were designed according to two SNPs (BG434 and BG170) of β-giardin (bg) gene, and specific PCR products were identified by inspection of a melting curve on real-time PCR thermocycler. A series of experiments on the stability, sensitivity, and accuracy of T m -shift method was tested, and clinical samples were also detected. The results showed that two sets of primers based on SNP could distinguish accurately between assemblages A and F. Coefficient of variation of T m values of assemblage A and F was 0.14 and 0.07% in BG434 and 0.10 and 0.11% in BG170, respectively. The lowest detection concentration was 4.52 × 10(-5) and 4.88 × 10(-5) ng/μL samples of assemblage A and F standard plasmids. The T m -shift genotyping results of ten DNA samples from the cat-derived G. lamblia were consistent with their known genotypes. The detection rate of clinical samples by T m -shift was higher than that by microscopy, and their genotyping results were in complete accordance with sequencing results. It is concluded that the T m -shift genotyping method is rapid, specific, and sensitive and may provide a new technological mean for molecular detection and epidemiological investigation of the cat-derived G. lamblia.

  11. Detection and differentiation of Japanese encephalitis virus genotype I and genotype III by reverse transcription loop-mediated isothermal amplification combined with restriction fragment length polymorphism.

    PubMed

    Zhang, Liang; Cao, Sanjie; Wu, Rui; Zhu, Shuquan; Liu, Hanyang; Yuan, Lei; Shi, Shuangyan; Zhang, Dan; Huang, Xiaobo; Wen, Xintian; Wen, Yiping; Yan, Qigui; Huang, Yong; Ma, Xiaoping

    2015-04-01

    Japanese encephalitis (JE), which is a mosquito-borne arboviral infection, is the leading cause of viral encephalitis in Asian countries. The causative agent of JE is Japanese encephalitis virus (JEV), in which the predominant genotype has changed from genotype III (G III) to genotype I (G I). However, a method for the rapid differentiation between JEV G I and G III remains unavailable. This study aimed to establish a rapid JEV genotyping method using reverse transcription loop-mediated isothermal amplification (RT-LAMP). An Spe I site, which was located in the target sequence (C gene) of JEV G III strains but not in JEV G I strains, was selected as the RT-LAMP target. After testing 64 specimens, results showed that RT-LAMP can detect and differentiate JEV G I and G III specifically. Thus, a novel RT-LAMP system for the rapid detection and differentiation of JEV G I and G III was developed successfully.

  12. Easy and fast detection and genotyping of high-risk human papillomavirus by dedicated DNA microarrays.

    PubMed

    Albrecht, Valérie; Chevallier, Anne; Magnone, Virginie; Barbry, Pascal; Vandenbos, Fanny; Bongain, André; Lefebvre, Jean-Claude; Giordanengo, Valérie

    2006-11-01

    Persistent cervical high-risk human papillomavirus (HPV) infection is correlated with an increased risk of developing a high-grade cervical intraepithelial lesion. A two-step method was developed for detection and genotyping of high-risk HPV. DNA was firstly amplified by asymmetrical PCR in the presence of Cy3-labelled primers and dUTP. Labelled DNA was then genotyped using DNA microarray hybridization. The current study evaluated the technical efficacy of laboratory-designed HPV DNA microarrays for high-risk HPV genotyping on 57 malignant and non-malignant cervical smears. The approach was evaluated for a broad range of cytological samples: high-grade squamous intraepithelial lesions (HSIL), low-grade squamous intraepithelial lesions (LSIL) and atypical squamous cells of high-grade (ASC-H). High-risk HPV was also detected in six atypical squamous cells of undetermined significance (ASC-US) samples; among them only one cervical specimen was found uninfected, associated with no histological lesion. The HPV oligonucleotide DNA microarray genotyping detected 36 infections with a single high-risk HPV type and 5 multiple infections with several high-risk types. Taken together, these results demonstrate the sensitivity and specificity of the HPV DNA microarray approach. This approach could improve clinical management of patients with cervical cytological abnormalities.

  13. Genotypic diversity of Theileria orientalis detected from cattle grazing in Kumamoto and Okinawa prefectures of Japan.

    PubMed

    Yokoyama, Naoaki; Ueno, Akio; Mizuno, Daisuke; Kuboki, Noritaka; Khukhuu, Altangerel; Igarashi, Ikuo; Miyahara, Tokuji; Shiraishi, Takashi; Kudo, Ryuta; Oshiro, Mamoru; Zakimi, Satoshi; Sugimoto, Chihiro; Matsumoto, Kotaro; Inokuma, Hisashi

    2011-03-01

    Theileria orientalis is a benign protozoan species that is widely distributed in Japan, yet sometimes causes serious economic losses in the livestock industry. In this study, we conducted a molecular survey based on genes encoding the major piroplasm surface protein (MPSP) and p23 for T. orientalis detected in cattle grazing in southern areas of Japan, consisting of 2 farms in Kumamoto prefecture (Aso and Kuma districts) and 3 farms in Okinawa prefecture (Ishigaki, Iriomote, and Yonaguni Islands). High prevalence rates of T. orientalis infection were shown in all the cattle populations using the diagnostic MPSP- and p23-PCR assays. Phylogenetic analyses revealed 4 MPSP genotypes and 3 p23 genotypes. Furthermore, MPSP genotype-specific PCR methods were developed in this study and wide distributions of 5-district genotypes of T. orientalis were observed for the examined farms. Our results indicate that at least 5 types of T. orientalis exist in Kumamoto and Okinawa prefectures of Japan and that genotype-specific PCR assays are highly applicable for the quarantine of transported cattle and for epidemiological surveys of bovine theileriosis in Japan.

  14. HPV detection methods and genotyping techniques in screening for cervical cancer.

    PubMed

    Eide, Maj Liv; Debaque, Hervé

    2012-12-01

    Human papillomaviruses (HPV), double-stranded DNA viruses, are causing many mucocutaneous diseases, benign or malignant, ranging from common warts to malignancies involving the upper aerodigestive tract and the anogenital sphere. The diagnosis of HPV infection is based primarily on the viral genome detection by molecular biological methods, given the difficulty in routine cultivation of these viruses. The current trend in screening against cervical cancer is to improve the sensitivity of screening with new methods and to propose new algorithms for diagnostic and therapeutic decisions. The development of liquid-based cytology facilitates the cytologic diagnosis and molecular assays from the same sample. There are two main types of HPV detection methods used on uterine cervical samples: signal amplification methods (hybridization techniques in liquid phase) and target amplification methods (the techniques of gene amplification or Polymerase Chain Reaction [PCR]). Genotyping techniques are also developed: they are based on an amplification technique followed by hybridization with probe specific types. In addition to the detection, genotyping techniques allow quantitative detection of viral DNA of HPV genotype and so monitor changes in viral load over time. Another approach relies on the detection of messenger RNA (mRNA) of HPV proteins E6 and E7 oncogenes, which would appear to be a relevant marker to identify and monitor women at risk of progression to a precancerous lesion or cervical cancer.

  15. Detecting and genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays

    SciTech Connect

    Call, Douglas R.; Brockman, Fred J.; Chandler, Darrell P.

    2001-07-05

    Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification. The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFUs ml-1 (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products and the system is amenable to automation.

  16. Detecting and Genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays

    SciTech Connect

    Call, Douglas R.; Brockman, Fred J. ); Chandler, Darrell P.

    2000-12-01

    Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification. The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFU ml-1 (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products and the system is amenable to automation.

  17. Colorimetric Detection of 23 Human Papillomavirus Genotypes by Loop-Mediated Isothermal Amplification.

    PubMed

    Lin, Junxiao; Ma, Biao; Fang, Jiehong; Wang, Ye; He, Haizhen; Lin, Wei; Su, Wei; Zhang, Mingzhou

    2017-03-01

    Human papillomavirus (HPV) infection is linked to cervical cancer. With the technological development of molecular biology and epidemiology, detection and treatment of HPV has become an important mean to prevent cervical cancer. A simple, rapid, and sensitive colorimetric loop-mediated isothermal amplification (LAMP) method was established herein to detect 23 HPV genotypes. The sequences of the primers for the LAMP reaction were located in the L1 gene of the HPV genome. As it is a fluorescent dye, calcein was added before the reaction. The reaction was run under isothermal conditions at 65°C for 40 minutes. A positive reaction was indicated by a color change from yellow to fluorescent green. The fluorescence curve diagram represents the monitoring of real time quantitative instrument. 450 cervical swab samples from patients with single infections of 23 different HPV genotypes were examined to evaluate the specificity. The results revealed no cross-reaction with other HPV genotypes. A serial dilution of a cloned plasmid containing 23 HPV L1 gene sequences was employed to evaluate the sensitivity. Different HPV subtypes have different detection capability. The sensitivity of different HPV subtypes tested by LAMP assay was in the range from 1.0 x10 to 4.0 x 103 copies per reaction. The LAMP assay and the RDB (reverse dot blot) were compared for detecting and genotyping HPV among the 450 clinical samples. There were 385 (85.6%) and 375 (83.3%) HPV positive specimens detected by LAMP and RDB, respectively, as well as 306 (68.0%) and 296 (65.8%) for HR-HPV positive specimens. The agreement between the LAMP and RDB assays was 93.3% (κ = 0.75) for HPV positivity and 94.7% (κ = 0.88) for HR-HPV positivity. It was concluded that this colorimetric LAMP assay had potential application for the rapid screening of the HPV infection in resource-limited hospitals or rural clinics.

  18. New universal primers for genotyping and resistance detection of low HBV DNA levels.

    PubMed

    Tong, Yongqing; Liu, Bei; Liu, Hui; Zheng, Hongyun; Gu, Jian; Liu, Hang; Lin, Min; Ding, Yali; Song, Chunhua; Li, Yan

    2016-08-01

    HBV (hepatitis B virus) genotyping is important in determining the clinical manifestation of disease and treatment response, particularly, in patients with low viral loads. Also, sensitive detection of HBV antiviral drug resistance mutations is essential for monitoring therapy response.Asensitive direct sequencing method for genotyping and the drug resistance mutation detection of low levels of HBV DNA in patients' plasma is developed by PCR amplification of the DNA with novel universal primers.The novel, common, and universal primers were identified by alignment of RT region of all the HBV DNA sequences in databases. These primers could efficiently amplify the RT region of HBV virus at low DNA levels by directly sequencing the resulting PCR products, and mapping with the reference sequence made it possible to clearly obtain the HBV subtypes and identify the resistance mutations in the samples with HBV DNA level as low as 20 IU/mL. We examined the reliability of the method in clinical samples, and found it could detect the HBV subtypes and drug resistance mutations in 80 clinical HBV samples with low HBV DNA levels ranging from 20 to 200 IU/mL.This method is a sensitive and reliable direct sequencing method for HBV genotyping and antiviral drug resistance mutation detection, and is helpful for efficiently monitoring the response to therapy in HBV patients.

  19. Highly specific and sensitive electrochemical genotyping via gap ligation reaction and surface hybridization detection.

    PubMed

    Huang, Yong; Zhang, Yan-Li; Xu, Xiangmin; Jiang, Jian-Hui; Shen, Guo-Li; Yu, Ru-Qin

    2009-02-25

    This paper developed a novel electrochemical genotyping strategy based on gap ligation reaction with surface hybridization detection. This strategy utilized homogeneous enzymatic reactions to generate molecular beacon-structured allele-specific products that could be cooperatively annealed to capture probes stably immobilized on the surface via disulfide anchors, thus allowing ultrasensitive surface hybridization detection of the allele-specific products through redox tags in close proximity to the electrode. Such a unique biphasic architecture provided a universal methodology for incorporating enzymatic discrimination reactions in electrochemical genotyping with desirable reproducibility, high efficiency and no interferences from interficial steric hindrance. The developed technique was demonstrated to show intrinsic high sensitivity for direct genomic analysis, and excellent specificity with discriminativity of single nucleotide variations.

  20. Linkage disequilibrium among commonly genotyped SNP variants detected from bull sequence().

    PubMed

    Snelling, W M; Kuehn, L A; Keel, B N; Thallman, R M; Bennett, G L

    2017-10-01

    Genomic prediction utilizing causal variants could increase selection accuracy above that achieved with SNPs genotyped by currently available arrays used for genomic selection. A number of variants detected from sequencing influential sires are likely to be causal, but noticeable improvements in prediction accuracy using imputed sequence variant genotypes have not been reported. Improvement in accuracy of predicted breeding values may be limited by the accuracy of imputed sequence variants. Using genotypes of SNPs on a high-density array and non-synonymous SNPs detected in sequence from influential sires of a multibreed population, results of this examination suggest that linkage disequilibrium between non-synonymous and array SNPs may be insufficient for accurate imputation from the array to sequence. In contrast to 75% of array SNPs being strongly correlated to another SNP on the array, less than 25% of the non-synonymous SNPs were strongly correlated to an array SNP. When correlations between non-synonymous and array SNPs were strong, distances between the SNPs were greater than separation that might be expected based on linkage disequilibrium decay. Consistently near-perfect whole-genome linkage disequilibrium between the full array and each non-synonymous SNP within the sequenced bulls suggests that whole-genome approaches to infer sequence variants might be more accurate than imputation based on local haplotypes. Opportunity for strong linkage disequilibrium between sequence and array SNPs may be limited by discrepancies in allele frequency distributions, so investigating alternate genotyping approaches and panels providing greater chances of frequency-matched SNPs strongly correlated to sequence variants is also warranted. Genotypes used for this study are available from https://www.animalgenome.org/repository/pub/;USDA2017.0519/. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  1. Two genotypes of canine coronavirus simultaneously detected in the fecal samples of dogs with diarrhea.

    PubMed

    Pratelli, Annamaria; Decaro, Nicola; Tinelli, Antonella; Martella, Vito; Elia, Gabriella; Tempesta, Maria; Cirone, Francesco; Buonavoglia, Canio

    2004-04-01

    Sixty-nine fecal samples from diarrheic puppies were examined by reverse transcription-PCR assays for the M and the S genes of canine coronaviruses (CCoVs). The isolates in 10 samples were recognized as CCoV type I, and the isolates in 6 samples were recognized as CCoV type II, while isolates of both genotypes were simultaneously detected in 53 samples.

  2. Detection of Aichi virus genotype B in two lines of wastewater treatment processes.

    PubMed

    Ibrahim, Chourouk; Hammami, Salah; Mejri, Selma; Mehri, Ines; Pothier, Pierre; Hassen, Abdennaceur

    2017-08-01

    Enteric viruses are released in important quantities into the environment where they can persist for a very long time. At very low doses, they can cause human gastroenteritis, and are responsible for a substantial number of waterborne diseases. The aims of this study were multiple: firstly, to study the circulation of Aichi viruses (AiV) in wastewater sampled at the scale of a pilot wastewater treatment plant; secondly, to evaluate the performance of two wastewater treatment procedures, as natural oxidizing lagoons and rotating Biodisks, concerning the AiV removal; and finally, to determine the different type of AiV genotype found during this study. Hence, the pilot wastewater treatment plant is principally irrigated by the wastewater of three neighbouring clinics. Wastewater samples were collected during 2011 from the two lines of biological treatment procedures. AiV detection in wastewater were achieved using the Reverse Transcription Polymerase Chain Reaction (RT-PCR) technique, and the identification of AiV genotype was realized by the direct sequencing of PCR products. The result revealed that AiV strains were identified in 50% (n = 51) of the wastewater samples. A significant increase of the AiV detection frequency was registered from upstream to downstream of the five ponds constituting the natural oxidizing lagoon process, and at the exit of the rotating Biodisks procedure. All detected AiV strains showed the highest nucleotide sequence identity to genotype B that has been recently observed in patients in Asia. This finding represented the first Tunisian survey that revealed and mentioned the first detection of AiV genotype B in sewage and by the same argued for a noticeable resistance or survival of this type of virus in the two lines of treatment considered. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Detection and genotyping of Toxoplasma gondii isolated from soil in Ahvaz, southwest of Iran.

    PubMed

    Saki, J; Khademvatan, S; Yousefi, E; Tavalla, M; Abdizadeh, R

    2017-03-01

    To detection and genotype of Toxoplasma gondii isolated from soil in Ahvaz, southwest of Iran. Between August 2011 and May 2012 at different sites located in the area of the Ahvaz city south west Iran. A total of 200 soil samples were taken from different points of the region. Oocysts were recovered using the flotation method. Then, PCR reactions targeting the GRA6 gene were performed for specific T. gondii detection. The positive samples were studied by RFLP (random amplified fragment length polymorphism) using MseI enzymes to confirm the parasite linage. Toxoplasma DNA was found in 18 samples. Among them, 12 samples were successfully genotyped as GRA6 type III and 6 as GRA6 Type II. This is the first investigation detecting and genotyping T. gondii oocyst in environmental soil samples of Ahvaz, South west of Iran. The results of this study indicated that soil contaminated with T. gondii oocysts especially in public park may play a role in the epidemiology of human toxoplasmosis in southwest of Iran.

  4. A Bayesian clustering approach for detecting gene-gene interactions in high-dimensional genotype data.

    PubMed

    Chen, Sui-Pi; Huang, Guan-Hua

    2014-06-01

    This paper uses a Bayesian formulation of a clustering procedure to identify gene-gene interactions under case-control studies, called the Algorithm via Bayesian Clustering to Detect Epistasis (ABCDE). The ABCDE uses Dirichlet process mixtures to model SNP marker partitions, and uses the Gibbs weighted Chinese restaurant sampling to simulate posterior distributions of these partitions. Unlike the representative Bayesian epistasis detection algorithm BEAM, which partitions markers into three groups, the ABCDE can be evaluated at any given partition, regardless of the number of groups. This study also develops permutation tests to validate the disease association for SNP subsets identified by the ABCDE, which can yield results that are more robust to model specification and prior assumptions. This study examines the performance of the ABCDE and compares it with the BEAM using various simulated data and a schizophrenia SNP dataset.

  5. Detecting Large Copy Number Variants Using Exome Genotyping Arrays In a Large Swedish Schizophrenia Sample

    PubMed Central

    Szatkiewicz, Jin P.; Neale, Benjamin M.; O'Dushlaine, Colm; Fromer, Menachem; Goldstein, Jacqueline I.; Moran, Jennifer L.; Chambert, Kimberly; Kähler, Anna; Magnusson, Patrik KE; Hultman, Christina M.; Sklar, Pamela; Purcell, Shaun; McCarroll, Steven A.; Sullivan, Patrick F.

    2014-01-01

    Although copy number variants (CNVs) are important in genomic medicine, CNVs have not been systematically assessed for many complex traits. Several large rare CNVs increase risk for schizophrenia (SCZ) and autism and often demonstrate pleiotropic effects; however, their frequencies in the general population and other complex traits are unknown. Genotyping large numbers of samples is essential for progress. Large cohorts from many different diseases are being genotyped using exome-focused arrays designed to detect uncommon or rare protein-altering sequence variation. Although these arrays were not designed for CNV detection, the hybridization intensity data generated in each experiment could, in principle, be used for gene-focused CNV analysis. Our goal was to evaluate the extent to which CNVs can be detected using data from one particular exome array (the Illumina Human Exome Bead Chip). We genotyped 9, 100 Swedish subjects (3, 962 cases with SCZ and 5, 138 controls) using both standard GWAS arrays and exome arrays. In comparison to CNVs detected using GWAS arrays, we observed high sensitivity and specificity for detecting genic CNVs ≥400 kb including known pathogentic CNVs along with replicating the literature finding that cases with SCZ had greater enrichment for genic CNVs. Our data confirm the association of SCZ with 16p11.2 duplications and 22q11.2 deletions and suggest a novel association with deletions at 11q12.2. Our results suggest the utility of exome focused arrays in surveying large genic CNVs in very large samples; and thereby open the door for new opportunities such as conducting well-powered CNV assessment and comparisons between different diseases. The use of a single platform also minimizes potential confounding factors that could impact accurate detection. PMID:23938935

  6. Detection of Hepatitis E Virus Genotype 1 Among Blood Donors From Southwest of Iran

    PubMed Central

    Parsa, Rahil; Adibzadeh, Setare; Behzad Behbahani, Abbas; Farhadi, Ali; Yaghobi, Ramin; Rafiei Dehbidi, Gholam Reza; Hajizamani, Saeideh; Rahbar, Sanaz; Nikouyan, Negin; Okhovat, Mohammad Ali; Naderi, Samaneh; Salehi, Saeede; Alizadeh, Marzieh; Ranjbaran, Reza; Zarnegar, Golnoosh; Alavi, Parnian

    2016-01-01

    Background Infection with hepatitis E virus (HEV) is endemic in developing countries and reveals significant regional differences. Several studies have reported virus transmission via blood transfusion. To date, however, no cases of HEV RNA detection in blood donors have been reported from Iran. Objectives The aim of this study was to determine the presence of HEV RNA in plasma samples of blood donors referred to a blood transfusion center in Shiraz in the southwest of Iran. The HEV genotypes were also investigated using nucleotide sequencing. Patients and Methods Blood samples were collected from 700 blood donors who were referred to Fars blood transfusion organization from January to March 2014. Plasma samples were screened for the presence of HEV IgG and IgM antibodies by standard enzyme immunoassay. Samples seroreactive to anti-HEV were further tested for the presence of HEV RNA using nested polymerase chain reaction (PCR) with universal primers for detection of all four HEV genotypes. Positive PCR samples were then subjected to DNA sequencing for further analysis. Results Fifty (50, 7.1%) out of 700 plasma samples tested positive for anti-HEV antibodies. HEV RNA was detected in 7/50 (12%) of the antibody-positive samples, the majority of which were IgM positive. Sequence analysis of seven isolates of the HEV RNA ORF 2 gene region revealed > 80% similarity with genotype 1. Conclusions The analysis indicates that the HEV isolated from blood donors in the southwest of Iran belongs to genotype 1. However, more samples from other geographic regions of Iran are needed to confirm these findings. Because transmission of HEV by administration of blood or blood components is likely to occur, it may be sensible to screen donor blood for HEV to eliminate transfusion-transmitted HEV infection when the recipient is immunocompromised. PMID:27630719

  7. HCV avidity as a tool for detection of recent HCV infection: Sensitivity depends on HCV genotype.

    PubMed

    Shepherd, Samantha J; McDonald, Scott A; Palmateer, Norah E; Gunson, Rory N; Aitken, Celia; Dore, Gregory J; Goldberg, David J; Applegate, Tanya L; Lloyd, Andrew R; Hajarizadeh, Behzad; Grebely, Jason; Hutchinson, Sharon J

    2017-08-26

    Accurate detection of incident hepatitis C virus (HCV) infection is required to target and evaluate public health interventions, but acute infection is largely asymptomatic and difficult to detect using traditional methods. Our aim was to evaluate a previously developed HCV avidity assay to distinguish acute from chronic HCV infection. Plasma samples collected from recent seroconversion subjects in two large Australian cohorts were tested using the avidity assay, and the avidity index (AI) was calculated. Demographic and clinical characteristics of patients with low/high AI were compared via logistic regression. Sensitivity and specificity of the assay for recent infection and the mean duration of recent infection (MDRI) were estimated stratified by HCV genotype. Avidity was assessed in 567 samples (from 215 participants), including 304 with viraemia (defined as ≥250 IU/mL). An inverse relationship between AI and infection duration was found in viraemic samples only. The adjusted odds of a low AI (<30%) decreased with infection duration (odds ratio [OR] per week of 0.93; 95% CI:0.89-0.97), and were lower for G1 compared with G3 samples (OR = 0.14; 95% CI:0.05-0.39). Defining recent infection as <26 weeks, sensitivity (at AI cut-off of 20%) was estimated at 48% (95% CI:39-56%), 36% (95% CI:20-52%), and 65% (95% CI:54-75%) and MDRI was 116, 83, and 152 days for all genotypes, G1, and G3, respectively. Specificity (≥52 weeks infection duration, all genotypes) was 96% (95% CI:90-98%). HCV avidity testing has utility for detecting recent HCV infection in patients, and for assessing progress in reaching incidence targets for eliminating transmission, but variation in assay performance across genotype should be recognized. © 2017 Wiley Periodicals, Inc.

  8. Molecular detection and genotypic characterization of Toxoplasma gondii in wild waterfowls in Jilin Province, Northeastern China.

    PubMed

    Zhang, Fu-Kai; Wang, Hai-Jun; Qin, Si-Yuan; Wang, Ze-Dong; Lou, Zhi-Long; Zhu, Xing-Quan; Liu, Quan

    2015-12-01

    Toxoplasma gondii can infect almost warm-blooded animals, including humans. Limited information about T. gondii infection in wild waterfowls is available in China. The present study was conducted to determine prevalence and genotype T. gondii infection in 11 wild waterfowl species in Jilin Province, northeastern China. A total of 249 wild waterfowls were sampled between April and July 2013 from Jilin Province, and the tissue samples were collected for the detection of T. gondii by a semi-nested PCR targeting the B1 gene. The positive samples were genotyped at 11 genetic markers (SAG1, 5'- and 3'-SAG2, alternative SAG2, SAG3, BTUB, GRA6, L358, PK1, c22-8, c29-2, and Apico) using multilocus polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technology. The overall prevalence of T. gondii in the wild waterfowls was 7.2% (18/249, 95% confidence interval [CI] 4.0-10.4), with the highest prevalence (22.0%, 95% CI 10.5-33.5) in Anas formosa, followed by Anas platyrhynchos (20.0%, 95% CI 6.0-44.0), Falcated teal (12.5%, 95% CI 0.0-35.4), and Fulica atra (4.0%, 95% CI 0.0-11.7). Of 18 positive samples, only 2 samples (TgWfjl1 and TgWfjl2) were genotyped completely, and one genotype, namely ToxoDB #9, was revealed. The result of this survey has implications for better understanding of the genetic diversity of T. gondii in China. This is the first report of prevalence and genotypic characterization of T. gondii in wild waterfowls in northeastern China. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  9. Detection and genotyping of Chlamydia species responsible for reproductive disorders in Algerian small ruminants.

    PubMed

    Merdja, Salah-Eddine; Khaled, Hamza; Aaziz, Rachid; Vorimore, Fabien; Bertin, Claire; Dahmani, Ali; Bouyoucef, Abdallah; Laroucau, Karine

    2015-02-01

    Chlamydiosis in small ruminants is a zoonotic disease mainly related to Chlamydia abortus. This bacterium is responsible for abortions and reproductive disorders in sheep and goats. Stillbirth and infertility, leading to important economic losses, are also associated with this pathology. In Algeria, abortion cases are frequently reported by veterinarians but, except for brucellosis which is a notifiable disease in this country, abortive diseases are in general poorly studied. In order to detect and genotype Chlamydia species in small ruminants in different areas of Algeria, a study was conducted on samples collected from females (164 blood samples and 199 vaginal swabs) between October 2011 and March 2013. Serum samples were tested with a C. abortus-specific indirect ELISA test. Fourteen samples (8.5 %), from six farms (6/20, 30 %) were tested positive. Vaginal swabs were analysed with a real-time PCR targeting all Chlamydiaceae spp. Thirty samples (15 %) were diagnosed positive in 16 farms (16/25, 64 %). Positive samples were all re-tested with a C. abortus- and a C. pecorum-specific real-time PCR. Finally, 13/30 (43.3 %) and 6/30 (20 %) were identified as C. abortus and C. pecorum, respectively. Enough concentrated C. abortus samples were genotyped by multi-loci variable number of tandem repeat (VNTR) analysis (MLVA), and all were related to the genotype [2] group which mainly includes French C. abortus isolates. C. pecorum-positive samples were genotyped by multi-locus sequence typing (MLST). Interestingly, two of them were successfully genotyped and showed identical MLST sequences to VB2, AB10, E58 and SBE, a group which includes C. pecorum isolates considered as highly pathogenic. These findings suggest a possible role of C. abortus and C. pecorum strains in the aetiology of abortion in Algerian small ruminants.

  10. [Comparison of three genotyping methods for the detection of HIV-1 resistance to antiretroviral drugs].

    PubMed

    Suárez, A; Picazo, J; Alonso, R; Bouza, E; Delgado, R; Rodríguez-Noriega, A; Bernal, A; García, A

    2002-03-01

    Highly active antiretroviral therapy has dramatically improved the life expectancy of human immunodeficiency virus (HIV)-infected patients, but mutations in the HIV-1 reverse transcriptase (RT) and protease (P) genes confer drug failure. Evaluation of drug resistance genotyping in HIV-1 has proven to be useful for the selection of drug combinations with maximum antiretroviral activity. The aim of this study was to evaluate the optimal procedure to determine the resistance profile in the laboratory. Plasma from 90 antiretroviral-treated patients was analyzed by reverse hybridization, which identifies the presence of wild-types or mutations at the 19 key codons for protease and RT regions, and was compared with two other methods of direct cDNA sequencing. A total of 408 mutations were detected by InnoLiPA HIV-1, (Line Probe Assay, Innogenetics, Belgium), 572 by TrueGene HIV-1 Genotyping System (Visible Genetics, Canada), and 721 by ViroSeq HIV-1 Genotyping System (Perkin Elmer/Applied Biosystems, California). Hybridization detected a significantly higher number of primary mutations which are associated with a high level of drug resistance (p <0.001). Hybridization also detected a higher number of mixtures of wild-type and mutant viruses. There was a good concordance among the three methods, although it was higher between the two sequencing methods. Sequencing determines a higher number of mutations, but hybridization better identifies primary mutations correlated with a high level of drug resistance. Hybridization is more suitable for detecting mixed populations and is easier to implement in clinical laboratories but does not eliminate the need for sequence analysis for detection of drug-resistant HIV.

  11. Development of a rapid HRM genotyping method for detection of dog-derived Giardia lamblia.

    PubMed

    Tan, Liping; Yu, Xingang; Abdullahi, Auwalu Yusuf; Wu, Sheng; Zheng, Guochao; Hu, Wei; Song, Meiran; Wang, Zhen; Jiang, Biao; Li, Guoqing

    2015-11-01

    Giardia lamblia is a zoonotic flagellate protozoan in the intestine of human and many mammals including dogs. To assess a threat of dog-derived G. lamblia to humans, the common dog-derived G. lamblia assemblages A, C, and D were genotyped by high-resolution melting (HRM) technology. According to β-giardin gene sequence, the qPCR-HRM primers BG5 and BG7 were designed. A series of experiments on the stability, sensitivity, and accuracy of the HRM method were also tested. Results showed that the primers BG5 and BG7 could distinguish among three assemblages A, C, and D, which Tm value differences were about 1 °C to each other. The melting curves of intra-assay reproducibility were almost coincided, and those of inter-assay reproducibility were much the same shape. The lowest detection concentration was about 5 × 10(-6)-ng/μL sample. The genotyping results from 21 G. lamblia samples by the HRM method were in complete accordance with sequencing results. It is concluded that the HRM genotyping method is rapid, stable, specific, highly sensitive, and suitable for clinical detection and molecular epidemiological survey of dog-derived G. lamblia.

  12. Neuroanatomical distribution of disease-associated prion protein in cases of bovine spongiform encephalopathy detected by fallen stock surveillance in Japan.

    PubMed

    Okada, Hiroyuki; Iwamaru, Yoshihumi; Imamura, Morikazu; Masujin, Kentaro; Matsuura, Yuichi; Shimizu, Yoshihisa; Kasai, Kazuo; Takata, Masuhiro; Fukuda, Shigeo; Nikaido, Satoshi; Fujii, Kei; Onoe, Sadao; Mohri, Shirou; Yokoyama, Takashi

    2011-11-01

    Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disorder of cattle characterized by accumulation of the disease-associated prion protein (PrP(Sc)) in the central nervous system (CNS). The immunohistochemical patterns and distribution of PrP(Sc) were investigated in the CNS, brains, and spinal cords of 7 naturally occurring BSE cases confirmed by the fallen stock surveillance program in Japan. No animals showed characteristic clinical signs of the disease. Coronal slices of 14 different brain areas in each case were immunohistochemically analyzed using an anti-prion protein antibody. Immunolabeled PrP(Sc) deposition was widely observed throughout each brain and spinal cord. Intense PrP(Sc) deposition was greater in the thalamus, brainstem, and spinal cord of the gray matter than in the neocortices. The topographical distribution pattern and severity of PrP(Sc) accumulation were mapped and plotted as immunohistochemical profiles of the different brain areas along the caudal-rostral axis of the brain. The distribution pattern and severity of the immunolabeled PrP(Sc) in the CNS were almost the same among the 7 cases analyzed, suggesting that the naturally occurring cases in this study were at the preclinical stage of the disease. Immunohistochemical mapping of the PrP(Sc) deposits will be used to clarify the different stages of BSE in cattle.

  13. Hepatitis E Virus Genotype 4 Sequences Detected in Sewage from Treatment Plants of China.

    PubMed

    Li, Heng; Li, Wei; She, Ruiping; Yu, Liang; Wu, Qiaoxing; Yang, Jingling; Hu, Fengjiao; Soomro, Majid Hussain; Shi, Ruihan; Hao, Wenzhuo; Zhao, Yue; Mao, Jingjing

    2017-06-01

    The aim of this study was to investigate the occurrence of hepatitis E virus (HEV) in sewage samples in Shen Zhen, China. Sewage samples were collected from 152 sewage plants including livestock sewage, domestic sewage and treated sewage from May to July of 2015. Two of 152 samples were HEV positive (1.32%) from the livestock sewage plants. Partial ORF2 fragments of HEV were sequenced and a phylogenetic tree was constructed using MEGA5.1. Blast and phylogenetic analyses showed that both of these two sequences belonged to HEV Genotype 4. To the best of our knowledge, this is the first study on the molecular characterization of HEV in wastewater in China and the first time to detect Genotype 4 in the sewage. Results from this study indicate that the possibilities of sporadic infections of HEV should be emphasized because virus still has the possibility to be circulating in the sewage in China.

  14. Detection of HCV genotypes 1b and 2a by a reverse transcription loop-mediated isothermal amplification assay.

    PubMed

    Zhao, Na; Liu, Jinxia; Sun, Dianxing

    2016-12-09

    Hepatitis C virus (HCV) genotypes 1b and 2a are the major cause of liver disease in northern China; however, conventional detection tools are labor-consuming, technically demanding, and costly. Here, we assessed the specificity, sensitivity, and clinical utility of reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detection of HCV genotypes 1b and 2a. Firstly, clinical samples were collected from HCV genotype 1b and 2a infected patients and the RNA were extracted. Secondly, specificity of RT-LAMP assay for detection HCV genotypes 1b and 2a were tested against viral genomes of other hepatitis viruses. Sensitivity of RT-LAMP assay was determined using serial dilutions of standard HCV genotypes 1b and 2a. The amplified products were detected by both electrophoresis and calcein/Mn(2+) -dependent visual methods. Finally, we compared the clinical detection rate of RT-LAMP to that of real-time PCR. RT-LAMP assay showed high specificity to detect HCV genotypes 1b and 2b since there was no cross-reactivity with other hepatitis viruses. Sensitivity of RT-LAMP was 100 IU/mL for both genotypes detected by either electrophoresis or calcein/Mn(2+) -dependent visual methods. The detection rate of RT-LAMP assay in clinical samples was also comparable to that of real-time PCR without significant difference between the both assays. This study proposes a newly developed RT-LAMP assay for detection of HCV genotypes 1b and 2a. RT-LAMP is highly specific, sensitive, and simple diagnostic tool which would be useful for screening and early diagnosis of HCV especially in resource-limited environments.

  15. Microarray for Hepatitis B Virus Genotyping and Detection of 994 Mutations along the Genome ▿ †

    PubMed Central

    Gauthier, Marie; Bonnaud, Bertrand; Arsac, Maud; Lavocat, Fabien; Maisetti, Jérôme; Kay, Alan; Simon, François; Zoulim, Fabien; Vernet, Guy

    2010-01-01

    Genome analysis of hepatitis B virus (HBV) in patient sera is helpful for monitoring treatment. We developed an improved version of a DNA microarray to identify HBV genotypes and to detect mutations of interest in the S, Pol, Core, and X genes. It includes an automated software analysis of fluorescence values for simpler, more robust data interpretation. In this version, probes were added to identify genotype H, to analyze 155 additional positions, and to detect 561 additional polymorphisms. Sequences were added to the alignments to resolve hybridization problems due to natural polymorphisms in the vicinity of important codons. The duplex PCR protocol allowed whole-genome analysis in a single tube. An alternative nested-PCR protocol allowed genotyping and mutations in S and reverse transcriptase (rt) genes in patients with low viral loads, as demonstrated in patients with less than 400 HBV genome copies/ml. Reproducibility was high, with variation coefficients lower than 3%. Only 0.57% of 20,771 codons from 253 samples could not be identified. The concordance with Sanger sequencing for the identification of codons improved from 92.8% to 95.7% with the improved version. Concordance was higher than 91% for codons associated with resistance to lamivudine, emtricitabine, telbivudine, famciclovir, entecavir, and tenofovir with vaccine escape and for pre-Core mutants. Concordance was lower for adefovir resistance mutations (68.6%) and mutations in the basal core promoter (60.3%), probably because hybridization efficiency was affected by the low GC content of the probes. A concordance of 93.7% with sequencing for genotype identification was observed in 190 specimens, lower than that obtained with the first version, possibly due to mixed virus populations. PMID:20826635

  16. A single-nucleotide-polymorphism-based genotyping assay for simultaneous detection of different carbendazim-resistant genotypes in the Fusarium graminearum species complex

    PubMed Central

    Zhang, Hao; Brankovics, Balázs; van der Lee, Theo A.J.; Waalwijk, Cees; van Diepeningen, Anne A.D.; Xu, Jin; Xu, Jingsheng

    2016-01-01

    The occurrence resistance to methyl benzimidazole carbamates (MBC)-fungicides in the Fusarium graminearum species complex (FGSC) is becoming a serious problem in the control of Fusarium head blight in China. The resistance is caused by point mutations in the β2-tubulingene. So far, five resistant genotypes (F167Y, E198Q, E198L, E198K and F200Y) have been reported in the field. To establish a high-throughput method for rapid detection of all the five mutations simultaneously, an efficient single-nucleotide-polymorphism-based genotyping method was developed based on the Luminex xMAP system. One pair of amplification primers and five allele specific primer extension probes were designed and optimized to specially distinguish the different genotypes within one single reaction. This method has good extensibility and can be combined with previous reported probes to form a highly integrated tool for species, trichothecene chemotype and MBC resistance detection. Using this method, carbendazim resistant FGSC isolates from Jiangsu, Anhui and Sichuan Province in China were identified. High and moderate frequencies of resistance were observed in Jiangsu and Anhui Province, respectively. Carbendazim resistance in F. asiaticum is only observed in the 3ADON genotype. Overall, our method proved to be useful for early detection of MBC resistance in the field and the result aids in the choice of fungicide type. PMID:27812414

  17. Toxoplasma gondii in free-ranging wild small felids from Brazil: molecular detection and genotypic characterization.

    PubMed

    Cañón-Franco, W A; Araújo, F A P; López-Orozco, N; Jardim, M M A; Keid, L B; Dalla-Rosa, C; Cabral, A D; Pena, H F J; Gennari, S M

    2013-11-08

    Brazil harbors the largest number of wild Neotropical felid species, with ten of the twelve species recorded in the American continent. Although these animals are considered to be definitive hosts for Toxoplasma gondii, there are few descriptions of the parasite in these species. Here, we performed a molecular detection of T. gondii by amplification of the marker ITS-1 from tissue samples obtained from 90 free-ranging wild small Neotropical felids from Rio Grande do Sul - Brazil. Of the sampled animals, 34.4% (n=31) were positive including the species Puma yagouaroundi - jaguarondi (9/22), Leopardus geoffroyi - Geoffroy's cat (6/22), Leopardus tigrinus - oncilla (8/28), Leopardus wiedii - margay (6/10), Leopardus pardalis - ocelot (1/1) and Leopardus colocolo - Pampas cat (1/7). Toxoplasma DNA was detected with a frequency of 14.6% (63/433) in primary samples of tongue (16/56), brain (8/43), skeletal muscle (15/83), heart (7/63), diaphragm (3/56), vitreous humor (2/44), eye muscle (6/44) and eyeball (6/44). Multilocus PCR-RFLP genotyping of eleven small Neotropical felids using the molecular markers SAG1, 5'3'SAG2, alt. SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico and CS3 allowed the partial characterization of eight genotypes. We fully characterized two new genotypes that have not been described previously in Brazil (Lw#31Tn from L. wiedii and Py#21Sm from P. yagouaroundi) and one genotype Py#56Br from P. yagouaroundi that has been described previously in isolates from cats, dogs and capybaras from São Paulo state. This study constitutes the first detection and genotypic characterization of T. gondii in free-ranging felids in Brazil, demonstrating the occurrence of the parasite in wild populations and suggesting its potential transmissibility to humans and other domestic and wild animals. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. [HPV genotypes prevalence in México and worldwide detected by Linear Array].

    PubMed

    Flores-Miramontes, María G; Torres-Reyes, Luis A; Aguilar-Lemarroy, Adriana; Vallejo-Ruíz, Verónica; Piña-Sánchez, Patricia; Cortés-Gutiérrez, Elva; Reyes-Leyva, Julio; Jave-Suárez, Luis Felipe

    2015-01-01

    Infection with human papillomavirus (HPV) is the main factor associated with the development of cervical cancer (CC). Knowing about the prevalence of HPVs at different stages in the development of CC is important for determining the HPV oncogenic risk, the development of screening strategies, the evaluation of prevention programs, and also for vaccine designing. This paper is a meta-analysis of HPV prevalence worldwide and in Mexico from studies using the Linear Array® HPV Genotyping Test as a diagnostic test (it is the commercial test that, up to date, identifies the largest number of HPV genotypes in a single sample) in DNA of cervical samples from women with normal cytology, with low grade squamous intraepithelial lesions (LGSIL), with high grade squamous intraepithelial lesions (HGSIL) and with CC. The most prevalent genotypes after HPV-16 and -18 in women with CC varies depending on geographic region, which supports the need to develop detection and prevention strategies according to the characteristics of the population.

  19. Rotavirus Diarrhea among Children in Taiz, Yemen: Prevalence—Risk Factors and Detection of Genotypes

    PubMed Central

    Al-Badani, Abdulmalik; Al-Areqi, Leena; Majily, Abdulatif; AL-Sallami, Saleh; AL-Madhagi, Anwar; Amood AL-Kamarany, Mohammed

    2014-01-01

    Diarrheal diseases are a great public health problem; they are among the most causes leading to morbidity and mortality of infants and children particularly in developing countries and even in developed countries. Rotavirus is the most common cause of severe gastroenteritis in infants and young children in both developed and developing countries. The purpose of this study was to determine the incidence rate of Rotavirus infection, its genotypes, and risk factors among children with diarrhea in Taiz, Yemen. 795 fecal samples were collected from children (less than 5 years old), suffering from diarrhea and attending the Yemeni-Swedish Hospital (YSH) in Taiz , Yemen, from November 2006 to February 2008. Rotavirus was detected by enzyme linkage immunosorbent assay (ELISA) on stool specimens of children. Genotypes of Rotavirus were characterized by reverse transcriptase-polymerase chain reaction (RT-PCR) and polyacrylamide gel electrophoresis (PAGE). The results showed that 358 (45.2%) were Rotavirus-positive and the most prevalent genotypes were G2P[4] (55%), followed by G1P[8] (15%). In addition, Rotavirus was found through the whole year; however, higher frequency during the summer season (53.4%) and lower frequency during the winter season (37.1%). PMID:25197286

  20. A rapid fluorescence polarization-based method for genotypic detection of drug resistance in Mycobacterium tuberculosis.

    PubMed

    Sun, Yisuo; Li, Shufen; Zhou, Lin; Zhou, Lin; Zhong, Qiu; Fang, Shisong; Chen, Tao; Bi, Lijun; Mat, Wai-Kin; Zhao, Cunyou; Xue, Hong

    2014-05-01

    Rapid detection of drug-resistant Mycobacterium tuberculosis is critical to the effective early treatment and prevention of the transmission of tuberculosis. However, conventional drug susceptibility tests for M. tuberculosis require up to several weeks. In the present study, the One Label Extension genotyping method was adapted for rapid detection of drug resistance-associated sequence variations in six genes of M. tuberculosis, viz. rpoB, rpsL, rrs, embB, katG, or inhA. The method utilizes polymerase chain reaction amplified fragments of the drug resistant genes as reaction templates, and proceeds with template-directed primer extension incorporating a fluorescence-labeled nucleotide, which is then measured by fluorescence polarization. A total of 121 M. tuberculosis isolates from clinical sputum specimens were examined by this genotyping method and verified by direct sequencing of polymerase chain reaction amplicons harboring previously reported mutational sites associated with M. tuberculosis drug resistance. Based on phenotyping results obtained from microbiology-based drug susceptibility tests, the sensitivity, specificity, and test efficiency estimated for One Label Extension assays were respectively 83.9 %, 95.5 %, and 92.4 % with ropB in rifampin resistance, 67.3 %, 97.1 %, and 84.3 % with rpsL and rrs in streptomycin resistance, 60.0 %, 96.0 %, and 91.4 % with embB in ethambutol resistance, 68.4 %, 94.9 %, and 86.3 % with inhA and katG in isoniazid resistance, and 74.1 %, 98.9 %, and 93.2 % in multiple drug resistance defined as resistance to at least both isoniazid and rifampin. In conclusion, examination of clinical sputum specimens by One Label Extension based genotyping provides a valid method for the rapid molecular detection of drug-resistant M. tuberculosis.

  1. Detection of clinically relevant genotypes of vancomycin-resistant enterococci in nosocomial surveillance specimens by PCR.

    PubMed

    Jayaratne, P; Rutherford, C

    1999-06-01

    This study evaluated a PCR method for the rapid detection of clinically significant genotypes of vancomycin-resistant enterococci (VRE) in nosocomial surveillance specimens. Detection of the vanA and vanB genes by multiplex PCR using 657 specimens that showed presumptive growth of VRE on bile esculin azide agar containing 6 mg of vancomycin/liter was compared to the conventional method. The diagnostic values for the PCR compared to the phenotypic method were as follows: 99.8% specificity, 95.4% sensitivity, 98.8% positive predictive value, and 99.3% negative predictive value. The average cost per test for PCR is $8.26, compared to $9.45 for the phenotypic method. The average turnaround time for detecting a VRE is 48 h for PCR, compared to 96 h for the conventional method.

  2. Genotyping of DNA using sequence-specific methyltransferases followed by immunochemical detection.

    PubMed

    López, Osvaldo J; Quintanar, André; Padhye, Nisha V; Nelson, Michael

    2003-01-01

    Modern molecular genetics relies on the ability to map the positions of genes on chromosomes, relative to known DNA markers. The first such DNA markers described were Restriction Fragment Length Polymorphisms, but any restriction endonuclease used for RFLP mapping is just one member of a restriction-modification pair. For each restriction endonuclease, there is a companion methyltransferase (MTase) that has the same DNA sequence specificity. Therefore, in principle, it should be possible to use MTases rather than restriction enzymes to detect polymorphic sites in DNA. We have used sequence-specific DNA MTases to detect polym orphisms in closely related viral pathogens. If at least one MTase recognition site is present in PCR-amplified DNA, then methyl groups are incorporated; if no MTase site is present, then methyl groups are not incorporated. When several different sequence-specific DNA MTase reactions are carried out, the pattern of methyl incorporation defines a DNA MTase genotype. DNA MTase Genotyping (DMG) can be used to rapidly diagnose heritable or infectious diseases, to immunochemically detect DNA at defined 2 to 8 base pair sites, or to characterize the amplicons by constructing ordered maps.

  3. Molecular Detection and Genotyping of Chlamydia psittaci in Captive Psittacines from Costa Rica

    PubMed Central

    Sheleby-Elías, Jessica; Solórzano-Morales, Ántony; Romero-Zuñiga, Juan José

    2013-01-01

    Oropharyngeal and cloacal swabs from 117 captive psittacine birds presented at veterinary clinics (88) and from shelters/rescue centers of wildlife (29) were collected to determine the prevalence of C. psittaci in captive birds in Costa Rica. Samples were collected during 2009 from a total of 19 different species of parrots, with Ara macao (33), Amazona autumnalis (24), Amazona ochrocephala (21), and Ara ararauna (8) being the most representative species sampled. C. psittaci was detected in four (3.4%) birds using molecular detection (PCR). The positive samples belonged to birds presented at veterinary clinics; three of them were Ara macao and one Amazona ochrocephala. Three birds were adults; all positive birds showed no symptoms of illness and lived in homes with other birds, two in San José and two in Heredia. Sequencing was used to confirm the PCR positive results, showing that two samples of C. psittaci belonged to genotype A, representing the first report of the presence of this genotype in Costa Rica. The detection of this bacterium in captive psittacine birds shows that there is a potential risk for people living or having contact with them and that there is a possibility of infecting other birds. PMID:24163776

  4. A broadly reactive monoclonal antibody detects multiple genotypes of hepatitis B virus X protein.

    PubMed

    Wei, Lili; Shen, Zhongliang; Zhao, Xue; Wu, Yanxin; Liu, Wei; Zhang, Junqi; Xie, Youhua; Liu, Jing

    2014-10-01

    A highly specific and broadly reactive monoclonal antibody against hepatitis B virus X (HBx) protein was developed that detected, in both immunoblotting and immunofluorescence, HBx proteins of seven of the eight currently known genotypes of HBV, which were overexpressed in cultured cells. Evaluation of HBx expression levels in cultured hepatocytes using this monoclonal antibody showed that cells transiently and stably transfected with HBV genomes expressed far less HBx protein than cells transiently transfected with an HBx overexpression plasmid routinely used for studying HBx functions. The availability of such sensitive and broadly reactive monoclonal antibodies against HBx will enable more-quantitative studies of HBx functions.

  5. [Investigation on HPV DNA detection and genotyping practices used in French laboratories in 2009].

    PubMed

    Heard, Isabelle; Favre, Michel; Fihman, Valentine

    2011-01-01

    The French National reference Laboratory for Human papillomavirus (HPV) performed in 2009 a national study in order to review the methods used to detect and identify HR HPV genotypes in microbiology laboratories. Results from this study show a great diversity in volumes of samples treated in laboratories. Among clinical indications, the most frequent is a result of ASC-US at a Pap smear. This indication in the only one covered by the National Public Insurance System and is mostly performed in laboratories from private sector. Other indications mainly correspond to research programs and are performed in public Hospitals. This study allowed also to review the adequacy between the liquid based cytology samples and the assays used for direct detection of HR HPV or identification of the genotypes present in the sample. The right tests were not carried in the right solution storage according to the recommendations from different HPV testing assays. National recommendations should be elaborated in order to improve the performance of the test used.

  6. Novel genotype of Ehrlichia canis detected in samples of human blood bank donors in Costa Rica.

    PubMed

    Bouza-Mora, Laura; Dolz, Gaby; Solórzano-Morales, Antony; Romero-Zuñiga, Juan José; Salazar-Sánchez, Lizbeth; Labruna, Marcelo B; Aguiar, Daniel M

    2017-01-01

    This study focuses on the detection and identification of DNA and antibodies to Ehrlichia spp. in samples of blood bank donors in Costa Rica using molecular and serological techniques. Presence of Ehrlichia canis was determined in 10 (3.6%) out of 280 blood samples using polymerase chain reaction (PCR) targeting the ehrlichial dsb conserved gene. Analysis of the ehrlichial trp36 polymorphic gene in these 10 samples revealed substantial polymorphism among the E. canis genotypes, including divergent tandem repeat sequences. Nucleotide sequences of dsb and trp36 amplicons revealed a novel genotype of E. canis in blood bank donors from Costa Rica. Indirect immunofluorescence assay (IFA) detected antibodies in 35 (35%) of 100 serum samples evaluated. Thirty samples showed low endpoint titers (64-256) to E. canis, whereas five sera yielded high endpoint titers (1024-8192); these five samples were also E. canis-PCR positive. These findings represent the first report of the presence of E. canis in humans in Central America. Copyright © 2016 Elsevier GmbH. All rights reserved.

  7. Molecular detection of Rickettsia felis, Rickettsia typhi and two genotypes closely related to Bartonella elizabethae.

    PubMed

    De Sousa, Rita; Edouard-Fournier, Pierre; Santos-Silva, Margarida; Amaro, Fatima; Bacellar, Fatima; Raoult, Didier

    2006-10-01

    A total of 56 fleas were collected from mice, rats, and one hedgehog in national parks of mainland Portugal and the Madeira Island. All fleas were tested for the presence of bacteria of the genera Rickettsia and Bartonella using PCR assays. In fleas from mainland Portugal, we detected Rickettsia felis in one Archaeopsylla erinacei maura flea and in one Ctenophtalmus sp. In five Leptopsylla segnis fleas taken from rats in the Madeira Island, we identified Rickettsia typhi. In addition, in four fleas from the genera Ornithophaga and Stenoponia collect from mice and a rat in mainland Portugal, we detected the presence of two new Bartonella genotypes closely related to Bartonella elizabethae. Our findings emphasize the potential risk of flea-transmitted infections in mainland Portugal and the Madeira archipelago, and extend our knowledge of the potential flea vectors of human pathogens.

  8. Detection and genotype analysis of human papillomavirus in non-small cell lung cancer patients.

    PubMed

    Sarchianaki, Emmanouela; Derdas, Stavros P; Ntaoukakis, Markos; Vakonaki, Elena; Lagoudaki, Eleni D; Lasithiotaki, Ismini; Sarchianaki, Anna; Koutsopoulos, Anastasios; Symvoulakis, Emmanouil K; Spandidos, Demetrios A; Antoniou, Katerina M; Sourvinos, George

    2014-04-01

    Although the role of human papillomavirus (HPV) in the development of uterine cervical cancer is well established, the role of HPV in lung carcinogenesis remains controversial. The detection rates of HPV DNA are subject to a wide variation from 0 to 100%. This is partly influenced by the detection techniques employed. To elucidate the impact of HPV infection on lung parenchyma, we analyzed 100 non-small cell lung cancer (NSCLC) specimens (39 squamous cell carcinomas, 50 adenocarcinomas, 5 samples with characteristics of both squamous cell and adenocarcinoma, 5 undifferentiated and 1 large cell carcinoma) from the region of Crete, Greece. Sixteen non-cancerous samples served as the negative controls. DNA was extracted from 100 paraffin-embedded tissue sections obtained from NSCLC patients. The specimens were examined for the detection of HPV DNA by Real-Time PCR using GP5+/GP6+ primers. Furthermore, the HPV-positive samples were subjected to genotyping. In contrast to the absence of viral genomes in the control samples, HPV DNA was detected in 19 NSCLC specimens (19%). In particular, 4 squamous cell carcinomas, 12 adenocarcinomas, 1 sample with characteristics of both squamous cell and adenocarcinoma, and 2 undifferentiated samples were HPV-positive. The distribution of HPV genotypes was as follows: HPV 16: eight cases (42.1%); HPV 11: three cases (15.8%); HPV 6: one case (5.2%); HPV 59: one case (5.2%); HPV 33: two cases (10.5%); HPV 31: two cases (10.5%) and HPV 18: two cases (10.5%). The presence of HPV in the tumor samples provides evidence of the potential role of HPV in NSCLC and strongly argues for additional research on this issue.

  9. Genotyping of Norovirus strains detected in outbreaks between April 2002 and March 2003 in Osaka City, Japan.

    PubMed

    Seto, Yoshiyuki; Iritani, Nobuhiro; Kubo, Hideyuki; Kaida, Atsushi; Murakami, Tsukasa; Haruki, Kosuke; Nishio, Osamu; Ayata, Minoru; Ogura, Hisashi

    2005-01-01

    Noroviruses (NVs) are the major cause of food- and waterborne nonbacterial gastroenteritis in Japan. Between April 2002 and March 2003, a total of 111 fecal specimens from 40 outbreaks of acute nonbacterial gastroenteritis in Osaka City, Japan were subject to NV detection. Seventy-two samples (64.9%) from 31 outbreaks (77.5%) were NV positive by a real time reverse transcription (RT)-PCR assay. To further determine the genotype of individual NV strains, we sequenced the capsid N-terminal/shell (N/S) domain of some representative strains from each outbreak. The 51 NV strains detected in this study were segregated into 15 genotypes (6 in genogroup I and 9 in genogroup II), and GII/5 genotype NV was a dominant outbreak genotype.

  10. Quick and sensitive SPR detection of prion disease-associated isoform (PrP(Sc)) based on its self-assembling behavior on bare gold film and specific interactions with aptamer-graphene oxide (AGO).

    PubMed

    Lou, Zhichao; Wan, Jinfeng; Zhang, Xiaohong; Zhang, Haiqian; Zhou, Xiaoyan; Cheng, Su; Gu, Ning

    2017-09-01

    Herein, we constructed a novel sandwich surface plasmon resonance (SPR) detection assay for sensitive prion disease-associated isoform (PrP(Sc)) detection, utilizing bare gold film and apatamer-graphene oxide (AGO). Due to the self-assembling behavior of PrP(Sc) on gold surface, the non-modified gold surface can be directly used as sensing surface for the quick detection, for the purpose to avoid the interference from the traditional, complex and changeable probe-modified sensing surface. And due to the highly specific affinity of AGO towards PrP(Sc), the sandwich type SPR sensor exhibits excellent analytical performance towards the discrimination and quantitation of PrP(Sc). A good linear relationship was obtained between SPR responses and the logarithm of PrP(Sc) concentrations over a range of 0.001-1ng/mL. The detection sensitivity for PrP(Sc) was improved by ∼156 orders of AGO compared with SPR direct detection format. Besides, morphological changes of the sensing film surfaces were investigated by high resolution AFM imaging, confirming the capture of PrP(Sc) molecules and their further specific recognition by AGO. The specificity of the present biosensor was also investigated by PrP(C) and other regents as controls. By compared with other reported methods, the AGO enhanced sandwich SPR assay was confirmed to be efficient, sensitive, and with wide working range. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Comparison of Cobas® HPV and Anyplex™ II HPV28 assays for detecting and genotyping human papillomavirus.

    PubMed

    Pasquier, Christophe; Sauné, Karine; Raymond, Stéphanie; Boisneau, Jérôme; Courtade, Monique; Izopet, Jacques

    2017-01-01

    Persistent infection with high-risk human papillomavirus (HPV-HR) is a recognized cause of cervical cancer. The aim of this study was to compare analytical and clinical performances of the Cobas® HPV and Anyplex™ II HPV28 assays for HPV detection and genotyping. A total of 94 cervical samples were tested. For HPV-HR, the results agreed very well (94.68%), with 100% agreement when detecting CIN2+. The Anyplex™ II HPV28 assay detected more genotypes than the Cobas® HPV Test, but their clinical performances were similar. Copyright © 2016. Published by Elsevier Inc.

  12. Molecular and statistical approaches to the detection and correction of errors in genotype databases

    SciTech Connect

    Brzustowicz, L.M.; Xie, X.; Merette, C.; Townsend, L.; Gilliam, T.C.; Ott, J. )

    1993-11-01

    Errors in genotyping data have been shown to have a significant effect on the estimation of recombination fractions in high-resolution genetic maps. Previous estimates of errors in existing databases have been limited to the analysis of relatively few markers and have suggested rates in the range 0.5%-1.5%. The present study capitalizes on the fact that within the Centre d'Etude du Polymorphisme Humain (CEPH) collection of reference families, 21 individuals are members of more than one family, with separate DNA samples provided by CEPH for each appearance of these individuals. By comparing the genotypes of these individuals in each of the families in which they occur, an estimated error rate of 1.4% was calculated for all loci in the version 4.0 CEPH database. Removing those individuals who were clearly identified by CEPH as appearing in more than one family resulted in a 3.0% error rate for the remaining samples, suggesting that some error checking of the identified repeated individuals may occur prior to data submission. An error rate of 3.0% for version 4.0 data was also obtained for four chromosome 5 markers that were retyped through the entire CEPH collection. The effects of these errors on a multipoint map were significant, with a total sex-averaged length of 36.09 cM with the errors, and 19.47 cM with the errors corrected. Several statistical approaches to detect and allow for errors during linkage analysis are presented. One method, which identified families containing possible errors on the basis of the impact on the maximum lod score, showed particular promise, especially when combined with the limited retyping of the identified families. The impact of the demonstrated error rate in an established genotype database on high-resolution mapping is significant, raising the question of the overall value of incorporating such existing data into new genetic maps. 15 refs., 8 tabs.

  13. The power of gene-based rare variant methods to detect disease-associated variation and test hypotheses about complex disease.

    PubMed

    Moutsianas, Loukas; Agarwala, Vineeta; Fuchsberger, Christian; Flannick, Jason; Rivas, Manuel A; Gaulton, Kyle J; Albers, Patrick K; McVean, Gil; Boehnke, Michael; Altshuler, David; McCarthy, Mark I

    2015-04-01

    Genome and exome sequencing in large cohorts enables characterization of the role of rare variation in complex diseases. Success in this endeavor, however, requires investigators to test a diverse array of genetic hypotheses which differ in the number, frequency and effect sizes of underlying causal variants. In this study, we evaluated the power of gene-based association methods to interrogate such hypotheses, and examined the implications for study design. We developed a flexible simulation approach, using 1000 Genomes data, to (a) generate sequence variation at human genes in up to 10K case-control samples, and (b) quantify the statistical power of a panel of widely used gene-based association tests under a variety of allelic architectures, locus effect sizes, and significance thresholds. For loci explaining ~1% of phenotypic variance underlying a common dichotomous trait, we find that all methods have low absolute power to achieve exome-wide significance (~5-20% power at α = 2.5 × 10(-6)) in 3K individuals; even in 10K samples, power is modest (~60%). The combined application of multiple methods increases sensitivity, but does so at the expense of a higher false positive rate. MiST, SKAT-O, and KBAC have the highest individual mean power across simulated datasets, but we observe wide architecture-dependent variability in the individual loci detected by each test, suggesting that inferences about disease architecture from analysis of sequencing studies can differ depending on which methods are used. Our results imply that tens of thousands of individuals, extensive functional annotation, or highly targeted hypothesis testing will be required to confidently detect or exclude rare variant signals at complex disease loci.

  14. The Power of Gene-Based Rare Variant Methods to Detect Disease-Associated Variation and Test Hypotheses About Complex Disease

    PubMed Central

    Fuchsberger, Christian; Flannick, Jason; Rivas, Manuel A.; Gaulton, Kyle J.; Albers, Patrick K.; McVean, Gil; Boehnke, Michael; Altshuler, David; McCarthy, Mark I.

    2015-01-01

    Genome and exome sequencing in large cohorts enables characterization of the role of rare variation in complex diseases. Success in this endeavor, however, requires investigators to test a diverse array of genetic hypotheses which differ in the number, frequency and effect sizes of underlying causal variants. In this study, we evaluated the power of gene-based association methods to interrogate such hypotheses, and examined the implications for study design. We developed a flexible simulation approach, using 1000 Genomes data, to (a) generate sequence variation at human genes in up to 10K case-control samples, and (b) quantify the statistical power of a panel of widely used gene-based association tests under a variety of allelic architectures, locus effect sizes, and significance thresholds. For loci explaining ~1% of phenotypic variance underlying a common dichotomous trait, we find that all methods have low absolute power to achieve exome-wide significance (~5-20% power at α=2.5×10-6) in 3K individuals; even in 10K samples, power is modest (~60%). The combined application of multiple methods increases sensitivity, but does so at the expense of a higher false positive rate. MiST, SKAT-O, and KBAC have the highest individual mean power across simulated datasets, but we observe wide architecture-dependent variability in the individual loci detected by each test, suggesting that inferences about disease architecture from analysis of sequencing studies can differ depending on which methods are used. Our results imply that tens of thousands of individuals, extensive functional annotation, or highly targeted hypothesis testing will be required to confidently detect or exclude rare variant signals at complex disease loci. PMID:25906071

  15. Immunohistochemical detection of disease-associated prion protein in the intestine of cattle naturally affected with bovine spongiform encephalopathy by using an alkaline-based chemical antigen retrieval method.

    PubMed

    Okada, Hiroyuki; Iwamaru, Yoshihumi; Imamura, Morikazu; Masujin, Kentaro; Yokoyama, Takashi; Mohri, Shirou

    2010-11-01

    An alkaline-based chemical antigen retrieval pretreatment step was used to enhance immunolabeling of disease-associated prion protein (PrP(Sc)) in formalin-fixed and paraffin-embedded tissue sections from cattle naturally affected with bovine spongiform encephalopathy (BSE). The modified chemical method used in this study amplified the PrP(Sc) signal by unmasking PrP(Sc) compared with the normal cellular prion protein. In addition, this method reduced nonspecific background immunolabeling that resulted from the destruction of the residual normal cellular form of prion protein, and reduced the treatment time compared with the usual autoclave pretreatment step. Immunolabeled PrP(Sc) was thereby clearly detected in the myenteric plexus of the ileum in naturally occurring BSE cattle.

  16. [Effectiveness of human papillomavirus genotyping for detection of high-grade anal intraepithelial neoplasia compared to anal cytology].

    PubMed

    Padilla-España, Laura; Repiso-Jiménez, Juan Bosco; Fernández-Sánchez, Fernando; Pereda, Teresa; Rivas-Ruiz, Francisco; Fernández-Morano, Teresa; de la Torre-Lima, Javier; Palma, Fermín; Redondo, Maximino; de Troya-Martín, Magdalena

    2016-01-01

    The incidence of high-grade anal intraepithelial neoplasia (HGAIN) -with an aetiological based on high-risk types of human papillomavirus- is increasing in some high-risk groups. Screening for HGAIN includes routine anal cytology and, more recently, HPV genotyping. The main objective of this study was to determine the sensitivity and specificity of anal cytology and HPV genotyping for the detection of HGAIN. This is a study to determine the correlation of cytological and microbiological findings with anal biopsy findings in a cohort of patients at high risk of developing AIN referred to the department of sexually transmitted infections of the Hospital Costa del Sol, Spain, between January 2008 and December 2014. Of the 151 patients subjected to screening, a total of 92 patients, all of them with the result of three screening test (anal cytology, genotyping and biopsy) were included in the study. Just under two-thirds (62%) of them were HIV-positive. The sensitivity and specificity of anal cytology to detect HGAIN were 52.8 and 85.7%, respectively (k: 0.328), and 78 and 62.8% to detect two or more HPV oncogenic genotypes (k: 0.417). The detection of oncogenic HPV genotypes allowed the identification of 23 new cases of HGAIN that had been underdiagnosed with anal cytology, with 14 cases containing at least three high-risk genotypes. Anal cytology did not show enough sensitivity in HGAIN screening. HPV genotyping has shown to be a useful tool to detect HGAIN cases, although it could lead to an over-diagnosis as a solitary screening procedure. Copyright © 2016 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  17. Detection of oncogenic human papillomavirus genotypes on spermatozoa from male partners of infertile couples.

    PubMed

    Schillaci, Rosaria; Capra, Giuseppina; Bellavia, Carmela; Ruvolo, Giovanni; Scazzone, Concetta; Venezia, Renato; Perino, Antonio

    2013-11-01

    To evaluate the prevalence of human papillomavirus (HPV) sperm infection and its correlation with sperm parameters in patients who attended a fertility clinic. Cross-sectional clinical study. University-affiliated reproductive medicine clinic. A total of 308 male partners of couples undergoing in vitro fertilization techniques. Specimens of semen were collected from all patients. Sperm parameters were evaluated according to the World Health Organization manual. The presence of HPV DNA was researched by the combined use of two HPV assays and a highly sensitive nested polymerase chain reaction assay followed by HPV genotyping. To examine whether HPV was associated with the sperm, in situ hybridization (ISH) analysis was performed. Results of HPV investigation were compared with sperm parameters and ISH analysis. Twenty-four out of 308 semen samples (7.8%) were HPV DNA positive, but HPV infection did not seem to affect semen quality. Moreover, ISH revealed a clear HPV localization at the equatorial region of sperm head in infected samples. Oncogenic HPV genotypes were detected on spermatozoa from asymptomatic subjects, but a role of the infection in male infertility was not demonstrated. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  18. Towards accurate detection and genotyping of expressed variants from whole transcriptome sequencing data.

    PubMed

    Duitama, Jorge; Srivastava, Pramod K; Măndoiu, Ion I

    2012-04-12

    Massively parallel transcriptome sequencing (RNA-Seq) is becoming the method of choice for studying functional effects of genetic variability and establishing causal relationships between genetic variants and disease. However, RNA-Seq poses new technical and computational challenges compared to genome sequencing. In particular, mapping transcriptome reads onto the genome is more challenging than mapping genomic reads due to splicing. Furthermore, detection and genotyping of single nucleotide variants (SNVs) requires statistical models that are robust to variability in read coverage due to unequal transcript expression levels. In this paper we present a strategy to more reliably map transcriptome reads by taking advantage of the availability of both the genome reference sequence and transcript databases such as CCDS. We also present a novel Bayesian model for SNV discovery and genotyping based on quality scores. Experimental results on RNA-Seq data generated from blood cell tissue of three Hapmap individuals show that our methods yield increased accuracy compared to several widely used methods. The open source code implementing our methods, released under the GNU General Public License, is available at http://dna.engr.uconn.edu/software/NGSTools/.

  19. Phenotypic- and Genotypic-Resistance Detection for Adaptive Resistance Management in Tetranychus urticae Koch

    PubMed Central

    Kwon, Deok Ho; Kang, Taek-Jun; Kim, Young Ho; Lee, Si Hyeock

    2015-01-01

    Rapid resistance detection is necessary for the adaptive management of acaricide-resistant populations of Tetranychus urticae. Detection of phenotypic and genotypic resistance was conducted by employing residual contact vial bioassay (RCV) and quantitative sequencing (QS) methods, respectively. RCV was useful for detecting the acaricide resistance levels of T. urticae, particularly for on-site resistance detection; however, it was only applicable for rapid-acting acaricides (12 out of 19 tested acaricides). QS was effective for determining the frequencies of resistance alleles on a population basis, which corresponded to 12 nonsynonymous point mutations associated with target-site resistance to five types of acaricides [organophosphates (monocrotophos, pirimiphos-methyl, dimethoate and chlorpyrifos), pyrethroids (fenpropathrin and bifenthrin), abamectin, bifenazate and etoxazole]. Most field-collected mites exhibited high levels of multiple resistance, as determined by RCV and QS data, suggesting the seriousness of their current acaricide resistance status in rose cultivation areas in Korea. The correlation analyses revealed moderate to high levels of positive relationships between the resistance allele frequencies and the actual resistance levels in only five of the acaricides evaluated, which limits the general application of allele frequency as a direct indicator for estimating actual resistance levels. Nevertheless, the resistance allele frequency data alone allowed for the evaluation of the genetic resistance potential and background of test mite populations. The combined use of RCV and QS provides basic information on resistance levels, which is essential for choosing appropriate acaricides for the management of resistant T. urticae. PMID:26545209

  20. Development of a Real-Time Quantitative PCR for Detecting Duck Hepatitis A Virus Genotype C

    PubMed Central

    Huang, Qiuxue; Yue, Hua; Zhang, Bin; Nie, Peiting

    2012-01-01

    Recently, duck hepatitis A virus genotype C (DHAV-C), a causative agent of duck viral hepatitis, has been responsible for increasing economic losses in the duck industry in China and South Korea. In this study, a real-time PCR assay targeting the 2C gene for detecting DHAV-C was developed. The assay was confirmed to be specific and sensitive, and the minimum detection limit was 3.36 × 103 copies per reaction, making this assay suitable for rapid diagnosis of DHAV-C infection from clinical samples. In addition, the dynamics of the viral loads in tissues of specific-pathogen-free (SPF) ducklings infected with DHAV-C were investigated using this method. The DHAV-C could be detected earliest in the liver within 12 h postinfection. Moreover, high viral loads were identified in the heart, liver, spleen, lung, kidney, bursa of Fabricius, thymus, pancreas, brain, and small intestine after 24 h postinfection. Taking the data collectively, the study described in this report is the first to have developed a real-time PCR method for detection of DHAV-C and thus contributes to pathogenicity research. PMID:22855514

  1. Development of a real-time quantitative PCR for detecting duck hepatitis a virus genotype C.

    PubMed

    Huang, Qiuxue; Yue, Hua; Zhang, Bin; Nie, Peiting; Tang, Cheng

    2012-10-01

    Recently, duck hepatitis A virus genotype C (DHAV-C), a causative agent of duck viral hepatitis, has been responsible for increasing economic losses in the duck industry in China and South Korea. In this study, a real-time PCR assay targeting the 2C gene for detecting DHAV-C was developed. The assay was confirmed to be specific and sensitive, and the minimum detection limit was 3.36 × 10(3) copies per reaction, making this assay suitable for rapid diagnosis of DHAV-C infection from clinical samples. In addition, the dynamics of the viral loads in tissues of specific-pathogen-free (SPF) ducklings infected with DHAV-C were investigated using this method. The DHAV-C could be detected earliest in the liver within 12 h postinfection. Moreover, high viral loads were identified in the heart, liver, spleen, lung, kidney, bursa of Fabricius, thymus, pancreas, brain, and small intestine after 24 h postinfection. Taking the data collectively, the study described in this report is the first to have developed a real-time PCR method for detection of DHAV-C and thus contributes to pathogenicity research.

  2. Simultaneous detection and differentiation of three genotypes of Brassica yellows virus by multiplex reverse transcription-polymerase chain reaction.

    PubMed

    Zhang, Xiaoyan; Peng, Yanmei; Wang, Ying; Zhang, Zongying; Li, Dawei; Yu, Jialin; Han, Chenggui

    2016-11-22

    Brassica yellows virus (BrYV), proposed to be a new polerovirus species, three distinct genotypes (BrYV-A, BrYV-B and BrYV-C) have been described. This study was to develop a simple, rapid, sensitive, cost-effective method for simultaneous detection and differentiation of three genotypes of BrYV. In this study, a multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for simultaneous detection and differentiation of the three genotypes of BrYV. The three genotypes of BrYV and Tunip yellows virus (TuYV) could be differentiated simultaneously using six optimized specific oligonucleotide primers, including one universal primer for detecting BrYV, three BrYV genotype-specific primers, and a pair of primers for specific detection of TuYV. Primers were designed from conserved regions of each virus and their specificity was confirmed by sequencing PCR products. The mRT-PCR products were 278 bp for BrYV-A, 674 bp for BrYV-B, 505 bp for BrYV-C, and 205 bp for TuYV. Amplification of three target genotypes was optimized by increasing the PCR annealing temperatures to 62 °C. One to three fragments specific for the virus genotypes were simultaneously amplified from infected samples and identified by their specific molecular sizes in agarose gel electrophoresis. No specific products could be amplified from cDNAs of other viruses which could infect crucifer crops. Detection limits of the plasmids for multiplex PCR were 100 fg for BrYV-A and BrYV-B, 10 pg for BrYV-C, and 1 pg for TuYV, respectively. The mRT-PCR was applied successfully for detection of three BrYV genotypes from field samples collected in China. The simple, rapid, sensitive, and cost-effective mRT-PCR was developed successfully for detection and differentiation of the three genotypes of BrYV.

  3. Evaluation of the accuracy of imputed sequence variant genotypes and their utility for causal variant detection in cattle.

    PubMed

    Pausch, Hubert; MacLeod, Iona M; Fries, Ruedi; Emmerling, Reiner; Bowman, Phil J; Daetwyler, Hans D; Goddard, Michael E

    2017-02-21

    provides accurate genotypes. However, the accuracy of imputation is low in regions where the genome contains large segmental duplications or the coverage with array-derived single nucleotide polymorphisms is poor. Using a reference population that includes individuals from many breeds increases the accuracy of imputation particularly at low-frequency variants. Considering allele dosages rather than best-guess genotypes as explanatory variables is advantageous to detect causal mutations in association studies with imputed sequence variants.

  4. Investigating the Epidemiology of Repeat Chlamydia trachomatis Detection after Treatment by Using C. trachomatis OmpA Genotyping

    PubMed Central

    Kapil, Richa; Press, Christen G.; Hwang, M. Lisa; Brown, LaDraka

    2014-01-01

    Repeat Chlamydia trachomatis detection frequently occurs within months after C. trachomatis infection treatment. The origins of such infection (persistence versus reinfection from untreated or new partners) are varied and difficult to determine. C. trachomatis strains can be differentiated by sequencing the ompA gene encoding the outer membrane protein A (OmpA). We used OmpA genotyping to investigate the epidemiology of repeat C. trachomatis detection after treatment in C. trachomatis-infected subjects seen at a sexually transmitted diseases clinic. Subjects were enrolled, tested for C. trachomatis, treated with azithromycin, and scheduled for a 6-month follow-up for repeat C. trachomatis testing. OmpA genotyping was performed on C. trachomatis-positive urogenital specimens obtained from patients at enrollment and follow-up. The enrollment visit OmpA genotypes for C. trachomatis were determined for 162 subjects (92% female, 94% African American). C. trachomatis was detected at follow-up in 39 subjects (24%). The OmpA genotype distribution at enrollment did not differ in those with versus those without repeat C. trachomatis detection. Of the 35 subjects with C. trachomatis strains genotyped at enrollment and follow-up, 7 (20%) had the same ompA sequence at both visits, while 28 (80%) had discordant sequences. A new sexual partner was reported more often in subjects with discordant C. trachomatis strains than in those with concordant strains (13 [46%] versus 1 [14%]; P = 0.195). Half of the subjects with discordant C. trachomatis strains who reported sexual activity since treatment denied a new sexual partner; 62% of these subjects reported that their partner was treated. Our study demonstrates that most repeat C. trachomatis detections after treatment were new infections with a different C. trachomatis strain rather than reinfection with the same strain. OmpA genotyping can be a useful tool in understanding the origins of repeat C. trachomatis detection after

  5. [Evaluation of GenoType MTBDRplus for the detection of multi-drug-resistant Mycobacterium tuberculosis strains].

    PubMed

    Chikamatsu, Kinuyo; Mizuno, Kazue; Aono, Akio; Yamada, Hiroyuki; Sugamoto, Tetsuhiro; Nishiyama, Hiroyuki; Mitarai, Satoshi

    2011-07-01

    To evaluate GenoType MTBDRplus (Hain Lifescience, Germany) for its capacity to detect the resistance of rifampicin (RFP) and isoniazid (INH). A total of 44 confirmed multi-drug resistant (MDR) and 67 susceptible M. tuberculosis strains were tested for susceptibility to RFP and INH by GenoType MTBDR plus. The core 81bp region of the rpoB gene and the 322bp region of the katG gene and the inhA gene (248bp of which included the promoter and the ORF of the 379bp inhA) were directly sequenced for both MDR-TB and susceptible M. tuberculosis strains, and the mutations were confirmed. Susceptibility was tested by standard proportion method with 1% Ogawa medium. The sensitivities of GenoType MTBDRplus for RFP and INH resistance were 97.7% and 65.9%, respectively. The specificity for RFP and INH was 100%. The sensitivity of GenoType MTBDRplus was almost equivalent to the sequencing method for RFP, but that for INH was slightly inferior to the sequencing without significant difference. Geno Type MTBDRplus detected 97.7% of the mutations of rpoB compared with the direct sequencing. It also detected 24 katG MUT1 (S315T1) (54.5%) and 5 inhA MUT1 (C15T) mutations (11.4%), while the direct sequencing detected an additional 2 (4.5%) katG mutants. The accuracy of GenoType MTBDRplus for the detection of RFP resistance was confirmed to be comparable to that of DST using conventional culture-based methods, while it was less accurate for detection of INH resistance. GenoType MTBDRplus is useful for early diagnosis and infection control for MDR-TB because it has a short turnaround time of approximately 6 hours.

  6. Molecular detection and genotyping of Aphanomyces astaci directly from preserved crayfish samples uncovers the Norwegian crayfish plague disease history.

    PubMed

    Vrålstad, Trude; Strand, David A; Grandjean, Frédéric; Kvellestad, Agnar; Håstein, Tore; Knutsen, Ann Kristin; Taugbøl, Trond; Skaar, Ida

    2014-09-17

    Aphanomyces astaci causes crayfish plague in European freshwater crayfish, but most historical epizootics lack agent isolation and identification. Although declared as crayfish plague outbreaks by the Norwegian Competent Authorities, only presumptive diagnoses without agent isolation exist from Norwegian epizootics until 2005. Molecular methods now allow both A. astaci detection and genotype determination from preserved samples. We therefore aimed to (1) investigate molecularly if A. astaci was involved in a selection of mass-mortality events in Norwegian noble crayfish populations from 1971 to 2004, and (2) determine the eventually involved A. astaci genotype groups both from these historical and also more recent mass-mortality events. DNA was extracted directly from presumptively infected crayfish tissues, and screened by A. astaci specific qPCR. A representative selection of positive samples was confirmed by ITS-sequencing. Finally, genotype determination was performed with microsatellite markers that distinguish all known A. astaci genotype groups. The molecular examination detected A. astaci in crayfish materials from all examined mass-mortality events. The first event in 1971-1974 was caused by the A. astaci genotype group A, presumably the first genotype group that entered Europe more than 150 years ago. All later outbreaks were caused by the A. astaci genotype group B which was introduced to Europe by importation of signal crayfish in the 1960s. The results suggest that molecular methods can verify the involvement of A. astaci in the vast majority of observed crayfish mass mortalities in Europe whenever preserved materials exist. Moreover, microsatellite genotyping can reveal at least parts of the underlying epidemiology.

  7. Toxoplasma gondii and Neospora caninum in wild small mammals: Seroprevalence, DNA detection and genotyping.

    PubMed

    Machačová, Tereza; Ajzenberg, Daniel; Žákovská, Alena; Sedlák, Kamil; Bártová, Eva

    2016-06-15

    Generally, rodents and other small mammals are considered as one of the sources of Toxoplasma gondii or Neospora caninum infection for cats and dogs as the definitive hosts of these two parasites, respectively. The aim of the study was to find out the prevalence of these two parasites in wild small mammals from the Czech Republic and to characterize T. gondii isolates by methods of molecular biology. A total of 621 wild small mammals were caught in the Czech Republic during years 2002-2014. Antibodies to T. gondii were detected by latex agglutination test in six (2.5%) of 240 small mammals (in two A. agrarius and four A. flavicollis). Antibodies to N. caninum were detected by commercially available competitive-inhibition enzyme-linked immunosorbent assay in one A. flavicolis (0.4%). Three of 427 (0.7%) liver samples were positive for T. gondii by PCR while negative for N. caninum. All embryo samples (n=102) were negative for both T. gondii and N. caninum. The three liver samples positive for T. gondii DNA (two from A. flavicollis and one from A. sylvaticus) were genotyped by 15 microsatellite markers and characterized as type II. To our knowledge, this is the first information about genetic characterization of T. gondii isolates in small mammals from Europe and the first detection of N. caninum antibodies in wild rodents from the Czech Republic.

  8. First detection of enteric adenoviruses genotype 41 in recreation spring areas of Taiwan.

    PubMed

    Shih, Yi-Jia; Tao, Chi-Wei; Tsai, Hsin-Chi; Huang, Wen-Chien; Huang, Tung-Yi; Chen, Jung-Sheng; Chiu, Yi-Chou; Hsu, Tsui-Kang; Hsu, Bing-Mu

    2017-06-22

    Human adenoviruses (HAdVs) are DNA viruses found in recreational water, such as water parks and swimming pools. Human adenovirus 41 (HAdV-41) is the most common serotype detected and is a leading cause of acute diarrheal disease. The focus of this study is to determine the prevalence of HAdVs in hot springs. Of 57 samples collected from four different geological sites, 16 samples have shown evidence of HAdVs (28.1%). HAdV-41 and porcine adenovirus 5 (PAdV-5) were the two types isolated, with a greater frequency of HAdV-41, which in other settings has been associated with acute diarrhea. The highest occurrence was found in private hot tubs/Yuya (37.5%), followed by an outlet of hot springs (30.8%); public pools and foot pools shared the same detection rate of 21.4% (3/14). However, there was no evidence supporting a link between water quality indicators and HAdV detection rate. From a phylogenic analysis and BLAST against the NCBI database, it was concluded that HAdV-41 obtained from hot spring areas are closely related to global environmental genotypes.

  9. Detection of tuberculosis drug resistance: a comparison by Mycobacterium tuberculosis MLPA assay versus Genotype®MTBDRplus.

    PubMed

    Santos, Paula Fernanda Gonçalves Dos; Costa, Elis Regina Dalla; Ramalho, Daniela M; Rossetti, Maria Lucia; Barcellos, Regina Bones; Nunes, Luciana de Souza; Esteves, Leonardo Souza; Rodenbusch, Rodrigo; Anthony, Richard M; Bergval, Indra; Sengstake, Sarah; Viveiros, Miguel; Kritski, Afrânio; Oliveira, Martha M

    2017-06-01

    To cope with the emergence of multidrug-resistant tuberculosis (MDR-TB), new molecular methods that can routinely be used to screen for a wide range of drug resistance related genetic markers in the Mycobacterium tuberculosis genome are urgently needed. To evaluate the performance of multiplex ligaton-dependent probe amplification (MLPA) against Genotype® MTBDRplus to detect resistance to isoniazid (INHr) and rifampicin (RIFr). 96 culture isolates characterised for identification, drug susceptibility testing (DST) and sequencing of rpoB, katG, and inhA genes were evaluated by the MLPA and Genotype®MTBDRplus assays. With sequencing as a reference standard, sensitivity (SE) to detect INHr was 92.8% and 85.7%, and specificity (SP) was 100% and 97.5%, for MLPA and Genotype®MTBDRplus, respectively. In relation to RIFr, SE was 87.5% and 100%, and SP was 100% and 98.8%, respectively. Kappa value was identical between Genotype®MTBDRplus and MLPA compared with the standard DST and sequencing for detection of INHr [0.83 (0.75-0.91)] and RIFr [0.93 (0.88-0.98)]. Compared to Genotype®MTBDRplus, MLPA showed similar sensitivity to detect INH and RIF resistance. The results obtained by the MLPA and Genotype®MTBDRplus assays indicate that both molecular tests can be used for the rapid detection of drug-resistant TB with high accuracy. MLPA has the added value of providing information on the circulating M. tuberculosis lineages.

  10. Detection of tuberculosis drug resistance: a comparison by Mycobacterium tuberculosis MLPA assay versus Genotype®MTBDRplus

    PubMed Central

    dos Santos, Paula Fernanda Gonçalves; Costa, Elis Regina Dalla; Ramalho, Daniela M; Rossetti, Maria Lucia; Barcellos, Regina Bones; Nunes, Luciana de Souza; Esteves, Leonardo Souza; Rodenbusch, Rodrigo; Anthony, Richard M; Bergval, Indra; Sengstake, Sarah; Viveiros, Miguel; Kritski, Afrânio; Oliveira, Martha M

    2017-01-01

    BACKGROUND To cope with the emergence of multidrug-resistant tuberculosis (MDR-TB), new molecular methods that can routinely be used to screen for a wide range of drug resistance related genetic markers in the Mycobacterium tuberculosis genome are urgently needed. OBJECTIVE To evaluate the performance of multiplex ligaton-dependent probe amplification (MLPA) against Genotype® MTBDRplus to detect resistance to isoniazid (INHr) and rifampicin (RIFr). METHOD 96 culture isolates characterised for identification, drug susceptibility testing (DST) and sequencing of rpoB, katG, and inhA genes were evaluated by the MLPA and Genotype®MTBDRplus assays. RESULTS With sequencing as a reference standard, sensitivity (SE) to detect INHr was 92.8% and 85.7%, and specificity (SP) was 100% and 97.5%, for MLPA and Genotype®MTBDRplus, respectively. In relation to RIFr, SE was 87.5% and 100%, and SP was 100% and 98.8%, respectively. Kappa value was identical between Genotype®MTBDRplus and MLPA compared with the standard DST and sequencing for detection of INHr [0.83 (0.75-0.91)] and RIFr [0.93 (0.88-0.98)]. CONCLUSION Compared to Genotype®MTBDRplus, MLPA showed similar sensitivity to detect INH and RIF resistance. The results obtained by the MLPA and Genotype®MTBDRplus assays indicate that both molecular tests can be used for the rapid detection of drug-resistant TB with high accuracy. MLPA has the added value of providing information on the circulating M. tuberculosis lineages. PMID:28591399

  11. Antibody Detection, Isolation, Genotyping, and Virulence of Toxoplasma gondii in Captive Felids from China

    PubMed Central

    Yang, Yu-Rong; Feng, Yong-Jie; Lu, Yao-Yao; Dong, Hui; Li, Tong-Yi; Jiang, Yi-Bao; Zhu, Xing-Quan; Zhang, Long-Xian

    2017-01-01

    The felids are the only definitive hosts of Toxoplasma gondii, which could excrete oocysts into the environment and provide an infection source for toxoplasmosis in various warm-blooded animal species, particularly the captive felids that live close to human communities. The infection rate of the captive felids is a perfect standard in detecting the presence of Toxoplasma gondii oocysts in the environment. In this study, sera or tissue samples from zoo (1 young tiger, 2 adult tigers, 6 young lions), farm (10 masked palm civets), and pet hospital (28 cats) from Henan Province (China) were collected. The sera (n = 47) were tested for immunoglobulin G (IgG) antibodies against T. gondii by using modified agglutination test (MAT), whereas the hearts tissue (n = 40) were bioassayed in mice to isolate T. gondii strains. The genotype was distinguished by using PCR-RFLP of 10 loci (SAG1, SAG2, SAG3, GRA6, BTUB, L358, c22-8, PK1, c29-2, and Apico). The detection rate for the T. gondii antibody in captive felids was 21.3% (10/47). One viable T. gondii strain (TgCatCHn4) was obtained from a cat heart tissue, and its genotype was ToxoDB#9. The oocysts of ToxoDB#9 were collected from a T. gondii-free cat. The virulence of TgCatCHn4 was low and no cysts were detected in the brain of mice at 60 days post-inoculation. The finding of the present study suggested a widespread exposure of T. gondii for felids in Henan Province of central China, particularly those from the zoological gardens and homes. ToxoDB#9 was the predominant strain in China. Preventive measures against T. gondii oocyst contamination of various components of the environment should thus be implemented, including providing pre-frozen meat, well-cooked cat food, cleaned fruits and vegetables, monitoring birds and rodents, inactive T. gondii oocysts in felids feces, and proper hygiene. PMID:28791002

  12. Genotypic and phenotypic detection of macrolide and lincosamide resistance in Streptococcus uberis.

    PubMed

    Schmitt-Van de Leemput, E; Zadoks, R N

    2007-11-01

    Streptococcus uberis isolates (n = 55) were obtained from milk samples of cases of mild clinical mastitis in 55 dairy cows from 35 herds serviced by one veterinary practice in Mayenne, France. Isolates were tested for macrolide and lincosamide resistance by using phenotypic and genotypic methods. Erythromycin resistance was detected in 12 of the 55 (22%) isolates based on agar disc diffusion testing and MIC measurements, and was encoded by ermB. This gene also conferred phenotypic resistance to pirlimycin based on MIC measurements, but the D-test was needed for detection of the resistance phenotype in the agar disc diffusion test. Isolates with ermB were also highly resistant to the macrolide antibiotic spiramycin. Seventeen of the 55 isolates (31%) were classified as resistant to spiramcyin only and as having intermediate susceptibility to spiramycin based on agar disc diffusion testing and MIC measurements, respectively. The genetic mechanism behind this phenotype and its clinical relevance are unknown. The efflux pump gene mefA was not detected in any of the 55 isolates in this study. Pirlimycin resistance without macrolide resistance was encoded by the lincosamide resistance gene linB in 4 isolates. Based on current guidelines, some linB-positive isolates would be classified as susceptible by using phenotypic tests, and alternative values for the interpretation of the agar disc diffusion test are suggested. We conclude that the agar disc diffusion test is a useful indicator for macrolide and lincosamide resistance of Strep. uberis in veterinary practice, provided that the D-test is used for detection of pirlimycin resistance.

  13. Rapid Detection of Rifampicin and Isoniazid Resistant Mycobacterium tuberculosis Using Genotype MTBDRplus Assay in Nepal

    PubMed Central

    Sharma, Bijay Kumar; Shrestha, Bhawana

    2014-01-01

    Rapid line probe assay (LPA) can be a practical and rapid alternative to the slow conventional phenotypic drug susceptibility testing (DST) for detection of drug resistant tuberculosis (TB). The purpose of this study is to determine the diagnostic accuracy of Genotype MTBDRplus, LPA for TB, and compare its performance with conventional DST. A total of 54 culture samples were analyzed for DST using both conventional proportion method and MTBDRplus, where conventional DST identified 43 isolates (79.6%) as drug resistant. Among these 43 drug resistant isolates, 30 isolates (69.7%) were found to be multidrug resistant (MDR). Of all observed mutations using MTBDRplus, codon 531 of rpoB gene and codon 315 of katG gene were found to have highest mutational frequency for RIF resistance (64.7%) and INH resistance (96.8%), respectively. In the present study, MTBDRplus assay was shown to have excellent specificity (100%) for both RIF and INH resistance while sensitivity of the assay was little lower with value of 89.4% for RIF resistance and 91.4% for INH resistance. Therefore, the assay can be a rapid, reliable, and promising molecular test for early detection of MDR-TB in Nepal. PMID:27419213

  14. Phenotypic and genotypic characterization of vancomycin-resistant Enterococcus faecium clinical isolates from two hospitals in Mexico: First detection of VanB phenotype-vanA genotype.

    PubMed

    Bocanegra-Ibarias, Paola; Flores-Treviño, Samantha; Camacho-Ortiz, Adrián; Morfin-Otero, Rayo; Villarreal-Treviño, Licet; Llaca-Díaz, Jorge; Martínez-Landeros, Erik Alan; Rodríguez-Noriega, Eduardo; Calzada-Güereca, Andrés; Maldonado-Garza, Héctor Jesús; Garza-González, Elvira

    2016-01-01

    Enterococcus faecium has emerged as a multidrug-resistant nosocomial pathogen involved in outbreaks worldwide. Our aim was to determine the antimicrobial susceptibility, biofilm production, and clonal relatedness of vancomycin-resistant E. faecium (VREF) clinical isolates from two hospitals in Mexico. Consecutive clinical isolates (n=56) were collected in two tertiary care hospitals in Mexico from 2011 to 2014. VREF isolates were characterized by phenotypic and molecular methods including pulsed-field gel electrophoresis (PFGE). VREF isolates were highly resistant to vancomycin, erythromycin, norfloxacin, high-level streptomycin, and teicoplanin, and showed lower resistance to tetracycline, nitrofurantoin and quinupristin-dalfopristin. None of the isolates were resistant to linezolid. The vanA gene was detected in all isolates. Two VanB phenotype-vanA genotype isolates, highly resistant to vancomycin and susceptible to teicoplanin, were detected. Furthermore, 17.9% of the isolates were classified as biofilm producers, and the espfm gene was found in 98.2% of the isolates. A total of 37 distinct PFGE patterns and 6 clones (25% of the isolates as clone A, 5.4% as clone B, and 3.6% each as clone C, D, E, and F) were detected. Clone A was detected in 5 different wards of the same hospital during 14 months of surveillance. The high resistance to most antimicrobial agents and the moderate cross-transmission of VREF detected accentuates the need for continuous surveillance of E. faecium in the hospital setting. This is also the first reported incidence of the E. faecium VanB phenotype-vanA genotype in the Americas. Copyright © 2015 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  15. Postnatal and non-invasive prenatal detection of β-thalassemia mutations based on Taqman genotyping assays.

    PubMed

    Breveglieri, Giulia; Travan, Anna; D'Aversa, Elisabetta; Cosenza, Lucia Carmela; Pellegatti, Patrizia; Guerra, Giovanni; Gambari, Roberto; Borgatti, Monica

    2017-01-01

    The β-thalassemias are genetic disorder caused by more than 200 mutations in the β-globin gene, resulting in a total (β0) or partial (β+) deficit of the globin chain synthesis. The most frequent Mediterranean mutations for β-thalassemia are: β039, β+IVSI-110, β+IVSI-6 and β0IVSI-1. Several molecular techniques for the detection of point mutations have been developed based on the amplification of the DNA target by polymerase chain reaction (PCR), but they could be labor-intensive and technically demanding. On the contrary, TaqMan® genotyping assays are a simple, sensitive and versatile method suitable for the single nucleotide polymorphism (SNP) genotyping affecting the human β-globin gene. Four TaqMan® genotyping assays for the most common β-thalassemia mutations present in the Mediterranean area were designed and validated for the genotype characterization of genomic DNA extracted from 94 subjects comprising 25 healthy donors, 33 healthy carriers and 36 β-thalassemia patients. In addition, 15 specimens at late gestation (21-39 gestational weeks) and 11 at early gestation (5-18 gestational weeks) were collected from pregnant women, and circulating cell-free fetal DNAs were extracted and analyzed with these four genotyping assays. We developed four simple, inexpensive and versatile genotyping assays for the postnatal and prenatal identification of the thalassemia mutations β039, β+IVSI-110, β+IVSI-6, β0IVSI-1. These genotyping assays are able to detect paternally inherited point mutations in the fetus and could be efficiently employed for non-invasive prenatal diagnosis of β-globin gene mutations, starting from the 9th gestational week.

  16. Postnatal and non-invasive prenatal detection of β-thalassemia mutations based on Taqman genotyping assays

    PubMed Central

    Breveglieri, Giulia; Travan, Anna; D’Aversa, Elisabetta; Cosenza, Lucia Carmela; Pellegatti, Patrizia; Guerra, Giovanni; Gambari, Roberto

    2017-01-01

    The β-thalassemias are genetic disorder caused by more than 200 mutations in the β-globin gene, resulting in a total (β0) or partial (β+) deficit of the globin chain synthesis. The most frequent Mediterranean mutations for β-thalassemia are: β039, β+IVSI-110, β+IVSI-6 and β0IVSI-1. Several molecular techniques for the detection of point mutations have been developed based on the amplification of the DNA target by polymerase chain reaction (PCR), but they could be labor-intensive and technically demanding. On the contrary, TaqMan® genotyping assays are a simple, sensitive and versatile method suitable for the single nucleotide polymorphism (SNP) genotyping affecting the human β-globin gene. Four TaqMan® genotyping assays for the most common β-thalassemia mutations present in the Mediterranean area were designed and validated for the genotype characterization of genomic DNA extracted from 94 subjects comprising 25 healthy donors, 33 healthy carriers and 36 β-thalassemia patients. In addition, 15 specimens at late gestation (21–39 gestational weeks) and 11 at early gestation (5–18 gestational weeks) were collected from pregnant women, and circulating cell-free fetal DNAs were extracted and analyzed with these four genotyping assays. We developed four simple, inexpensive and versatile genotyping assays for the postnatal and prenatal identification of the thalassemia mutations β039, β+IVSI-110, β+IVSI-6, β0IVSI-1. These genotyping assays are able to detect paternally inherited point mutations in the fetus and could be efficiently employed for non-invasive prenatal diagnosis of β-globin gene mutations, starting from the 9th gestational week. PMID:28235086

  17. Birth of correctly genotyped calves after multiplex marker detection from bovine embryo microblade biopsies.

    PubMed

    Peippo, Jaana; Viitala, Sirja; Virta, Jouni; Räty, Mervi; Tammiranta, Niina; Lamminen, Terttu; Aro, Johanna; Myllymäki, Hannu; Vilkki, Johanna

    2007-11-01

    We report a method for multiplex genotyping of bovine embryo microblade biopsies. We have tested the reliability of the method and the viability of the embryos in vitro and in vivo. Two polymorphic gene markers (GHR F279Y and PRLR S18N) associated with milk production traits and one marker for sex diagnosis (ZFX/ZFY) were genotyped simultaneously with a method that combines nested PCR and allelic discrimination. To test the accuracy of genotyping, in the first experiment the genotypes of 134 biopsies from in vitro produced embryos were compared to genotypes determined from the corresponding embryos after biopsy. The method proved to be highly accurate as only in three cases (two for PRLR S18N and one for GHR F279Y) out of 395 genotypes the genotype was in disagreement between the two samples. The viability of similarly biopsied embryos was tested in parallel: after 24-hr culture 94.6% of embryos recovered in vitro. In the second experiment, a total of 150 in vivo-produced embryos were biopsied on Day 7 and genotyped. After the genotyping results were obtained on Day 8, female embryos were selected for transfer. From a total of 57 selected embryos 43 were transferred individually and 14 as pairs. After single embryo transfers, 19 recipients became pregnant and after embryo transfers in pairs one became pregnant. The success of genotyping was tested with the genotypes of donors and bulls and also from the hair samples of born calves. All calves were females and of the same genotypes determined from the biopsy.

  18. Detection of the Cryptosporidium parvum "human" genotype in a dugong (Dugong dugon).

    PubMed

    Morgan, U M; Xiao, L; Hill, B D; O'Donoghue, P; Limor, J; Lal, A; Thompson, R C

    2000-12-01

    The Cryptosporidium "human" genotype was identified in a paraffin-embedded tissue section from a dugong (Dugong dugon) by 2 independent laboratories. DNA sequencing and polymerase chain reaction/restriction fragment length polymorphism analysis of the 18S ribosomal RNA gene and the acetyl CoA synthethase gene clearly identified the genotype as that of the Cryptosporidium variant that infects humans. This is the first report of the human Cryptosporidium genotype in a nonprimate host.

  19. Molecular Detection and Genotypic Characterization of Toxoplasma gondii in Paraffin-Embedded Fetoplacental Tissues of Women with Recurrent Spontaneous Abortion

    PubMed Central

    Abdoli, Amir; Dalimi, Abdolhossein; Soltanghoraee, Haleh; Ghaffarifar, Fatemeh

    2017-01-01

    Background Congenital toxoplasmosis is an important cause of spontaneous abortion worldwide. However, there is limited information on detection and genotypic characterization of Toxoplasma gondii (T. gondii) in women with recurrent spontaneous abortion (RSA). The aim of this study is the molecular detection and genotypic characterization of T. gondii in formalin-fixed, paraffin-embedded fetoplacental tissues (FFPTs) of women with RSA that have referred to the Avicenna Research Institute in Tehran, Iran. Materials and Methods This experimental research was undertaken on 210 FFPTs of women with RSA. The information of the patients was collected from the archives of Avicenna Research Institute in Tehran, Iran. After DNA extraction, the presence of T. gondii was examined by nested polymerase chain reaction targeting the GRA6 gene. Genotyping was performed on positive samples using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) that targeted the GRA6 and SAG3 genes. Sequencing was conducted on two GRA6 positive samples. Results T. gondii DNA was detected in 3.8% (8/210) of the samples. Genotyping showed that all positive samples belonged to type III of the T. gondii genotype. Sequencing two genomic DNAs of the GRA6 gene revealed 99% similarity with each other and 99-100% similarity with T. gondii sequences deposited in GenBank. There were six patients with histories of more than three abortions; one patient had a healthy girl and another patient had two previous abortions. Abortions occurred in the first trimester of pregnancy in seven patients and in the second trimester of pregnancy in one patient. Conclusion The results of this study have indicated that genotype III is the predominant type of T. gondii in women with RSA in Tehran, Iran. Also, our findings suggest that toxoplasmosis may play a role in the pathogenesis of RSA. However, further studies are needed to elucidate a clear relationship between T. gondii infection and RSA. PMID

  20. Detecting small-scale genotype-environment interactions in apomictic dandelion (Taraxacum officinale) populations.

    PubMed

    McLeod, K A; Scascitelli, M; Vellend, M

    2012-08-01

    Studies of genotype × environment interactions (G × E) and local adaptation provide critical tests of natural selection's ability to counter opposing forces such as gene flow. Such studies may be greatly facilitated in asexual species, given the possibility for experimental replication at the level of true genotypes (rather than populations) and the possibility of using molecular markers to assess genotype-environment associations in the field (neither of which is possible for most sexual species). Here, we tested for G × E in asexual dandelions (Taraxacum officinale) by subjecting six genotypes to experimental drought, mown and benign (control) conditions and subsequently using microsatellites to assess genotype-environment associations in the field. We found strong G × E, with genotypes that performed poorly under benign conditions showing the highest performance under stressful conditions (drought or mown). Our six focal genotypes comprise > 80% of plants in local populations. The most common genotype in the field showed its highest relative performance under mown conditions (the most common habitat in our study area), and almost all plants of this genotype in the field were found growing in mowed lawns. Genotypes performing best under benign experimental conditions were found most frequently in unmown conditions in the field. These results are strongly indicative of local adaptation at a very small scale, with unmown microsites of only a few square metres typically embedded within larger mown lawns. By studying an asexual species, we were able to map genotypes with known ecological characteristics to environments with high spatial precision. © 2012 The Authors. Journal of Evolutionary Biology © 2012 European Society For Evolutionary Biology.

  1. HPV detection and genotyping in males from the city of Córdoba, Argentina.

    PubMed

    Venezuela, F; Kremer, L E; Kiguen, X; Cuffini, C

    2010-01-01

    A wide range of human papillomavirus (HPV) types can infect the anogenital region of males. Although there is a vast knowledge on HPV infections in women as well as on their association with cervical cancer, the study of HPV infections in males is scarce and controversial. The aim of the present work was to detect and typify HPV infections of the anogenital region in males and analyze the associated risk factors in the population studied. Anogenital samples from 37 patients (30 of whom were HIV carriers) attending the Infectology Service at the Hospital Nacional de Clinicas in C6rdoba, Argentina, were studied. Nine of these patients tested HPV-positive and five out of these nine were found to have mixed infections, being 18 and 61 the most frequent genotypes. There was a significant correlation between the HPV-positive patients and those having an HPV-compatible lesion or AIDS. The present work is the first study in the city of Cordoba which contributes relevant results to the knowledge of HPV infection and to the possible implementation of measures for its prevention.

  2. Evaluation of DNA extraction methods suitable for PCR-based detection and genotyping of Clostridium botulinum.

    PubMed

    Auricchio, Bruna; Anniballi, Fabrizio; Fiore, Alfonsina; Skiby, Jeffrey E; De Medici, Dario

    2013-09-01

    Sufficient quality and quantity of extracted DNA is critical to detecting and performing genotyping of Clostridium botulinum by means of PCR-based methods. An ideal extraction method has to optimize DNA yield, minimize DNA degradation, allow multiple samples to be extracted, and be efficient in terms of cost, time, labor, and supplies. Eleven botulinum toxin-producing clostridia strains and 25 samples (10 food, 13 clinical, and 2 environmental samples) naturally contaminated with botulinum toxin-producing clostridia were used to compare 4 DNA extraction procedures: Chelex(®) 100 matrix, Phenol-Cloroform-Isoamyl alcohol, NucliSENS(®) magnetic extraction kit, and DNeasy(®) Blood & Tissue kit. Integrity, purity, and amount of amplifiable DNA were evaluated. The results show that the DNeasy(®) Blood & Tissue kit is the best extraction method evaluated because it provided the most pure, intact, and amplifiable DNA. However, Chelex(®) 100 matrix seems to be suitable for PCR-based methods intended for laboratory diagnosis of suspected outbreaks of botulism, because it is faster and cheaper compared to DNeasy(®) Blood & Tissue kit, and for samples in which the mean of Ct values obtained are statistically different (P>0.05) with respect to the best method, no lack of PCR amplification was shown. In addition, molecular methods for laboratory diagnosis currently are based on a microbial enrichment step prior to PCR, and so the differences in amplification seem to not influence the analytical results.

  3. Genetic analysis of attachment glycoprotein (G) gene in new genotype ON1 of human respiratory syncytial virus detected in Japan.

    PubMed

    Tsukagoshi, Hiroyuki; Yokoi, Hajime; Kobayashi, Miho; Kushibuchi, Izumi; Okamoto-Nakagawa, Reiko; Yoshida, Ayako; Morita, Yukio; Noda, Masahiro; Yamamoto, Norio; Sugai, Kazuko; Oishi, Kazunori; Kozawa, Kunihisa; Kuroda, Makoto; Shirabe, Komei; Kimura, Hirokazu

    2013-09-01

    We studied the evolution of the G gene in the new genotype ON1 of RSV detected from patients with acute respiratory infection in Japan. Phylogenetic analyses and the evolutionary timescale were obtained by the Bayesian MCMC method. We also analyzed p-distance and positive selection sites. A new genotype ON1 emerged around 2001. The evolution rate was rapid (3.57 × 10(-3) substitutions/site per year). The p-distance was short and no positive selection site was found in the present strains. These results suggested that a new genotype ON1 of RSV-A emerged approximately10 years ago and spread to some countries with a high evolution rate.

  4. A new sieving matrix for DNA sequencing, genotyping and mutation detection and high-throughput genotyping with a 96-capillary array system

    SciTech Connect

    Gao, David

    1999-11-08

    Capillary electrophoresis has been widely accepted as a fast separation technique in DNA analysis. In this dissertation, a new sieving matrix is described for DNA analysis, especially DNA sequencing, genetic typing and mutation detection. A high-throughput 96 capillary array electrophoresis system was also demonstrated for simultaneous multiple genotyping. The authors first evaluated the influence of different capillary coatings on the performance of DNA sequencing. A bare capillary was compared with a DB-wax, an FC-coated and a polyvinylpyrrolidone dynamically coated capillary with PEO as sieving matrix. It was found that covalently-coated capillaries had no better performance than bare capillaries while PVP coating provided excellent and reproducible results. The authors also developed a new sieving Matrix for DNA separation based on commercially available poly(vinylpyrrolidone) (PVP). This sieving matrix has a very low viscosity and an excellent self-coating effect. Successful separations were achieved in uncoated capillaries. Sequencing of M13mp18 showed good resolution up to 500 bases in treated PVP solution. Temperature gradient capillary electrophoresis and PVP solution was applied to mutation detection. A heteroduplex sample and a homoduplex reference were injected during a pair of continuous runs. A temperature gradient of 10 C with a ramp of 0.7 C/min was swept throughout the capillary. Detection was accomplished by laser induced fluorescence detection. Mutation detection was performed by comparing the pattern changes between the homoduplex and the heteroduplex samples. High throughput, high detection rate and easy operation were achieved in this system. They further demonstrated fast and reliable genotyping based on CTTv STR system by multiple-capillary array electrophoresis. The PCR products from individuals were mixed with pooled allelic ladder as an absolute standard and coinjected with a 96-vial tray. Simultaneous one-color laser-induced fluorescence

  5. Detection and genotyping of human rotavirus VP4 and VP7 genes by reverse transcriptase PCR and reverse hybridization.

    PubMed

    van Doorn, Leen-Jan; Kleter, Bernhard; Hoefnagel, Evert; Stainier, Isabelle; Poliszczak, Annick; Colau, Brigitte; Quint, Wim

    2009-09-01

    Rotavirus infections can be diagnosed in stool samples by serological and molecular methods. We developed a novel reverse transcriptase PCR (RT-PCR) method for the amplification of rotavirus RNA and a reverse hybridization assay on a strip to detect amplimers and identify the specific G and P genotypes present in human stool specimens. An additional aim was to permit specific identification of the rotavirus G1P[8] strain, used in the Rotarix vaccine. Novel broad-spectrum PCR primers were developed for both VP4 and VP7, permitting the amplification of a wide range of rotavirus genotypes. Primer sets comprise mixtures of defined primer sequences. For the identification of G and P genotypes, two reverse hybridization strip assays were developed. Both the VP4 and the VP7 strip contain universal probes for the detection of VP4 and VP7 sequences, irrespective of the G or P genotype. The VP4 strip contains type-specific probes for P[4], P[6], P[8], P[9], and P[10]. The VP7 strip contains type-specific probes for G1, G2, G3, G4, G5, G6, G8, and G9. In addition, probes to distinguish between wild-type G1 and G1 vaccine strain sequences were present. Testing by analysis of multiple reference strains confirmed that both RT-PCR methods allowed the detection of a broad spectrum of genotypes. RT-PCR for VP7 was more sensitive than RT-PCR for VP4, but all samples identified as positive for rotavirus antigen by an enzyme-linked immunosorbent assay (ELISA) were also positive for both VP4 and VP7. The high specificity of the reverse hybridization method was confirmed by sequence analysis as well as by type-specific PCR, and the vaccine strain could also be specifically identified. The reverse hybridization method permits accurate identification of mixed infections with different genotypes. Rotavirus genotypes for which no type-specific probes were present on the strip were adequately identified by the universal detection probes. The assay was formally validated by analyses of

  6. Molecular Detection of the Virulent ExoU Genotype of Pseudomonas aeruginosa Isolated from Infected Surgical Incisions.

    PubMed

    Hassuna, Noha A

    2016-10-01

    Pseudomonas aeruginosa is one of the major pathogens responsible for hospital-acquired infections, which harbor a wide array of virulence factors. The main aim of this study was to determine the frequency of the virulent ExoU genotype in relation to the ExoS genotype among isolated P. aeruginosa from infected surgical incisions, followed by phylogenetic analysis. A total of 66 P. aeruginosa isolates were identified by cultural and biochemical characteristics. All isolates were tested for antimicrobial susceptibility against the following antimicrobial agents: imipenem, amikacin, gentamicin, amoxycillin, cefotaxime, cefepime, and levofloxacin. Molecular detection of the ExoS and ExoU as well as two other virulence genes was done by polymerase chain reaction (PCR). Sequencing of ExoU gene and phylogenetic analysis was performed. Approximately 81% of the isolated P. aeruginosa were multi-drug resistant. The ExoS genotype was more prevalent (63%) among the isolates than the ExoU genotype (18%), with 9% of the isolates possessing both toxins. LasB and AprA were detected in 63.6% and 27.2% of the isolates, respectively. An association was observed between the number of virulence genes and the presence of multi-drug resistance. All the ExoU were multi-drug resistant (MDR), whereas 71% of the ExoS were MDR. Phylogenetic analysis of ExoU gene showed a 99% similarity with four different strains. Despite the greater frequency of the ExoS genotype, the presence of the virulent MDR ExoU genotype isolates from surgical site infections is an alarming sign requiring further intervention and investigations.

  7. HPV genotypes detected in the oropharyngeal mucosa of HIV-infected men who have sex with men in Northern Italy.

    PubMed

    Martinelli, M; Mazza, F; Frati, E R; Fasolo, M M; Colzani, D; Bianchi, S; Fasoli, E; Amendola, A; Orlando, G; Tanzi, E

    2016-09-01

    The aim of this study was to investigate the epidemiological profile of HPV oropharyngeal infections in HIV-infected men who have sex with men. A total of 135 subjects were enrolled at the L. Sacco University Hospital (Milan, Italy) to evaluate their HPV oropharyngeal infection status at baseline and at a follow-up visit at least 12 months later. HPV DNA was detected from oropharyngeal swabs using an in-house nested PCR that amplifies a segment of the L1 gene. The PCR products were then sequenced and genotyped. A greater percentage of high-risk genotypes was identified compared to low-risk genotypes (13·7% vs. 6·9%, P < 0·05), and two uncommon alpha-HPV genotypes were detected, i.e. HPV-102 and HPV-114. HPV infection prevalence was 24·4% and the cumulative incidence was 24·1%. During the follow-up period, one case of HPV infection (HPV-33) persisted, while the overall rate of infection clearance was 58·3%. HPV oropharyngeal infection was widespread in the cohort examined, and most of the infections were transient and cleared within 12 months. These results may help to clarify the role of HPV in the oropharynx and may also improve our understanding of the need to implement preventive strategies in at-risk populations.

  8. [Detection of ABO genotype genetic polymorphism by multiplex-PCR based sequencing and application in forensic medicine].

    PubMed

    Chen, Feng; Chen, Teng; Yan, Chun-Xia; Dang, Yong-Hui; Mu, Hao-Fang; Yu, Xiao-Guang; Zhang, Bo; Deng, Ya-Jun

    2008-06-01

    Multiplex PCR-direct sequencing method was established to detect 9 different SNPs in exon 6 and exon 7 of ABO genes and could identify at least 28 different ABO genotypes. Population study was carried out in a sample of 80 unrelated Chinese Tibetan minority individual dwelled in Qinghai Province. The method was also applied to forensic cases. A variety of degeneration forensic samples, including blood stain, hair root, swab, bone and mixed stain were successfully identified by this efficient method and in conformance with serological typing. There were no significant deviations from Hardy-Weinberg equilibrium in ABO genotypes of Tibetan population. The heterozygosity, polymorphic information content, discrimination power, paternity of exclusion, and probability of genetic identity were 0.675, 0.672, 0.874, 0.391, and 0.126 respectively. The gene frequency of ABO was O>B>A. The multiplex PCR-directed sequencing method can accurately and reliably detect ABO genotypes in many kinds of samples, and it improves personal identification efficiency. The ABO genotype is high variance in Qinghai Tibetan minority group, and it can be applied in forensic medicine and population genetic study.

  9. Genotyping and detection of common avian and human origin-influenza viruses using a portable chemiluminescence imaging microarray.

    PubMed

    Zhang, Yingjie; Liu, Qiqi; Wang, Dou; Chen, Suhong; Wang, Xiaobo; Wang, Shengqi

    2016-01-01

    Influenza viruses are divided into three types, A, B, and C. Human influenza A and B viruses can cause seasonal epidemics, but influenza C causes only a mild respiratory illness. Influenza A virus can infect various host species. In 2013, human-infectious avian influenza A (H7N9) was first reported in China. By the second week of 2014, there were 210 laboratory-confirmed human cases in the country, and the mortality rate eventually reached 22 %. Rapid and accurate diagnosis of influenza viruses is important for clinical management and epidemiology. In this assay, a cost-effective chemiluminescence (CL) detection oligonucleotide microarray was developed to genotype and detect avian influenza A (H7N9), avian influenza A (H5N1), 2009 influenza A (H1N1), seasonal influenza A (H1N1), and seasonal influenza A (H3N2). Influenza A viruses and influenza B viruses were also generally detected using this microarray. The results of detection of 40 cultivated influenza virus strains showed that the microarray was able to distinguish the subtypes of these influenza viruses very well. The microarray possessed similar or 10 fold higher limit of detection than the real-time RT-PCR method. Sixty-six clinical swab samples were detected using this microarray and verified with real time RT-PCR to evaluate the efficiency of this microarray for clinical testing. A reliable CL detection oligonucleotide microarray had been developed to genotype and detected these influenza viruses.

  10. Individual Differences Among Children in Sucrose Detection Thresholds: Relationship With Age, Gender, and Bitter Taste Genotype.

    PubMed

    Joseph, Paule Valery; Reed, Danielle R; Mennella, Julie A

    2016-01-01

    Little research has focused on whether there are individual differences among children in their sensitivity to sweet taste and, if so, the biological correlates of such differences. Our goal was to understand how variations in children's sucrose detection thresholds relate to their age and gender, taste genotype, body composition, and dietary intake of added sugars. Sucrose detection thresholds in 7- to 14-year-old children were tested individually using a validated, two-alternative, forced-choice, paired-comparison tracking method. Five genetic variants of taste genes were assayed: TAS1R3 and GNAT3 (sweet genes; one variant each) and the bitter receptor gene TAS2R38 (three variants). All children were measured for body weight and height. A subset of these children were measured for the percentage of body fat and waist circumference and provided added sugar intake by 24-hour dietary recall. Sucrose thresholds ranged from 0.23 to 153.8 mM with most of the children completing the threshold task (216/235; 92%). Some children were biologically related (i.e., siblings), and for the genetic analysis, one sibling from each family was studied. Variants in the bitter but not the sweet genes were related to sucrose threshold and sugar intake; children with two bitter-sensitive alleles could detect sucrose at lower concentrations (F(2,165) = 4.55, p = .01; rs1726866) and reported eating more added sugar (% kcal; F(2, 62) = 3.64, p = .03) than did children with less sensitive alleles. Age, gender, and indices of obesity also were related to child-to-child differences in sucrose threshold; girls were more sensitive than boys (t(214) = 2.0, p = .05), older children were more sensitive than younger children (r(214) = -.16, p = .02), and fatter (r(84) = -.22, p = .05) or more centrally obese children (r(84) = -.26, p = .02) were more sensitive relative to others. Inborn differences in bitter sensitivity may affect childhood dietary sugar intake with long-term health consequences

  11. Comparison of methods to detect copy number alterations in cancer using simulated and real genotyping data

    PubMed Central

    2012-01-01

    Background The detection of genomic copy number alterations (CNA) in cancer based on SNP arrays requires methods that take into account tumour specific factors such as normal cell contamination and tumour heterogeneity. A number of tools have been recently developed but their performance needs yet to be thoroughly assessed. To this aim, a comprehensive model that integrates the factors of normal cell contamination and intra-tumour heterogeneity and that can be translated to synthetic data on which to perform benchmarks is indispensable. Results We propose such model and implement it in an R package called CnaGen to synthetically generate a wide range of alterations under different normal cell contamination levels. Six recently published methods for CNA and loss of heterozygosity (LOH) detection on tumour samples were assessed on this synthetic data and on a dilution series of a breast cancer cell-line: ASCAT, GAP, GenoCNA, GPHMM, MixHMM and OncoSNP. We report the recall rates in terms of normal cell contamination levels and alteration characteristics: length, copy number and LOH state, as well as the false discovery rate distribution for each copy number under different normal cell contamination levels. Assessed methods are in general better at detecting alterations with low copy number and under a little normal cell contamination levels. All methods except GPHMM, which failed to recognize the alteration pattern in the cell-line samples, provided similar results for the synthetic and cell-line sample sets. MixHMM and GenoCNA are the poorliest performing methods, while GAP generally performed better. This supports the viability of approaches other than the common hidden Markov model (HMM)-based. Conclusions We devised and implemented a comprehensive model to generate data that simulate tumoural samples genotyped using SNP arrays. The validity of the model is supported by the similarity of the results obtained with synthetic and real data. Based on these results and

  12. Evaluation of the GenoType MTBDR assay for detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis complex isolates.

    PubMed

    Saglik, I; Oz, Y; Kiraz, N

    2014-01-01

    Detection of drug resistance plays a critical role in tuberculosis treatment. The aim of this study was to evaluate the performance of GenoType Mycobacteria Drug Resistance (MTBDR) assay (Hain Lifescience, Germany) and to compare it with radiometric BACTEC 460 TB system (Becton Dickinson, USA) for the detection of rifampicin (RIF) and isoniazid (INH) resistance in 84 Mycobacterium tuberculosis complex (MTBC) isolates. RIF resistance was identified in 6 of 7 (85.7%) isolates and INH resistance was identified in 8 of 14 (57.1%) isolates by the GenoType MTBDR assay. Compared with BACTEC system, the sensitivity, specificity, positive predictive value and negative predictive values were 85.7%, 98.7%, 85.7% and 98.7% for RIF resistance; and 57.1%, 100%, 100% and 92.1% for INH resistance, respectively. GenoType MTBDR assay is reliable when tested specimen is resistant to the tested drugs. Although test was more successful in the detection of RIF resistance, it exhibited low sensitivity for the detection of INH resistance.

  13. Bias Characterization in Probabilistic Genotype Data and Improved Signal Detection with Multiple Imputation

    PubMed Central

    Palmer, Cameron; Pe’er, Itsik

    2016-01-01

    Missing data are an unavoidable component of modern statistical genetics. Different array or sequencing technologies cover different single nucleotide polymorphisms (SNPs), leading to a complicated mosaic pattern of missingness where both individual genotypes and entire SNPs are sporadically absent. Such missing data patterns cannot be ignored without introducing bias, yet cannot be inferred exclusively from nonmissing data. In genome-wide association studies, the accepted solution to missingness is to impute missing data using external reference haplotypes. The resulting probabilistic genotypes may be analyzed in the place of genotype calls. A general-purpose paradigm, called Multiple Imputation (MI), is known to model uncertainty in many contexts, yet it is not widely used in association studies. Here, we undertake a systematic evaluation of existing imputed data analysis methods and MI. We characterize biases related to uncertainty in association studies, and find that bias is introduced both at the imputation level, when imputation algorithms generate inconsistent genotype probabilities, and at the association level, when analysis methods inadequately model genotype uncertainty. We find that MI performs at least as well as existing methods or in some cases much better, and provides a straightforward paradigm for adapting existing genotype association methods to uncertain data. PMID:27310603

  14. Nested PCR for detection and genotyping of Ehrlichia ruminantium: use in genetic diversity analysis.

    PubMed

    Martinez, Dominique; Vachiéry, Nathalie; Stachurski, Frederic; Kandassamy, Yane; Raliniaina, Modestine; Aprelon, Rosalie; Gueye, Arona

    2004-10-01

    Ehrlichia ruminantium, the agent of cowdriosis transmitted by Amblyomma ticks, presents an extensive genetic and antigenic diversity of key importance for vaccine formulation. Two means of nested polymerase chain reaction (PCR) targeting were developed to conduct molecular epidemiology studies in the Caribbean and Africa. The first used a conserved DNA fragment for detection of the pathogen in animals and vectors, and the second relied on the polymorphic map1 gene for genotyping. As compared to a PCR, the nested PCR showed a 2-Log10 improvement of sensitivity and allowed amplification from ticks, blood, brain, and lungs from infected animals, providing a more accurate picture of the tick infection rate. In Guadeloupe, this rate reached 36% (N = 212) instead of 1.7% (N = 224), as previously estimated. Genetic typing was done by restriction fragment length polymorphism or sequencing of map1 amplification products. Molecular epidemiology studies conducted in field sites selected for vaccination trials with inactivated vaccine, revealed the circulation of genetically divergent strains in limited geographical areas. It is known, then, that genetic clustering based on map1 has no predictive value regarding the protective value of a given strain against a new strain. However, tracing the strains by this technique revealed the extent of E. ruminantium diversity that one can expect in a given region, and the method allows differentiation between an inadequate immune response and the challenge by a breakthrough strain on animals dying despite vaccination. Up to now, genetic typing does not avoid cross-protection studies, which were conducted in parallel, although on a more limited scale. The importance of pathogen diversity studies for optimization of vaccine design is discussed as well as the research for new polymorphic genes. These genes may allow better predictions on cross-protection, given the recent completion of the sequence of the full genome of two E. ruminantium

  15. Detection and genotyping of Toxoplasma gondii DNA in the blood and milk of naturally infected donkeys (Equus asinus).

    PubMed

    Mancianti, Francesca; Nardoni, Simona; Papini, Roberto; Mugnaini, Linda; Martini, Mina; Altomonte, Iolanda; Salari, Federica; D'Ascenzi, Carlo; Dubey, Jitender P

    2014-04-03

    Toxoplasma gondii is a worldwide zoonotic protozoan. Consumption of raw milk from infected animals is considered a risk factor for acquiring toxoplasmosis in humans. Recently, donkey milk has been indicated for therapeutic and nutritional purposes and T. gondii infection is common in donkeys. The purpose of the present paper was to detect the presence of parasite DNA in milk of T. gondii positive donkeys. Antibodies to T. gondii were found in 11 out of 44 healthy lactating donkeys by IFAT. T. gondii DNA was detected by PCR in blood of 6 and milk of 3 seropositive jennies. Results of limited RFLP-PCR genotyping indicated the presence of T. gondii genotype II or III, commonly found in Europe. The occurrence of T. gondii DNA in milk suggests that the consumption of raw milk from seropositive donkeys could be a potential source of human infection.

  16. Lactase persistence genotyping: rapid detection of seven sequence variants in a single tube with melting curve analyses.

    PubMed

    Strand, Harald; Sørensen, Liv Karin; Ingebretsen, Ole Christian

    2014-09-01

    Lactase persistence is an inherited autosomal dominant trait that confers the ability to digest lactose after weaning. Lactose persistence is caused by single nucleotide variants in a regulatory element for the lactase gene (LCT). In Europeans, lactase persistence is associated with the nucleotide variant LCT -13910C>T. Ethnic groups from Africa and the Arabian Peninsula have other lactase persistence variants in close proximity to the -13910C>T variant. Current hybridisation probe analysis methods have been limited by the inability to detect all the known nucleotide variants. The aim of this study was to devise a method based on hybridisation probes and melting curve analysis for rapidly detecting LCT variants. We designed hybridisation probes for producing melting curve profiles that could identify seven LCT nucleotide variants: -13907C>G, -13910C>T, -13913T>C, -13914G>A, -13915T>G, -14009T>G, and -14010G>C. To validate the assay, we generated plasmid standards of all the alleles and mixed them to create artificial heterozygote samples. All genotypes could be detected in a single tube assay. Rare genotypes were confirmed in a second assay with probes that had different complementary sequences. This assay can be used for rapidly genotyping lactase persistence in multi-ethnic populations.

  17. Molecular prevalence of different genotypes of Theileria orientalis detected from cattle and water buffaloes in Thailand.

    PubMed

    Altangerel, Khukhuu; Sivakumar, Thillaiampalam; Inpankaew, Tawin; Jittapalapong, Sathaporn; Terkawi, Mohamad Alaa; Ueno, Akio; Xuan, Xuenan; Igarashi, Ikuo; Yokoyama, Naoaki

    2011-12-01

    Here we report on an epidemiological study regarding the molecular prevalence of different genotypes of Theileria orientalis present among domestic cattle and water buffalo populations bred in Thailand. A phylogenetic analysis based on the parasitic gene encoding a major piroplasm surface protein revealed the presence of 5 genotypes (Types 1, 3, 5, 7, and N-3) in cattle and 7 genotypes (Types 1, 3, 4, 5, 7, N-2, and N-3) in water buffaloes. Types 4, 7, and N-3 of T. orientalis were reported for the first time in water buffaloes. The previously reported C and Thai types from Thailand clustered as types 7 and 6, respectively, in the present analysis. Great similarities were observed among nucleotide sequences of isolates of the same genotype from cattle and water buffaloes, and, therefore, water buffaloes were considered to serve as a reservoir for these genotypes of T. orientalis in Thailand. In conclusion, T. orientalis parasites circulating in Thailand are more diverse in their genetic characters than previously anticipated.

  18. Rapid Detection of Rare Deleterious Variants by Next Generation Sequencing with Optional Microarray SNP Genotype Data.

    PubMed

    Watson, Christopher M; Crinnion, Laura A; Gurgel-Gianetti, Juliana; Harrison, Sally M; Daly, Catherine; Antanavicuite, Agne; Lascelles, Carolina; Markham, Alexander F; Pena, Sergio D J; Bonthron, David T; Carr, Ian M

    2015-09-01

    Autozygosity mapping is a powerful technique for the identification of rare, autosomal recessive, disease-causing genes. The ease with which this category of disease gene can be identified has greatly increased through the availability of genome-wide SNP genotyping microarrays and subsequently of exome sequencing. Although these methods have simplified the generation of experimental data, its analysis, particularly when disparate data types must be integrated, remains time consuming. Moreover, the huge volume of sequence variant data generated from next generation sequencing experiments opens up the possibility of using these data instead of microarray genotype data to identify disease loci. To allow these two types of data to be used in an integrated fashion, we have developed AgileVCFMapper, a program that performs both the mapping of disease loci by SNP genotyping and the analysis of potentially deleterious variants using exome sequence variant data, in a single step. This method does not require microarray SNP genotype data, although analysis with a combination of microarray and exome genotype data enables more precise delineation of disease loci, due to superior marker density and distribution.

  19. Diseases associated with genomic imprinting.

    PubMed

    Wilkins, Jon F; Úbeda, Francisco

    2011-01-01

    Genomic imprinting is the phenomenon where the expression of a locus differs between the maternally and paternally inherited alleles. Typically, this manifests as transcriptional silencing of one of the alleles, although many genes are imprinted in a tissue- or isoform-specific manner. Diseases associated with imprinted genes include various cancers, disorders of growth and metabolism, and disorders in neurodevelopment, cognition, and behavior, including certain major psychiatric disorders. In many cases, the disease phenotypes associated with dysfunction at particular imprinted loci can be understood in terms of the evolutionary processes responsible for the origin of imprinting. Imprinted gene expression represents the outcome of an intragenomic evolutionary conflict, where natural selection favors different expression strategies for maternally and paternally inherited alleles. This conflict is reasonably well understood in the context of the early growth effects of imprinted genes, where paternally inherited alleles are selected to place a greater demand on maternal resources than are maternally inherited alleles. Less well understood are the origins of imprinted gene expression in the brain, and their effects on cognition and behavior. This chapter reviews the genetic diseases that are associated with imprinted genes, framed in terms of the evolutionary pressures acting on gene expression at those loci. We begin by reviewing the phenomenon and evolutionary origins of genomic imprinting. We then discuss diseases that are associated with genetic or epigenetic defects at particular imprinted loci, many of which are associated with abnormalities in growth and/or feeding behaviors that can be understood in terms of the asymmetric pressures of natural selection on maternally and paternally inherited alleles. We next described the evidence for imprinted gene effects on adult cognition and behavior, and the possible role of imprinted genes in the etiology of certain

  20. Comparing Human Metapneumovirus and Respiratory Syncytial Virus: Viral Co-Detections, Genotypes and Risk Factors for Severe Disease.

    PubMed

    Moe, Nina; Krokstad, Sidsel; Stenseng, Inger Heimdal; Christensen, Andreas; Skanke, Lars Høsøien; Risnes, Kari Ravndal; Nordbø, Svein Arne; Døllner, Henrik

    2017-01-01

    It is unclarified as to whether viral co-detection and human metapneumovirus (HMPV) genotypes relate to clinical manifestations in children with HMPV and lower respiratory tract infection (LRTI), and if the clinical course and risk factors for severe LRTI differ between HMPV and respiratory syncytial virus (RSV). We prospectively enrolled hospitalized children aged <16 years with LRTI from 2006 to 2015. Children were clinically examined, and nasopharyngeal aspirates were analyzed using semi-quantitative, real-time polymerase chain reaction tests for HMPV, RSV and 17 other pathogens. HMPV-positive samples were genotyped. A total of 171 children had HMPV infection. HMPV-infected children with single virus (n = 106) and co-detections (n = 65) had similar clinical manifestations. No clinical differences were found between HMPV genotypes A (n = 67) and B (n = 80). The HMPV-infected children were older (median 17.2 months) than RSV-infected children (median 7.3 months, n = 859). Among single virus-infected children, no differences in age-adjusted LRTI diagnoses were found between HMPV and RSV. Age was an important factor for disease severity among single virus-infected children, where children <6 months old with HMPV had a milder disease than those with RSV, while in children 12-23 months old, the pattern was the opposite. In multivariable logistic regression analysis for each virus type, age ≥12 months (HMPV), and age <6 months (RSV), prematurity, ≥1 chronic disease and high viral loads of RSV, but not high HMPV viral loads, were risk factors for severe disease. Among hospitalized children with LRTI, HMPV manifests independently of viral co-detections and HMPV genotypes. Disease severity in HMPV- and RSV-infected children varies in relation to age. A history of prematurity and chronic disease increases the risk of severe LRTI among HMPV- and RSV-infected children.

  1. Interest of human papillomavirus DNA quantification and genotyping in paired cervical and urine samples to detect cervical lesions.

    PubMed

    Ducancelle, A; Legrand, M C; Pivert, A; Veillon, P; Le Guillou-Guillemette, H; De Brux, M A; Beby-Defaux, A; Agius, G; Hantz, S; Alain, S; Catala, L; Descamps, P; Postec, E; Caly, H; Charles-Pétillon, F; Labrousse, F; Lunel, F; Payan, C

    2014-08-01

    Cervical cancer is caused by persistent infection with high-risk human papillomavirus (HR-HPV). Conventional human papillomavirus (HPV) testing requires cervical sampling. However, vaginal and urine self-sampling methods are more acceptable for patients and result in increased participation when they are available in screening programs. In this context, we have developed a non-invasive screening method via the detection of HPV DNA in urine samples. To compare HPV viral loads and genotypes in paired cervical and urine samples, and to assess correlation between virological and cytological results in women seeking gynecological consultation. Paired urine and cervical specimens were collected and analyzed from 230 of 245 women participating in the previously described prospective PapU study. HPV DNA detection and quantification were performed using a real-time PCR method with short fragment PCR primers. Genotyping was carried out using the INNO-LiPA HPV genotyping assay. The prevalence of HPV in the 230 paired urine and cervical smear samples was 42 and 49 %, respectively. Overall agreement for HPV positivity and negativity between the paired samples was 90 % (κ = 0.80). High HPV viral load in both cervical and urine samples was associated with cytological abnormalities. HPV-positive women were mostly infected with HR-HPV types. The agreement between high- and low-risk HPV (LR-HPV) detection in both samples was 97 % (κ = 0.95 for HR-HPV and κ = 0.97 for LR-HPV). High concordance rates for HPV-DNA quantification and high/low-risk HPV genotyping in paired urine/cervical samples suggest that urinary HPV DNA testing could be useful for cervical lesion screening.

  2. Comparing Human Metapneumovirus and Respiratory Syncytial Virus: Viral Co-Detections, Genotypes and Risk Factors for Severe Disease

    PubMed Central

    Moe, Nina; Krokstad, Sidsel; Stenseng, Inger Heimdal; Christensen, Andreas; Skanke, Lars Høsøien; Risnes, Kari Ravndal; Nordbø, Svein Arne; Døllner, Henrik

    2017-01-01

    Background It is unclarified as to whether viral co-detection and human metapneumovirus (HMPV) genotypes relate to clinical manifestations in children with HMPV and lower respiratory tract infection (LRTI), and if the clinical course and risk factors for severe LRTI differ between HMPV and respiratory syncytial virus (RSV). Methods We prospectively enrolled hospitalized children aged <16 years with LRTI from 2006 to 2015. Children were clinically examined, and nasopharyngeal aspirates were analyzed using semi-quantitative, real-time polymerase chain reaction tests for HMPV, RSV and 17 other pathogens. HMPV-positive samples were genotyped. Results A total of 171 children had HMPV infection. HMPV-infected children with single virus (n = 106) and co-detections (n = 65) had similar clinical manifestations. No clinical differences were found between HMPV genotypes A (n = 67) and B (n = 80). The HMPV-infected children were older (median 17.2 months) than RSV-infected children (median 7.3 months, n = 859). Among single virus-infected children, no differences in age-adjusted LRTI diagnoses were found between HMPV and RSV. Age was an important factor for disease severity among single virus-infected children, where children <6 months old with HMPV had a milder disease than those with RSV, while in children 12–23 months old, the pattern was the opposite. In multivariable logistic regression analysis for each virus type, age ≥12 months (HMPV), and age <6 months (RSV), prematurity, ≥1 chronic disease and high viral loads of RSV, but not high HMPV viral loads, were risk factors for severe disease. Conclusions Among hospitalized children with LRTI, HMPV manifests independently of viral co-detections and HMPV genotypes. Disease severity in HMPV- and RSV-infected children varies in relation to age. A history of prematurity and chronic disease increases the risk of severe LRTI among HMPV- and RSV-infected children. PMID:28095451

  3. Detection of bovine leukemia virus and identification of its genotype in Mongolian cattle.

    PubMed

    Ochirkhuu, Nyamsuren; Konnai, Satoru; Odbileg, Raadan; Nishimori, Asami; Okagawa, Tomohiro; Murata, Shiro; Ohashi, Kazuhiko

    2016-04-01

    Epidemiological studies have indicated that bovine leukemia virus (BLV) infection is globally distributed. However, no information regarding the disease and genetic diversity of the virus in the cattle of Mongolia is currently available. In this study, the prevalence of BLV was assessed using PCR, and the genetic diversity was analyzed through DNA sequencing. Of the 517 samples tested, 20 positives were identified. Phylogenetic analysis showed that six, one, and four isolates were classified into genotype 4, 7, and 1, respectively. Most isolates were clustered with isolates from Eastern Europe and Russia. This study is the first to investigate the BLV genotype in Mongolia.

  4. Detection of mumps virus genotype H in two previously vaccinated patients from Mexico City.

    PubMed

    Del Valle, Alberto; García, Alí A; Barrón, Blanca L

    2016-06-01

    Infections caused by mumps virus (MuV) have been successfully prevented through vaccination; however, in recent years, an increasing number of mumps outbreaks have been reported within vaccinated populations. In this study, MuV was genotyped for the first time in Mexico. Saliva samples were obtained from two previously vaccinated patients in Mexico City who had developed parotitis. Viral isolation was carried out in Vero cells, and the SH and HN genes were amplified by RT-PCR. Amplicons were sequenced and compared to a set of reference sequences to identify the MuV genotype.

  5. DNA Detection and Genotypic Identification of Potentially Human-Pathogenic Microsporidia from Asymptomatic Pet Parrots in South Korea as a Risk Factor for Zoonotic Emergence ▿

    PubMed Central

    Lee, So-Young; Lee, Sung-Seok; Lyoo, Young S.; Park, Hee-Myung

    2011-01-01

    We detected and identified genotypes of human-pathogenic microsporidia in fecal samples from 51 asymptomatic captive-bred pet parrots in South Korea. Microsporidia were identified in 8 samples (15.7%); 7 parrots tested positive for Encephalitozoon hellem, and 1 parrot tested positive for both E. hellem and Encephalitozoon cuniculi. In genotypic identifications, E. hellem was present in genotypes 1A and 2B and E. cuniculi was present in genotype II. Pet parrots might be a source of human microsporidian infection. PMID:21965400

  6. DNA detection and genotypic identification of potentially human-pathogenic microsporidia from asymptomatic pet parrots in South Korea as a risk factor for zoonotic emergence.

    PubMed

    Lee, So-Young; Lee, Sung-Seok; Lyoo, Young S; Park, Hee-Myung

    2011-12-01

    We detected and identified genotypes of human-pathogenic microsporidia in fecal samples from 51 asymptomatic captive-bred pet parrots in South Korea. Microsporidia were identified in 8 samples (15.7%); 7 parrots tested positive for Encephalitozoon hellem, and 1 parrot tested positive for both E. hellem and Encephalitozoon cuniculi. In genotypic identifications, E. hellem was present in genotypes 1A and 2B and E. cuniculi was present in genotype II. Pet parrots might be a source of human microsporidian infection.

  7. Evaluation of genotype MTBDRplus assay for rapid detection of isoniazid and rifampicin resistance in Mycobacterium tuberculosis clinical isolates from Pakistan.

    PubMed

    Javed, Hasnain; Jamil, Nazia; Jagielski, Tomasz; Bakuła, Zofia; Tahir, Zarfishan

    2016-12-01

    GenoType MTBDRplus is a molecular assay for detection of Mycobacterium tuberculosis resistance to isoniazid (INH) and rifampicin (RMP), the two major anti-tuberculosis (TB) drugs. Identification of INH resistance is largely based on the occurrence of mutations in the katG gene, coding for the catalase-peroxidase, or in the promoter region of the inhA gene, coding for the NADH-dependent enoyl-ACP reductase. For testing the RMP resistance, mutations in the rpoB gene, coding for the RNA polymerase β subunit, particularly in the RMP resistance determining region (RRDR) of the gene are investigated. The GenoType MTBDRplus assay has been validated in several countries. The aim of this study was to evaluate the ability of the assay to detect INH and RMP resistance among strains of M. tuberculosis, isolated from Pakistani TB patients, and phenotypically identified as multidrug-resistant (MDR), that is resistant to both INH and RMP. The study included a collection of 100 MDR M. tuberculosis strains isolated from as many Pakistani TB patients over a 9-month period (i.e. between January and September 2014). Drug susceptibility testing was performed using the standard 1% proportion method on Löwenstein-Jensen medium, with INH and RMP critical concentrations of 0.2mg/L and 40mg/L, respectively. Genomic DNA was extracted by the cetyl-trimethyl ammonium bromide (CTAB) method. The GenoType MTBDRplus assay (Hain Lifescience, Germany) was done following the manufacturer's instructions. In the katG gene, with MTBDRplus assay, a specific mutation associated with INH resistance (i.e. G944C transition, conferring Ser315Thr amino acid change) was detected in 66 (66%) of the strains. Thirty-four (34%) strains did not carry the katG mutation detected by the assay. Mutations in the mabA-inhA promoter region were detected in 10 (10%) strains (C-15T - in 10 strains, and T-8C - in 2 strains). Seventy-seven (77%) strains tested harboured a mutation in rpoB gene. Mutations in the rpoB gene

  8. Molecular Detection and Genotyping of Male-Specific Coliphages by Reverse Transcription-PCR and Reverse Line Blot Hybridization

    PubMed Central

    Vinjé, Jan; Oudejans, Sjon J. G.; Stewart, Jill R.; Sobsey, Mark D.; Long, Sharon C.

    2004-01-01

    In recent years, there has been increased interest in the use of male-specific or F+ coliphages as indicators of microbial inputs to source waters. Sero- or genotyping of these coliphages can also be used for microbial source tracking (MST). Among the male-specific coliphages, the F+ RNA (FRNA) viruses are well studied, while little is known about the F+ DNA (FDNA) viruses. We have developed a reverse line blot hybridization (RLB) assay which allows for the simultaneous detection and genotyping of both FRNA as well as FDNA coliphages. These assays included a novel generic duplex reverse transcription-PCR (RT-PCR) assay for FRNA viruses as well as a generic PCR for FDNA viruses. The RT-PCR assays were validated by using 190 field and prototype strains. Subsequent DNA sequencing and phylogenetic analyses of RT-PCR products revealed the classification of six different FRNA clusters, including the well-established subgroups I through IV, and three different FDNA clusters, including one (CH) not previously described. Within the leviviruses, a potentially new subgroup (called JS) including strains having more than 40% nucleotide sequence diversity with the known levivirus subgroups (MS2 and GA) was identified. We designed subgroup-specific oligonucleotides that were able to genotype all nine (six FRNA, three FDNA) different clusters. Application of the method to a panel of 351 enriched phage samples from animal feces and wastewater, including known prototype strains (MS2, GA, Qβ, M11, FI, and SP for FRNA and M13, f1, and fd for FDNA), resulted in successful genotyping of 348 (99%) of the samples. In summary, we developed a novel method for standardized genotyping of F+ coliphages as a useful tool for large-scale MST studies. PMID:15466543

  9. High performance of a new PCR-based urine assay for HPV-DNA detection and genotyping.

    PubMed

    Tanzi, Elisabetta; Bianchi, Silvia; Fasolo, Maria Michela; Frati, Elena R; Mazza, Francesca; Martinelli, Marianna; Colzani, Daniela; Beretta, Rosangela; Zappa, Alessandra; Orlando, Giovanna

    2013-01-01

    Human papillomavirus (HPV) testing has been proposed as a means of replacing or supporting conventional cervical screening (Pap test). However, both methods require the collection of cervical samples. Urine sample is easier and more acceptable to collect and could be helpful in facilitating cervical cancer screening. The aim of this study was to evaluate the sensitivity and specificity of urine testing compared to conventional cervical smear testing using a PCR-based method with a new, designed specifically primer set. Paired cervical and first voided urine samples collected from 107 women infected with HIV were subjected to HPV-DNA detection and genotyping using a PCR-based assay and a restriction fragment length polymorphism method. Sensitivity, specificity, Positive Predictive Value (PPV), and Negative Predictive Value (NPV) were calculated using the McNemar's test for differences. Concordance between tests was assessed using the Cohen's unweighted Kappa (k). HPV DNA was detected in 64.5% (95% CI: 55.1-73.1%) of both cytobrush and urine samples. High concordance rates of HPV-DNA detection (k = 0.96; 95% CI: 0.90-1.0) and of high risk-clade and low-risk genotyping in paired samples (k = 0.80; 95% CI: 0.67-0.92 and k = 0.74; 95% CI: 0.60-0.88, respectively) were observed. HPV-DNA detection in urine versus cervix testing revealed a sensitivity of 98.6% (95% CI: 93.1-99.9%) and a specificity of 97.4% (95% CI: 87.7-99.9%), with a very high NPV (97.4%; 95% CI: 87.7-99.9%). The PCR-based assay utilized in this study proved highly sensitive and specific for HPV-DNA detection and genotyping in urine samples. These data suggest that a urine-based assay would be a suitable and effective tool for epidemiological surveillance and, most of all, screening programs.

  10. Detecting High-Order Epistasis in Nonlinear Genotype-Phenotype Maps.

    PubMed

    Sailer, Zachary R; Harms, Michael J

    2017-03-01

    High-order epistasis has been observed in many genotype-phenotype maps. These multi-way interactions between mutations may be useful for dissecting complex traits and could have profound implications for evolution. Alternatively, they could be a statistical artifact. High-order epistasis models assume the effects of mutations should add, when they could in fact multiply or combine in some other nonlinear way. A mismatch in the "scale" of the epistasis model and the scale of the underlying map would lead to spurious epistasis. In this article, we develop an approach to estimate the nonlinear scales of arbitrary genotype-phenotype maps. We can then linearize these maps and extract high-order epistasis. We investigated seven experimental genotype-phenotype maps for which high-order epistasis had been reported previously. We find that five of the seven maps exhibited nonlinear scales. Interestingly, even after accounting for nonlinearity, we found statistically significant high-order epistasis in all seven maps. The contributions of high-order epistasis to the total variation ranged from 2.2 to 31.0%, with an average across maps of 12.7%. Our results provide strong evidence for extensive high-order epistasis, even after nonlinear scale is taken into account. Further, we describe a simple method to estimate and account for nonlinearity in genotype-phenotype maps.

  11. Detecting High-Order Epistasis in Nonlinear Genotype-Phenotype Maps

    PubMed Central

    Sailer, Zachary R.; Harms, Michael J.

    2017-01-01

    High-order epistasis has been observed in many genotype-phenotype maps. These multi-way interactions between mutations may be useful for dissecting complex traits and could have profound implications for evolution. Alternatively, they could be a statistical artifact. High-order epistasis models assume the effects of mutations should add, when they could in fact multiply or combine in some other nonlinear way. A mismatch in the “scale” of the epistasis model and the scale of the underlying map would lead to spurious epistasis. In this article, we develop an approach to estimate the nonlinear scales of arbitrary genotype-phenotype maps. We can then linearize these maps and extract high-order epistasis. We investigated seven experimental genotype-phenotype maps for which high-order epistasis had been reported previously. We find that five of the seven maps exhibited nonlinear scales. Interestingly, even after accounting for nonlinearity, we found statistically significant high-order epistasis in all seven maps. The contributions of high-order epistasis to the total variation ranged from 2.2 to 31.0%, with an average across maps of 12.7%. Our results provide strong evidence for extensive high-order epistasis, even after nonlinear scale is taken into account. Further, we describe a simple method to estimate and account for nonlinearity in genotype-phenotype maps. PMID:28100592

  12. Detection of East/Central/South African Genotype of Chikungunya Virus in Myanmar, 2010

    PubMed Central

    Tun, Mya Myat Ngwe; Thant, Kyaw Zin; Inoue, Shingo; Nabeshima, Takeshi; Aoki, Kotaro; Kyaw, Aung Kyaw; Myint, Tin; Tar, Thi; Maung, Kay Thwe Thwe; Hayasaka, Daisuke

    2014-01-01

    In 2010, chikungunya virus of the East Central South African genotype was isolated from 4 children in Myanmyar who had dengue-like symptoms. Phylogenetic analysis of the E1 gene revealed that the isolates were closely related to isolates from China, Thailand, and Malaysia that harbor the A226V mutation in this gene. PMID:25062511

  13. Microsporidia Detection and Genotyping Study of Human Pathogenic E. bieneusi in Animals from Spain

    PubMed Central

    Galván-Díaz, Ana Luz; Magnet, Angela; Fenoy, Soledad; Henriques-Gil, Nuno; Haro, María; Gordo, Francisco Ponce; Miró, Guadalupe; del Águila, Carmen; Izquierdo, Fernando

    2014-01-01

    Microsporidia are ubiquitous parasites infecting all animal phyla and we present evidence that supports their zoonotic potential. Fecal samples taken from domestic (cats and dogs), farm (pigs, rabbits and ostriches) and wild animals (foxes) from different provinces of Spain were evaluated for microsporidia infection by light microscopy and PCR. After Microsporidia species identification, E. bieneusi genotypes were additionally studied by sequence analysis of the ITS region. Eighty-five samples out of 159 exhibited structures that were compatible with microsporidia spores by Webeŕs stain with 37 of them being confirmed by PCR. Microsporidia species identified included E. bieneusi, E. intestinalis and A. algerae. We report the first diagnosis of E. intestinalis and E. bieneusi in ostriches and A. algerae in pigs. We also provide new information on the molecular characterization of E. bieneusi isolates both in rabbits and ostriches. All of the E. bieneusi genotypes identified belonged to the zoonotic group of genotypes (Group I) including genotypes A (dogs), I (pigs), D (rabbits and foxes) and type IV (ostriches). Our results demonstrate that microsporidia are present in domestic, farm and wild animals in Spain, corroborating their potential role as a source of human infection and environmental contamination. PMID:24651457

  14. Development of a single nucleotide polymorphism DNA microarray for the detection and genotyping of the SARS coronavirus.

    PubMed

    Guo, Xi; Geng, Peng; Wang, Quan; Cao, Boyang; Liu, Bin

    2014-10-01

    Severe acute respiratory syndrome (SARS), a disease that spread widely in the world during late 2002 to 2004, severely threatened public health. Although there have been no reported infections since 2004, the extremely pathogenic SARS coronavirus (SARS-CoV), as the causative agent of SARS, has recently been identified in animals, showing the potential for the re-emergence of this disease. Previous studies showed that 27 single nucleotide polymorphism (SNP) mutations among the spike (S) gene of this virus are correlated closely with the SARS pathogenicity and epidemicity. We have developed a SNP DNA microarray in order to detect and genotype these SNPs, and to obtain related information on the pathogenicity and epidemicity of a given strain. The microarray was hybridized with PCR products amplified from cDNAs obtained from different SARS-CoV strains. We were able to detect 24 SNPs and determine the type of a given strain. The hybridization profile showed that 19 samples were detected and genotyped correctly by using our microarray, with 100% accuracy. Our microarray provides a novel method for the detection and epidemiological surveillance of SARS-CoV.

  15. Detection and molecular characterisation of Giardia lamblia genotypes in Sharjah, United Arab Emirates.

    PubMed

    ElBakri, Ali; Samie, Amidou; Bessong, Pascal; Potgieter, Natasha; Odeh, Ra'ed Abu

    2014-08-01

    No data are available on Giardia lamblia genotypes in the United Arab Emirates (UAE). This study aimed to identify G. lamblia from DNA extracted from human stool samples to gain information on its prevalence and to perform molecular analysis on isolates collected from expatriates from different localities residing in Sharjah, UAE. In total, 111 healthy expatriates residing in Sharjah were screened for G. lamblia using nested PCR amplification of the small subunit ribosomal RNA (ssu-rRNA) gene. Positive samples were genotyped using a nested PCR amplifying the triosephosphate isomerase (tpi) gene to differentiate between the two human assemblages (A and B). A subset of the PCR products (n=23) were sequenced and their phylogenetic relationships were determined. Of the 111 participants, 67 (60.4%) were identified as positive for the ssu-rRNA gene. When genotyped for the tpi gene, 18.9% (21/111) were of assemblage A, 17.1% (19/111) belonged to assemblage B and 5.4% (6/111) showed patterns compatible with mixed infections. A strong correlation between the presence of diarrhoea and assemblage B was observed (χ(2)=10.553; p=0.001). Moreover, an association was also observed between mixed infections (A+B) and diarrhoea (χ(2)=8.899; p=0.003). No correlation between age, gender and geographic origin of the infected individual was noted. Phylogenetic analysis showed three clusters for the tpi gene. No relationship between the clusters and the origin of samples was noted. This study is the first to determine the infection rate and genotypic composition of Giardia in Sharjah, UAE. © The Author 2014. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Genotyping approach for non-invasive foetal RHD detection in an admixed population

    PubMed Central

    Boggione, Carolina Trucco; Luján Brajovich, Melina E.; Mattaloni, Stella M.; Di Mónaco, René A.; García Borrás, Silvia E.; Biondi, Claudia S.; Cotorruelo, Carlos M.

    2017-01-01

    Background Non-invasive foetal RHD genotyping can predict haemolytic disease of the foetus and the newborn in pregnancies with anti-D alloantibodies and also avoid antenatal anti-D prophylaxis in pregnant women carrying an RHD negative foetus. Considering that the Argentine genetic background is the result of generations of intermixing between several ethnic groups, we evaluated the diagnostic performance of a non-invasive foetal RHD determination strategy to guide targeted antenatal RhD immunoprophylaxis. This algorithm is based on the analysis of four regions of the RHD gene in cell-free foetal DNA in maternal plasma and maternal and paternal RHD genotyping. Materials and methods DNA from 298 serologically D negative pregnant women between 19–28 weeks gestation were RHD genotyped. Foetal RHD status was determined by real-time PCR in 296 maternal plasma samples. In particular cases, RHDΨ and RHD-CE-Ds alleles were investigated in paternal DNA. Umbilical cord blood was collected at birth, and serological and molecular studies were performed. Results Of the 298 maternal samples, 288 were D−/RHD− and 10 D−/RHD+ (2 RHD*DAR; 5 RHD-CE-Ds; 3 RHDΨ). Plasma from RHD*DAR carriers was not analysed. Real-time PCR showed 210 RHD+ and 78 RHD− foetuses and 8 inconclusive results. In this latter group, paternal molecular studies were useful to report a RHD negative status in 5 foetuses while only 3 remained inconclusive. All the results, except one false positive due to a silent allele (RHD[581insG]), agreed with the neonatal typing performed in cord blood. Discussion The protocol used for non-invasive prenatal RHD genotyping proved to be suitable to determine foetal RHD status in our admixed population. The knowledge of the genetic background of the population under study and maternal and paternal molecular analysis can reduce the number of inconclusive results when investigating foetal RHD status. PMID:27136427

  17. GenoType MTBDRplus Assay for Rapid Detection of Multidrug Resistance in Mycobacterium tuberculosis: A Meta-Analysis.

    PubMed

    Bai, Yuanyuan; Wang, Yueling; Shao, Chunhong; Hao, Yingying; Jin, Yan

    2016-01-01

    There is an urgent demand for rapid and accurate drug-susceptibility testing for the detection of multidrug-resistant tuberculosis. The GenoType MTBDRplus assay is a promising molecular kit designed for rapid identification of resistance to first-line anti-tuberculosis drugs, isoniazid and rifampicin. The aim of this meta-analysis was to evaluate the diagnostic accuracy of GenoType MTBDRplus in detecting drug resistance to isoniazid and rifampicin in comparison with the conventional drug susceptibility tests. We searched PubMed, EMBASE, and Cochrane Library databases to identify studies according to predetermined criteria. A total of 40 studies were included in the meta-analysis. QUADAS-2 was used to assess the quality of included studies with RevMan 5.2. STATA 13.0 software was used to analyze the tests for sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio, and area under the summary receiver operating characteristic curves. Heterogeneity in accuracy measures was tested with Spearman correlation coefficient and Chi-square. Patient selection bias was observed in most studies. The pooled sensitivity (95% confidence intervals were 0.91 (0.88-0.94) for isoniazid, 0.96 (0.95-0.97) for rifampicin, and 0.91(0.86-0.94) for multidrug-resistance. The pooled specificity (95% CI) was 0.99 (0.98-0.99) for isoniazid, 0.98 (0.97-0.99) for rifampicin and 0.99 (0.99-1.00) for multidrug-resistance, respectively. The area under the summary receiver operating characteristic curves ranged from 0.99 to 1.00. This meta-analysis determined that GenoType MTBDRplus had good accuracy for rapid detection of drug resistance to isoniazid and/or rifampicin of M. tuberculosis. MTBDRplus method might be a good alternative to conventional drug susceptibility tests in clinical practice.

  18. Detection of Japanese Encephalitis Virus Genotype V in Culex orientalis and Culex pipiens (Diptera: Culicidae) in Korea

    PubMed Central

    Kim, Hyunwoo; Cha, Go-Woon; Jeong, Young Eui; Lee, Wook-Gyo; Chang, Kyu Sik; Roh, Jong Yul; Yang, Sung Chan; Park, Mi Yeoun; Park, Chan; Shin, E-Hyun

    2015-01-01

    Japanese encephalitis virus (JEV) causes significant viral encephalitis and is distributed throughout the Asian countries. The virus is known to be transmitted by Culex tritaeniorhynchus, which mainly breeds in rice paddies in Korea. In this study, we investigated the presence of other mosquito species that can transmit JEV as a second or regional vector. We selected five cities where patients have experienced JE in the last 5 years as mosquito-collecting locations and subdivided them into four collection sites according to the mosquito habitats (cowshed, downtown area, forest, and swamp). Mosquitoes were caught using the BG-Sentinel trap, CDC black-light trap, Fay-Prince trap, and Gravid trap. A total of 993 pools from 22,774 mosquitoes were prepared according to their species, collection date, and site. We performed a SYBR Green 1-based real-time RT-PCR assay to detect JEV from the mosquito pools. A total of six JEV-positive pools were detected from Culex orientalis and Culex pipiens caught in the Gangwon-do and Gyeonngi-do provinces. All the detected JEVs were revealed as genotype V by phylogenetic analysis of the envelope gene. Our findings confirm that a new genotype of JEV was introduced in Korea and suggest that two mosquito species may play a role in JEV transmission. PMID:25658839

  19. External quality assessment of human neutrophil antigen (HNA)-specific antibody detection and HNA genotyping from 2000 to 2012.

    PubMed

    Lucas, G; Porcelijn, L; Fung, Y L; Green, F; Reil, A; Hopkins, M; Schuller, R; Green, A; de Haas, M; Bux, J

    2013-10-01

    Since 2000, Quality Assurance (QA) exercises for the detection and identification of granulocyte antibodies and DNA typing for human neutrophil antigens (HNA) have been distributed within the International Granulocyte Immunobiology Workshops, which are linked to International Society of Blood Transfusion. The exercises were standardised at the outset to enable laboratory performance to be monitored. Between 2000 and 2012, nine exercises were distributed to 20 laboratories. Overall, 45 examples of 42 unique samples containing defined granulocyte reactive antibodies were distributed for serological analysis together with 20 samples for HNA genotyping. The level of satisfactory serological performance was initially set at 50% and later increased to 70%, while the 'cut-off' for HNA genotyping was set at 100% after 2008. Failure to achieve the minimum score in the QA exercises in consecutive years resulted in temporary exclusion. In 2000, the 15 participating laboratories had a mean score of 56.1% for serological analysis and 13 laboratories attempted HNA-1a and -1b genotyping, while 11 attempted HNA-1c typing. Steady improvements in proficiency for serological testing and HNA typing occurred in subsequent exercises. In 2012, the mean score for serology was 88.5% and 12/13 laboratories scored 100% for HNA-1a, -1b, -1c, -3a, -3b, -4a, -4bw, -5a and -5bw genotyping. These QA exercises have provided an invaluable tool to monitor and improve the standard of granulocyte immunology investigations for participating laboratories, thereby enhancing performance for both clinical investigations and donor screening programmes to reduce the incidence of TRALI. © 2013 International Society of Blood Transfusion.

  20. Detection of Genotype 4 Swine Hepatitis E Virus in Systemic Tissues in Cross-Species Infected Rabbits

    PubMed Central

    Wu, Qiaoxing; An, Junqing; She, Ruiping; Shi, Ruihan; Hao, Wenzhuo; Soomro, MajidHussain; Yuan, Xuerui; Yang, Jinling; Wang, Jingyuan

    2017-01-01

    Increasing evidence demonstrates that hepatitis E virus (HEV) can be transmitted across species. According to previous reports, swine HEV has two genotypes, genotype 3 and 4, and both can infect humans by the fecal-oral route. Thus, it is crucial for the control of HEV zoonotic transmission to evaluate the dynamics of viral shedding and distribution in different tissues during cross-species infection by HEV. In this study, rabbits were infected with genotype 4 swine HEV by the intraperitoneal route. The results showed that HEV RNA not only shed in the feces but also in the saliva of some rabbits during infection with swine HEV. Viremia appeared late after infection, and anti-HEV IgG was not obvious until the appearance of high viremia levels. After the rabbits were euthanized, a histopathological examination showed that the livers developed overt hepatitis accompanied by an elevation of alanine aminotransferase (ALT) and aspartate transaminase (AST). Furthermore, HEV RNA was detected in various tissues, especially in the salivary glands and tonsils. Subsequently, negative-stranded HEV RNA was practiced in tissues with positive HEV RNA, which demonstrated that HEV replicated in the tissues. Next, we harvested additional tissues from the liver, salivary gland, tonsil, spleen, thymus gland, lymph node and intestine, which are known as replication sites of swine HEV. Additionally, we also observed the HEV antigen distributed in the organs above through immunohistochemical staining. These results demonstrate that rabbits could be used as an animal model for researching cross-species infection of genotype 4 HEV. It is also noteworthy that HEV can shed in the saliva and presents the risk of droplet transmission. These new data provide valuable information for understanding cross-species infection by HEV. PMID:28129390

  1. GenoType MTBDRplus assay for molecular detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis strains and clinical samples.

    PubMed

    Lacoma, A; Garcia-Sierra, N; Prat, C; Ruiz-Manzano, J; Haba, L; Rosés, S; Maldonado, J; Domínguez, J

    2008-11-01

    The purpose of this study was to evaluate the GenoType MTBDRplus assay (Hain Lifescience GmbH, Nehren, Germany) for its ability to detect resistance to rifampin (RIF) and isoniazid (INH) in Mycobacterium tuberculosis clinical strains and directly in clinical samples. A total of 62 clinical strains characterized with the Bactec 460TB system were included. For the INH-resistant strains, the MIC was measured and sequencing was performed. Sixty-five clinical samples from 28 patients (39 smear-positive samples and 26 smear-negative samples) were also tested directly. The corresponding isolates of the clinical specimens were studied with the Bactec 460TB system. The overall rates of concordance of the MTBDRplus assay and the Bactec 460TB system for the detection of RIF and INH susceptibility in clinical strains were 98.3% (61/62) and 79% (49/62), respectively. The rate of concordance between the Bactec 460TB system and the MTBDRplus test for the detection of INH resistance in the group of 27 strains with low-level resistance was 62.9% (17/27), and that for the detection of INH resistance in the group of 21 strains with high-level resistance was 85.71% (18/21). Valid test results were obtained for 78.45% (51/65) of the clinical samples tested. The rates of concordance between both assays for the detection of drug resistance in these samples were 98% (50/51) for RIF and 96.2% (49/51) for INH. Taking into account only one sample per patient, the overall rate of concordance between both tests was 92.85% (26/28). The GenoType MTBDRplus assay is easy to perform and is a useful tool for the management of tuberculosis, as it allows the detection of resistance to RIF and INH in M. tuberculosis strains and also in clinical samples.

  2. GenoType MTBDRplus Assay for Molecular Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Strains and Clinical Samples▿

    PubMed Central

    Lacoma, A.; Garcia-Sierra, N.; Prat, C.; Ruiz-Manzano, J.; Haba, L.; Rosés, S.; Maldonado, J.; Domínguez, J.

    2008-01-01

    The purpose of this study was to evaluate the GenoType MTBDRplus assay (Hain Lifescience GmbH, Nehren, Germany) for its ability to detect resistance to rifampin (RIF) and isoniazid (INH) in Mycobacterium tuberculosis clinical strains and directly in clinical samples. A total of 62 clinical strains characterized with the Bactec 460TB system were included. For the INH-resistant strains, the MIC was measured and sequencing was performed. Sixty-five clinical samples from 28 patients (39 smear-positive samples and 26 smear-negative samples) were also tested directly. The corresponding isolates of the clinical specimens were studied with the Bactec 460TB system. The overall rates of concordance of the MTBDRplus assay and the Bactec 460TB system for the detection of RIF and INH susceptibility in clinical strains were 98.3% (61/62) and 79% (49/62), respectively. The rate of concordance between the Bactec 460TB system and the MTBDRplus test for the detection of INH resistance in the group of 27 strains with low-level resistance was 62.9% (17/27), and that for the detection of INH resistance in the group of 21 strains with high-level resistance was 85.71% (18/21). Valid test results were obtained for 78.45% (51/65) of the clinical samples tested. The rates of concordance between both assays for the detection of drug resistance in these samples were 98% (50/51) for RIF and 96.2% (49/51) for INH. Taking into account only one sample per patient, the overall rate of concordance between both tests was 92.85% (26/28). The GenoType MTBDRplus assay is easy to perform and is a useful tool for the management of tuberculosis, as it allows the detection of resistance to RIF and INH in M. tuberculosis strains and also in clinical samples. PMID:18784319

  3. Simultaneous Detection and Genotype Determination of HSV 1 and 2 by Real-time PCR Using Melting Curve Analysis and a Unique Pair of Primers.

    PubMed

    Paryan, Mahdi; Mohammadi-Yeganeh, Samira; Rezvan, Houri; Kia, Vahid; Mansouri, Ardalan; Mirab Samiee, Siamak

    2017-02-01

    Herpes simplex virus (HSV) is a human pathogen that causes different pathologic manifestations. Rapid and feasible detection and discrimination methods for HSV genotyping is a challenge in clinical laboratories, especially in children suffering from herpetic encephalitis. A quantitative real-time polymerase chain reaction (PCR)-based genotyping assay using SYBR Green I was established. We designed only 1 pair of primer for HSV 1 and 2, targeting thymidine kinase gene conserved region. HSV genotypes were determined by PCR using melting curve analysis with LightCycler. Different HSV genotypes were successfully detected in all clinical samples. The melting temperature for HSV 1 and 2 was 85.5±0.78°C and 89±0.53°C, respectively. These 2 genotypes were completely distinguished by means of the accurate melting assay. Importantly, detection was reliably performed within only 1 hour. The assay had no cross-reactivity across species, an excellent dynamic range from 10 to 10 copies per reaction, a good intra-assay and interassay reproducibility, and a detection limit of a single copy per reaction. Our homebrew designed and validated quantitative real-time PCR followed by a melting curve analysis provided a rapid and convenient screening test for differential identification of HSV genotypes 1 and 2. We recommend the large-scale application of this method for HSV 1 and 2 detection.

  4. Detection and Genotyping of Human Papillomaviruses from Archival Formalin-Fixed Tissue Samples.

    PubMed

    Van Doorslaer, Koenraad; Chen, Zigui; McBride, Alison A

    2016-11-18

    Pathology departments routinely process and store formalin-fixed, paraffin-embedded (FFPE) tissue samples for clinical diagnosis. These collections often contain decades' worth of samples and represent a treasure trove of specimens that can be analyzed for retrospective epidemiological studies, diagnostics, and pathogen discovery. Accurate amplification and sequencing of DNA from these samples is critical for the usability of these FFPE samples. Here we present a collection of protocols that describe extraction of DNA from FFPE tissues, PCR amplification of human papillomavirus DNA, and subsequent genotyping of the infecting virus. © 2016 by John Wiley & Sons, Inc.

  5. Molecular detection and genotyping of Acanthamoeba spp. among stray dogs using conjunctival swab sampling.

    PubMed

    Karakuş, Mehmet; Aykur, Mehmet; Özbel, Yusuf; Töz, Seray; Dağcı, Hande

    2016-12-01

    Acanthamoeba is one of the most common free-living amoebas (FLA) that present in environment. In humans, Acanthamoeba can cause an infection of the eye termed Acanthamoeba keratitis, which mostly occurs in contact lens wearers. In the present study, we aimed to screen the presence of Acanthamoeba DNA in stray dogs using previously collected conjunctival swab samples in a hyper-endemic area for canine leishmaniasis. Totally, 184 dogs were included in the study and 27 of them (14.6%) were found positive for Acanthamoeba according to the 18s rRNA gene sequencing. Two different genotypes (T4 and T5) were identified and T5 was firstly reported in Turkey in the present study. Statistical analysis was performed and no correlation was found between Leishmania and Acanthamoeba positivity (P<0.05). To best of our knowledge, this is the first study conducted to screen Acanthamoeba among stray dogs. Further studies are necessary to reveal the infection status and genotypes among dogs and its possible correlation with leishmaniasis.

  6. Magnetic nanowires for rapid and ultrasensitive isolation of DNA from cervical specimens for the detection of multiple human papillomaviruses genotypes.

    PubMed

    Lee, HyungJae; Hwang, Na Rae; Hwang, Sang-Hyun; Cho, Youngnam

    2016-12-15

    Detecting human papillomavirus (HPV) is central in diagnosing and monitoring HPV-related disease. However, limited sensitivity and the wide variability of the HPV genome pose challenges in the identification of HPV genes, particularly high-risk types. This study reports the development of polyethyleneimine-conjugated magnetic nanowires (PEI-MNWs) and their use in the isolation, identification, and analysis of multiple genotypes of HPV DNA from cervical cancer specimens. The nanowires are electrochemically doped with a high density of magnetic nanoparticles and biotin moieties during potentiostatic deposition, thereby allowing conjugating cationic branched polymers to direct the attachment of negatively charged DNA molecules with strong magnetic response. For proof of concept, the rapid and ultrasensitive isolation of HPV DNA is performed at concentrations as low as 10pg/mL with an efficiency of >95%. For clinical optimization, the analytical and clinical sensitivity of PEI-MNWs is compared with that of the Roche Cobas 4800 HPV Test and demonstrates excellent correlation for multiple HPV DNA genotypes with superior threshold cycle values. The high sensitivity, specificity, and good reproducibility of PEI-MNWs are particularly well suited for the recovery of DNA and provide significant and clinically meaningful evidence for the early detection and treatment of HPV-associated cancers. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Genotypic detection of acyclovir-resistant HSV-1: characterization of 67 ACV-sensitive and 14 ACV-resistant viruses.

    PubMed

    Frobert, Emilie; Cortay, Jean-Claude; Ooka, Tadamasa; Najioullah, Fatiha; Thouvenot, Danielle; Lina, Bruno; Morfin, Florence

    2008-07-01

    Infections due to herpes simplex virus (HSV) resistant to acyclovir (ACV) represent an important clinical concern in immunocompromised patients. In order to switch promptly to an appropriate treatment, rapid viral susceptibility assays are required. We developed herein a genotyping analysis focusing on thymidine kinase gene (TK) mutations in order to detect acyclovir-resistant HSV in clinical specimens. A total of 85 HSV-1 positive specimens collected from 69 patients were analyzed. TK gene could be sequenced directly for 81 clinical specimens (95%) and 68 HSV-1 specimens could be characterized as sensitive or resistant by genotyping (84%). Genetic characterization of 67 susceptible HSV-1 specimens revealed 10 polymorphisms never previously described. Genetic characterization of 14 resistant HSV-1 revealed 12 HSV-1 with either TK gene additions/deletions (8 strains) or substitutions (4 strains) and 2 HSV-1 with no mutation in the TK gene. DNA polymerase gene was afterwards explored. With this rapid PCR-based assay, ACV-resistant HSV could be detected directly in clinical specimens within 24 h.

  8. Evaluation of a Probe-Based PCR-ELISA System for Simultaneous Semi Quantitative Detection and Genotyping of Human Cytomegalovirus (HCMV) Infection in Clinical Specimens.

    PubMed

    Talkhabifard, Majid; Javid, Naeme; Moradi, Abdolvahab; Ghaemi, Amir; Tabarraei, Alijan

    2017-01-01

    Human cytomegalovirus (HCMV) is a common opportunistic pathogen that causes serious complications in immunosuppressed patients and infected newborns. In this study, PCR-ELISA was optimized for semi-quantitative detection of infection in clinical specimens and simultaneous genotyping of glycoprotein B for 4 major genotypes, due to its significance. During DIG-labeling PCR, a pair of primers amplifies a fragment of variable region of the glycoprotein B encoding sequence. Under optimized conditions, labeled Target amplicons hybridize to biotinated specific probes and are detected in an ELISA system. PCR-ELISA system showed specific performance with detection limit of approximately 100 copies of CMV DNA. The linear correlation was observed between the PCR-ELISA results (OD) and logarithmic scale of CMV (r=0.979). Repeatability of PCR-ELISA detection system for intra-assay and inter-assay was evaluated for negative and positive samples. In optimized conditions of hybridization, differentiation between genotypes of glycoprotein B was feasible using genotype-specific probes in PCR-ELISA genotyping system. In comparison with sequencing method, genotyping system was confirmed with kappa index of 1. PCR-ELISA is proposed as an applicable and reliable technique for semi-quantitative diagnosis and typing of the infection. This technique is flexible to apply in a variety of molecular fields.

  9. Predicting disease associations via biological network analysis.

    PubMed

    Sun, Kai; Gonçalves, Joana P; Larminie, Chris; Przulj, Nataša

    2014-09-17

    Understanding the relationship between diseases based on the underlying biological mechanisms is one of the greatest challenges in modern biology and medicine. Exploring disease-disease associations by using system-level biological data is expected to improve our current knowledge of disease relationships, which may lead to further improvements in disease diagnosis, prognosis and treatment. We took advantage of diverse biological data including disease-gene associations and a large-scale molecular network to gain novel insights into disease relationships. We analysed and compared four publicly available disease-gene association datasets, then applied three disease similarity measures, namely annotation-based measure, function-based measure and topology-based measure, to estimate the similarity scores between diseases. We systematically evaluated disease associations obtained by these measures against a statistical measure of comorbidity which was derived from a large number of medical patient records. Our results show that the correlation between our similarity measures and comorbidity scores is substantially higher than expected at random, confirming that our similarity measures are able to recover comorbidity associations. We also demonstrated that our predicted disease associations correlated with disease associations generated from genome-wide association studies significantly higher than expected at random. Furthermore, we evaluated our predicted disease associations via mining the literature on PubMed, and presented case studies to demonstrate how these novel disease associations can be used to enhance our current knowledge of disease relationships. We present three similarity measures for predicting disease associations. The strong correlation between our predictions and known disease associations demonstrates the ability of our measures to provide novel insights into disease relationships.

  10. Detection of Sickle Cell Hemoglobin in Haiti by Genotyping and Hemoglobin Solubility Tests

    PubMed Central

    Carter, Tamar E.; von Fricken, Michael; Romain, Jean R.; Memnon, Gladys; St. Victor, Yves; Schick, Laura; Okech, Bernard A.; Mulligan, Connie J.

    2014-01-01

    Sickle cell disease is a growing global health concern because infants born with the disorder in developing countries are now surviving longer with little access to diagnostic and management options. In Haiti, the current state of sickle cell disease/trait in the population is unclear. To inform future screening efforts in Haiti, we assayed sickle hemoglobin mutations using traditional hemoglobin solubility tests (HST) and add-on techniques, which incorporated spectrophotometry and insoluble hemoglobin separation. We also generated genotype data as a metric for HST performance. We found 19 of 202 individuals screened with HST were positive for sickle hemoglobin, five of whom did not carry the HbS allele. We show that spectrophotometry and insoluble hemoglobin separation add-on techniques could resolve false positives associated with the traditional HST approach, with some limitations. We also discuss the incorporation of insoluble hemoglobin separation observation with HST in suboptimal screening settings like Haiti. PMID:24957539

  11. Multiplex detection and genotyping of pathogenic bacteria on paper-based biosensor with a novel universal primer mediated asymmetric PCR.

    PubMed

    Liu, Fang; Liu, Hongxing; Liao, Yuhui; Wei, Jitao; Zhou, Xiaoming; Xing, Da

    2015-12-15

    Traditionary multiplex asymmetric polymerase chain reaction (PCR) can be applied to detect multiplex target organisms simultaneously, but complex optimizations of primer concentrations and staggered additions of primers are required to achieve equal amplification of multiplex genes. To overcome this shortcoming, we propose a novel method based on multiplex asymmetric PCR and paper-based nucleic acid diagnostics (PBNAD). In the asymmetric PCR, a universal primer was introduced to break the bottlenecks of low sensitivity and self-inhibition among different sets of primers. Amplification using the novel multiplex asymmetric PCR boosted the quantity of single-stranded amplicons, and the amplified products contained the same sequence at the 5' end. Therefore, only one gold nanoparticle-based signal probe was needed for the simultaneous detection of three genes using the PBNAD platform, and the detection signals could be observed with the naked eye. With this highly efficient, novel multiplex asymmetric PCR, as little as 1 pg/μL genomic DNA can be detected. This method can also be applied to genotyping for reliable epidemiological investigations. This proof-of-concept study highlights the potential of the PBNAD platform for cost- and labor-effective applications in the detection of pathogenic bacteria.

  12. PCR detection and PFGE genotype analyses of streptococcal clinical isolates from tilapia in China.

    PubMed

    Chen, Ming; Li, Li-Ping; Wang, Rui; Liang, Wan-Wen; Huang, Yan; Li, Jian; Lei, Ai-Ying; Huang, Wei-Yi; Gan, Xi

    2012-10-12

    Large-scale streptococcal outbreaks occurred continuously in tilapia farms of China from 2009 to 2011. The objective of this study was to characterize the prevalent strains of tilapia streptococci from the main cultured areas of China through species specific PCR and pulse field gel electrophoresis (PFGE). A total of 105 prevalent strains were isolated from Guangdong, Guangxi, Hainan and Fujian provinces between 2006 and 2011, 85 of which were identified as Streptococcus agalactiae while the rest were all identified as Streptococcus iniae. The prevalent stains in 2006 and 2007 were S. iniae (94.7%, 18/19), with S. agalactiae account for only 5.3% (1/19); The prevalent strains in 2009 and 2011 however changed to S. agalactiae (97.7%, 84/86), with only 2.3% (2/86) was S. iniae. Of these 105 strains, a total of 13 PFGE types (A-M) were characterized, among which D, F, G and K genotypes were predominant, accounting for 81.90% (86/105). The cluster analysis of PFGE electropherograms separated S. iniae and S. agalactiae to two distinctive branches, 20 strains of S. iniae exhibiting 3 types of PFGE band patterns with a similarity of 94.8-100%, and the 85 strains of S. agalactiae producing 10 types of PFGE band patterns with a similarity between 48.4% and 100%. Data suggested that the prevalent strains of tilapia streptococci in China have shifted from the former (before 2008) dominant strains of S. iniae to the current (2009-2011) dominant strains of S. agalactiae. Moreover, PFGE genotypes of the prevalent strains demonstrated geographic differences and temporal changes.

  13. Rapid detection of apolipoprotein E genotypes in Alzheimer's disease using polymerase chain reaction-single strand conformation polymorphism.

    PubMed

    Kamruecha, Worawan; Chansirikarnjana, Sirintorn; Nimkulrat, Ekapot; Udommongkol, Chesda; Wongmek, Wanna; Thangnipon, Wipawan

    2006-07-01

    Apolipoprotein E (APOE) gene on chromosome 19q13.2 is encoded by three common alleles designated as epsilon2, epsilon3 and epsilon4. In Alzheimer's disease (AD) the epsilon4 allele is over-represented and is considered to be a major genetic risk factor. Several methods have been developed to determine APOE genotypes. Among them, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) appears to be highly reliable. In this study, we improved the nonisotopic PCR-SSCP method for determining APOE genotypes in 42 cases of AD patients, 40 cases of non-AD dementia patients, and 49 cases of age-matched controls. DNA from the target sequence on APOE was amplified by PCR from peripheral blood genomic DNA. PCR products were electrophoresed in a non-denaturing polyacrylamide gel and visualized by silver staining. We found that the epsilon4 allele had a significantly high frequency of occurrence in AD patients (33.3%) compared with age-matched controls (13.3%) (chi(2) = 10.43, p = 0.001) and non-AD dementia (10%) (chi(2) = 13.02, p<0.001) whereas the epsilon3 allele was of high frequency in non-AD dementia (90%) compared with age-matched controls (85.7%) and AD patients (66.7%). APOE epsilon4 homozygotes were found only in AD groups. On the other hand, the epsilon2 allele was found only in an age-matched control. This study confirmed that the APOE psilon4 allele is a risk factor in Thai AD subjects and that the PCR-SSCP method is a rapid and useful means of detecting the APOE genotype in AD.

  14. Ovine plasma prion protein levels show genotypic variation detected by C-terminal epitopes not exposed in cell-surface PrPC.

    PubMed

    Thackray, Alana M; Fitzmaurice, Tim J; Hopkins, Lee; Bujdoso, Raymond

    2006-12-01

    Ovine PBMCs (peripheral blood mononuclear cells) express PrP(C) [cellular PrP (prion-related protein)] and have the potential to harbour and release disease-associated forms of PrP during scrapie in sheep. Cell-surface PrP(C) expression by PBMCs, together with plasma PrP(C) levels, may contribute to the regulatory mechanisms that determine susceptibility and resistance to natural scrapie in sheep. Here, we have correlated cell-surface PrP(C) expression on normal ovine PBMCs by FACS with the presence of PrP(C) in plasma measured by capture-detector immunoassay. FACS showed similar levels of cell-surface PrP(C) on homozygous ARR (Ala136-Arg154-Arg171), ARQ (Ala136-Arg154-Gln171) and VRQ (Val136-Arg154-Gln171) PBMCs. Cell-surface ovine PrP(C) showed modulation of N-terminal epitopes, which was more evident on homozygous ARR cells. Ovine plasma PrP(C) levels showed genotypic variation and the protein displayed C-terminal epitopes not available in cell-surface PrP(C). Homozygous VRQ sheep showed the highest plasma PrP(C) level and homozygous ARR animals the lowest. For comparison, similar analyses were performed on normal bovine PBMCs and plasma. PrP(C) levels in bovine plasma were approx. 4-fold higher than ovine homozygous ARQ plasma despite similar levels of PBMC cell-surface PrP(C) expression. Immunoassays using C-terminal-specific anti-PrP monoclonal antibodies as capture and detector reagents revealed the highest level of PrP(C) in both ovine and bovine plasma, whilst lower levels were detected using N-terminal-specific monoclonal antibody FH11 as the capture reagent. This suggested that a proportion of plasma PrP(C) was N-terminally truncated. Our results indicate that the increased susceptibility to natural scrapie displayed by homozygous VRQ sheep correlates with a higher level of plasma PrP(C).

  15. Detection of Mutually Exclusive Mosaicism in a Girl with Genotype-Phenotype Discrepancies

    PubMed Central

    Luo, Minjie; Mulchandani, Surabhi; Dubbs, Holly A.; Swarr, Daniel; Pyle, Louise; Zackai, Elaine H.; Spinner, Nancy B.; Conlin, Laura K.

    2015-01-01

    Discordance between clinical phenotype and genotype has multiple causes, including mosaicism. Phenotypes can be modified due to tissue distribution, or the presence of multiple abnormal cell lines with different genomic contributions. We have studied a 20-month-old female whose main phenotypes were failure to thrive, developmental delay, and patchy skin pigmentation. Initial chromosome and SNP microarray analysis of her blood revealed a non-mosaic ~24 Mb duplication of 15q25.1q26.3 resulting from the unbalanced translocation of terminal 15q to the short arm of chromosome 15. The most common feature associated with distal trisomy 15q is prenatal and postnatal overgrowth, which was not consistent with this patient’s phenotype. The phenotypic discordance, in combination with the patchy skin pigmentation, suggested the presence of mosaicism. Further analysis of skin biopsies from both hyper- and hypopigmented regions confirmed the presence of an additional cell line with the short arm of chromosome X deleted and replaced by the entire long arm of chromosome 15. The Xp deletion, consistent with a variant Turner Syndrome diagnosis, better explained the patient’s phenotype. Parental studies revealed that the alterations in both cell lines were de novo and the duplicated distal 15q and the deleted Xp were from different parental origins, suggesting a mitotic event. The possible mechanism for the occurrence of two mutually exclusive structural rearrangements with both involving the long arm of chromosome 15 is discussed. PMID:26198585

  16. Detection of Self Incompatibility Genotypes in Prunus africana: Characterization, Evolution and Spatial Analysis

    PubMed Central

    Nantongo, Judith Ssali; Eilu, Gerald; Geburek, Thomas; Schueler, Silvio; Konrad, Heino

    2016-01-01

    In flowering plants, self-incompatibility is an effective genetic mechanism that prevents self-fertilization. Most Prunus tree species exhibit a homomorphic gametophytic self-incompatibility (GSI) system, in which the pollen phenotype is encoded by its own haploid genome. To date, no identification of S-alleles had been done in Prunus africana, the only member of the genus in Africa. To identify S-RNase alleles and hence determine S-genotypes in African cherry (Prunus africana) from Mabira Forest Reserve, Uganda, primers flanking the first and second intron were designed and these amplified two bands in most individuals. PCR bands on agarose indicated 26 and 8 different S-alleles for second and first intron respectively. Partial or full sequences were obtained for all these fragments. Comparison with published S-RNase data indicated that the amplified products were S-RNase alleles with very high interspecies homology despite the high intraspecific variation. Against expectations for a locus under balancing selection, frequency and spatial distribution of the alleles in a study plot was not random. Implications of the results to breeding efforts in the species are discussed, and mating experiments are strongly suggested to finally prove the functionality of SI in P. africana. PMID:27348423

  17. Newly Identified Enterovirus C Genotypes, Identified in the Netherlands through Routine Sequencing of All Enteroviruses Detected in Clinical Materials from 2008 to 2015.

    PubMed

    Van Leer-Buter, Coretta C; Poelman, Randy; Borger, Renze; Niesters, Hubert G M

    2016-09-01

    Enteroviruses (EVs) are a group of human and animal viruses that are capable of causing a variety of clinical syndromes. Different genotypes classified into species can be distinguished on the basis of sequence divergence in the VP1 capsid-coding region. Apparently new genotypes are discovered regularly, often as incidental findings in studies investigating respiratory syndromes or as part of poliovirus surveillance. Recently, some EVs have become recognized as significant respiratory pathogens, and a number of new genotypes belonging to species C have been identified. The circulation of these newly identified species C EVs, such as EV-C104, EV-C105, EV-C109, and EV-C117, nevertheless appears to be limited. In this report, we show the results of routine genotyping of all enteroviruses detected in our tertiary care hospital between January 2008 and April 2015. We detected 365 EVs belonging to 40 genotypes. Interestingly, several newly identified species C EVs were detected during the study period. Sequencing of the 5' untranslated region (5' UTR) of these viruses shows divergence in this region, which is a target region in many detection assays.

  18. Newly Identified Enterovirus C Genotypes, Identified in the Netherlands through Routine Sequencing of All Enteroviruses Detected in Clinical Materials from 2008 to 2015

    PubMed Central

    Poelman, Randy; Borger, Renze; Niesters, Hubert G. M.

    2016-01-01

    Enteroviruses (EVs) are a group of human and animal viruses that are capable of causing a variety of clinical syndromes. Different genotypes classified into species can be distinguished on the basis of sequence divergence in the VP1 capsid-coding region. Apparently new genotypes are discovered regularly, often as incidental findings in studies investigating respiratory syndromes or as part of poliovirus surveillance. Recently, some EVs have become recognized as significant respiratory pathogens, and a number of new genotypes belonging to species C have been identified. The circulation of these newly identified species C EVs, such as EV-C104, EV-C105, EV-C109, and EV-C117, nevertheless appears to be limited. In this report, we show the results of routine genotyping of all enteroviruses detected in our tertiary care hospital between January 2008 and April 2015. We detected 365 EVs belonging to 40 genotypes. Interestingly, several newly identified species C EVs were detected during the study period. Sequencing of the 5′ untranslated region (5′ UTR) of these viruses shows divergence in this region, which is a target region in many detection assays. PMID:27358467

  19. Detection and Genotyping of Human Papillomavirus DNA in Formalin-Fixed Paraffin-Embedded Specimens with the HPV Direct Flow CHIP System.

    PubMed

    Herraez-Hernandez, Elsa; Preda, Ovidiu; Alonso, Sonia; Pardo, Rosario Serrano; Olmo, Asuncion

    2013-01-01

    The novel HPV Direct Flow CHIP commercial system for Human Papillomavirus (HPV) genotyping is based on rapid PCR and automatic reverse dot blot hybridization to genotype-specific probes, allowing the detection of 36 HPV genotypes. This study examined the performance of HPV Direct Flow CHIP in formalin-fixed paraffin-embedded (FFPE) samples (n= 99). Each sample was analyzed both by Direct PCR, using crude cell extracts without DNA purification, and by conventional PCR, using purified DNA. Pair-wise analysis of the results demonstrated strong concordance between the results obtained with the two protocols, although a slightly higher rate of multiple infections was detected by conventional PCR. In summary, HPV Direct Flow CHIP achieves effective HPV detection from FFPE samples with both Direct PCR and Conventional PCR protocols.

  20. Detection and genetic characterization of hepatitis E virus (HEV) genotype 3 subtype c in wild boars in Italy.

    PubMed

    Di Profio, Federica; Melegari, Irene; Sarchese, Vittorio; Robetto, Serena; Marruchella, Giuseppe; Bona, Maria Cristina; Orusa, Riccardo; Martella, Vito; Marsilio, Fulvio; Di Martino, Barbara

    2016-10-01

    Hepatitis E virus (HEV) was detected in stools collected from wild boars in Italy, with an overall prevalence of 1.5 % (3/196). The sequence of a ~3.0-kb portion at the 3' end of the genome of one such strain, HEV/WB/P6-15/ITA, was determined. In the full-length ORF2, which encodes the capsid protein, the virus was genetically closest to wild boar and human HEV strains currently classified as genotype 3 subtype c. Interestingly, the 3' end of ORF2 of the WB/P6-15/ITA matched the 340-nucleotide (nt) sequence (94.0 % nt identity) of the human strain PeGe, identified in 2015 from a patient with acute hepatitis E in Genoa, Italy, suggesting that similar HEV strains are circulating in the same geographical setting in humans and animals.

  1. Limitations of vitek GPS-418 cards in exact detection of vancomycin-resistant enterococci with the vanB genotype.

    PubMed

    Okabe, T; Oana, K; Kawakami, Y; Yamaguchi, M; Takahashi, Y; Okimura, Y; Honda, T; Katsuyama, T

    2000-06-01

    The susceptibilities of 20 strains of vancomycin-resistant enterococci (VRE) with the vanB genotype obtained by using Vitek GPS-418 cards were compared with those obtained by the broth dilution method of the National Committee for Clinical Laboratory Standards (NCCLS) (approved standard M7-A4) and with those obtained by the agar screen method using bile esculin azide agar containing 6 microgram of vancomycin per ml. Although both the broth dilution and agar screen methods disclosed no discordance, Vitek GPS-418 cards yielded a very major error compared with the results obtained by the reference broth dilution method of the NCCLS. Vitek GPS-418 cards were therefore found to have considerable room for improvement for the accurate detection of vanB VRE strains.

  2. Detection of Leptospira spp. in wildlife reservoir hosts in Ontario through comparison of immunohistochemical and polymerase chain reaction genotyping methods

    PubMed Central

    Shearer, Karen E.; Harte, Michael J.; Ojkic, Davor; DeLay, Josepha; Campbell, Douglas

    2014-01-01

    A total of 460 kidney samples from wildlife (beavers, coyotes, deer, foxes, opossums, otters, raccoons, skunks) were obtained from road-kill and hunter/trapper donations in Ontario between January 2010 and November 2012. The objectives of the study were to detect Leptospira spp. by immunohistochemistry and polymerase chain reaction (PCR), to map presence of leptospires in wildlife relative to livestock and human populations, and to characterize positive samples by sequencing and comparison to leptospires known to affect domestic animals and humans. The proportion of samples that tested positive ranged from 0% to 42%, with the highest rates in skunks and raccoons. Leptospira spp. were present in kidneys of wildlife across Ontario, particularly in areas of high human density, and areas in which livestock populations are abundant. The PCR was too weak in most samples to permit genotyping and examination of the relationship between the leptospires found in this study and those affecting domestic animals and humans. PMID:24587507

  3. Evaluation of a new-generation line-probe assay that detects 5' untranslated and core regions to genotype and subtype hepatitis C virus.

    PubMed

    Nadarajah, Rohan; Khan, G Yasmeen; Miller, Steven A; Brooks, Geo F

    2007-08-01

    The VERSANT HCV Genotype 2.0 Assay (LiPA 2.0; Innogenetics, Ghent, Belgium; distributed by Siemens Medical Solutions Diagnostics, Tarrytown, NY) is a new-generation line-probe assay that simultaneously detects sequences in the 5' untranslated (5'UTR) and core regions to genotype and subtype hepatitis C virus (HCV). We tested 60 specimens of known genotype and subtype and 2 specimens with mixed infections with the LiPA 2.0 assay. After arbitration based on genotype and subtype determined by sequencing, there was concordance in 58 of 60 specimens (specificity, 96.7%). Computer-assisted typing yielded comparable results, but much more rapidly. Of 67 clinical specimens, 64 readily yielded genotype and subtype; 3 indeterminate specimens were typed by sequencing and were uncommon types not in the database. The newgeneration line-probe assay that detects the 5'UTR and core regions to genotype and subtype HCV is applicable to more than 95% of specimens. Interpretation is facilitated by computer-assisted analysis.

  4. Detection of Porphyromonas gingivalis fimA Type I Genotype in Gingivitis by Real-Time PCR–A Pilot Study

    PubMed Central

    Krishnan, Mahalakshmi; Chandrasekaran, S.C.

    2016-01-01

    Introduction Published literature till date reveals a high prevalence of Porphyromonas gingivalis fimA type I genotype among healthy subjects. Quite a few studies have reported its prevalence also in periodontitis patients. Nevertheless incidence of this genotype in gingivitis is lacking in adult population. Aim The present study was chosen to detect P. gingivalis fimA type I genotype among chronic gingivitis patients. Materials and Methods A total of 46 subgingival plaque samples collected from chronic marginal gingivitis (n=23) and chronic periodontitis subjects (control group) (n=23) were subjected to Real-Time Polymerase Chain Reaction to detect the P. gingivalis fimA type I gene. Statistical analysis was performed using chi-square test. Results Prevalence of P. gingivalis fimA type I gene among chronic periodontitis and chronic gingivitis patients were 8.7% and 30.4% respectively. P. gingivalis fimA type I genotype prevalence was found to be statistically insignificant between the two study groups (p=0.135). Conclusion The avirulent P. gingivalis fimA type I genotype, occurred in high prevalence among chronic gingivitis patients, while its presence was low in chronic periodontitis patients. Presence of this avirulent genotype in chronic marginal gingivitis signifies its reversible condition. PMID:27504406

  5. Molecular characterization of human respiratory syncytial virus NA1 and GA5 genotypes detected in Assam in northeast India, 2009-2012.

    PubMed

    Biswas, Dipankar; Yadav, Kaushal; Borkakoty, Biswajyoti; Mahanta, Jagadish

    2013-09-01

    A significant number of children die each year from acute respiratory tract infections especially in developing countries. Human respiratory syncytial virus (RSV) is the most common virus identified in such cases. Genetic characterization and the circulation pattern of RSV is important for future selection of appropriate vaccine strains. Limited information is available on the circulation of RSV in developing countries including India. The present study aimed to provide baseline information on the genetic variability of RSV in the Dibrugarh district of Assam, northeast India. Clinical specimens collected from children aged ≤6 years for routine influenza surveillance in the Dibrugarh district of Assam during the period 2009-2012, were screened for RSV by real-time reverse transcription-polymerase chain reaction. Genotyping was based on partial sequencing of the RSV attachment glycoprotein gene. RSV was detected in 7.9% (39/493) of cases. Only RSV group A viruses were detected during the study period with predominance of NA1 genotypes (89%). Two RSV GA5 genotypes were found to be co-circulating during 2012. The specific amino acid substitutions characteristics of the NA1 genotypes were distinct from RSV strains reported from the rest of India. It is concluded that the circulating genotypes of RSV in Assam, northeast India are NA1 and GA5. To our knowledge this is the first report of circulation of the NA1 genotype in India.

  6. Comparison of the clinical performance of restriction fragment mass polymorphism (RFMP) and Roche linear array HPV test assays for HPV detection and genotyping.

    PubMed

    Lee, Hyo-Pyo; Cho, Woojae; Bae, Jae-Man; Shin, Ji Young; Shin, Soo-Kyung; Hwang, Sun Young; Min, Kyung Tae; Kim, Soo Nyung; Lee, Sun Joo; Kim, Soo-Ok; Yoo, Wang Don; Hong, Sun Pyo

    2013-06-01

    The need for accurate genotyping of human papillomavirus (HPV) infections is becoming increasingly important as HPV is the primary cause of cervical cancer worldwide. The matrix-assisted laser desorption ionization time-of-flight mass spectrometry-based restriction fragment mass polymorphism (RFMP) assay provides accurate, broad-spectrum, high-throughput genotyping of HPV. We evaluated the clinical performance of the RFMP assay compared to a commercially available Roche linear array HPV genotyping test (LA) for detecting and genotyping of HPV. The RFMP assay and the LA were compared for detecting and genotyping HPV among a cohort of 244 liquid-based cytology samples. Overall, 216 specimens (93.1%, κ = 0.86) generated concordant results for the presence or absence of high-risk HPV (HR-HPV) by the two assays. The RFMP assay and the LA assay generated concordant, compatible, and discordant genotyping results for 79.3, 9.9, and 10.8%, respectively. The diagnostic sensitivity and specificity of RFMP and LA for the cervical lesions of squamous cell carcinoma (SCC) were similar, at 92.9 and 85.0% (RFMP) and 92.9 and 83.8% (LA), respectively. In addition, the odds ratio for SCC with HR-HPV positivity estimated by the RFMP assay (73.7, 95% CI: 8.9-3173.3) was higher than the LA assay (67.0, 95% CI: 8.2-2887.0). The RFMP and the LA assays were highly comparable with regard to detection and genotyping analysis of HPV. The sensitivity and specificity of RFMP assay for the detection of HR-HPV in various levels of cervical lesions seems to be valuable in the monitoring of HPV-associated cervical cancer. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Whole genome analysis of porcine astroviruses detected in Japanese pigs reveals genetic diversity and possible intra-genotypic recombination.

    PubMed

    Ito, Mika; Kuroda, Moegi; Masuda, Tsuneyuki; Akagami, Masataka; Haga, Kei; Tsuchiaka, Shinobu; Kishimoto, Mai; Naoi, Yuki; Sano, Kaori; Omatsu, Tsutomu; Katayama, Yukie; Oba, Mami; Aoki, Hiroshi; Ichimaru, Toru; Mukono, Itsuro; Ouchi, Yoshinao; Yamasato, Hiroshi; Shirai, Junsuke; Katayama, Kazuhiko; Mizutani, Tetsuya; Nagai, Makoto

    2017-06-01

    Porcine astroviruses (PoAstVs) are ubiquitous enteric virus of pigs that are distributed in several countries throughout the world. Since PoAstVs are detected in apparent healthy pigs, the clinical significance of infection is unknown. However, AstVs have recently been associated with a severe neurological disorder in animals, including humans, and zoonotic potential has been suggested. To date, little is known about the epidemiology of PoAstVs among the pig population in Japan. In this report, we present an analysis of nearly complete genomes of 36 PoAstVs detected by a metagenomics approach in the feces of Japanese pigs. Based on a phylogenetic analysis and pairwise sequence comparison, 10, 5, 15, and 6 sequences were classified as PoAstV2, PoAstV3, PoAstV4, and PoAstV5, respectively. Co-infection with two or three strains was found in individual fecal samples from eight pigs. The phylogenetic trees of ORF1a, ORF1b, and ORF2 of PoAstV2 and PoAstV4 showed differences in their topologies. The PoAstV3 and PoAstV5 strains shared high sequence identities within each genotype in all ORFs; however, one PoAstV3 strain and one PoAstV5 strain showed considerable sequence divergence from the other PoAstV3 and PoAstV5 strains, respectively, in ORF2. Recombination analysis using whole genomes revealed evidence of multiple possible intra-genotype recombination events in PoAstV2 and PoAstV4, suggesting that recombination might have contributed to the genetic diversity and played an important role in the evolution of Japanese PoAstVs. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Accurate detection of Neisseria gonorrhoeae ciprofloxacin susceptibility directly from genital and extragenital clinical samples: towards genotype-guided antimicrobial therapy.

    PubMed

    Pond, Marcus J; Hall, Catherine L; Miari, Victoria F; Cole, Michelle; Laing, Ken G; Jagatia, Heena; Harding-Esch, Emma; Monahan, Irene M; Planche, Timothy; Hinds, Jason; Ison, Catherine A; Chisholm, Stephanie; Butcher, Philip D; Sadiq, Syed Tariq

    2016-04-01

    Increasing use of nucleic acid amplification tests (NAATs) as the primary means of diagnosing gonococcal infection has resulted in diminished availability of Neisseria gonorrhoeae antimicrobial susceptibility data. We conducted a prospective diagnostic assessment of a real-time PCR assay (NGSNP) enabling direct detection of gonococcal ciprofloxacin susceptibility from a range of clinical sample types. NGSNP, designed to discriminate an SNP associated with ciprofloxacin resistance within the N. gonorrhoeae genome, was validated using a characterized panel of geographically diverse isolates (n = 90) and evaluated to predict ciprofloxacin susceptibility directly on N. gonorrhoeae-positive NAAT lysates derived from genital (n = 174) and non-genital (n = 116) samples (n = 290), from 222 culture-confirmed clinical episodes of gonococcal infection. NGSNP correctly genotyped all phenotypically susceptible (n = 49) and resistant (n = 41) panel isolates. Ciprofloxacin-resistant N. gonorrhoeae was responsible for infection in 29.7% (n = 66) of clinical episodes evaluated. Compared with phenotypic susceptibility testing, NGSNP demonstrated sensitivity and specificity of 95.8% (95% CI 91.5%-98.3%) and 100% (95% CI 94.7%-100%), respectively, for detecting ciprofloxacin-susceptible N. gonorrhoeae, with a positive predictive value of 100% (95% CI 97.7%-100%). Applied to urogenital (n = 164), rectal (n = 40) and pharyngeal samples alone (n = 30), positive predictive values were 100% (95% CI 96.8%-100%), 100% (95% CI 87.2%-100%) and 100% (95% CI 82.4%-100%), respectively. Genotypic prediction of N. gonorrhoeae ciprofloxacin susceptibility directly from clinical samples was highly accurate and, in the absence of culture, will facilitate use of tailored therapy for gonococcal infection, sparing use of current empirical treatment regimens and enhancing acquisition of susceptibility data for surveillance. © The Author 2016. Published by Oxford

  9. Molecular epidemiology of group A rotavirus among children admitted to hospital in Salto, Uruguay, 2011-2012: first detection of the emerging genotype G12.

    PubMed

    Tort, Luis Fernando López; Victoria, Matías; Lizasoain A, Andrés; Castells, Matías; Maya, Leticia; Gómez, Mariela Martínez; Arreseigor, Edit; López, Patricia; Cristina, Juan; Leite, Jose Paulo Gagliardi; Colina, Rodney

    2015-05-01

    Group A rotavirus (RVA) is the most important etiologic agent of infant acute gastroenteritis (AGE) worldwide. Detection and molecular characterization of RVA in Salto department, Northwestern region of Uruguay, was conducted on 175 clinical samples, being 153 stool and 22 vomit samples, collected from hospitalized children with AGE, between 0-15 years old, from two hospitals of Salto city during 2011 and 2012. RVA was detected and genotyped by seminested multiplex RT-PCR in order to determine G- and P-genotypes. Positive samples were sequenced and phylogenetic analyses were carried out in order to determine lineages and sub-lineages. RVA were detected in 64 (37%) of the samples and the G and P genotypes observed were: 6% G1P[8], 23% G2P[4]/G2P[X]/GXP[4], 23% G3P[8]/G3P[X], 14% G12P[8]/G12P[X], 16% GXP[8], 1,5% G12P[9], 3% G2P[4]/[8], and 16% non-typeable. VP7 and VP4 genotypes related to DS-1 like gene constellation were prevalent during 2011 and those VP7 and VP4 genotypes related to Wa-like constellation were prevalent during 2012 (mainly represented by G3P[8]). Interestingly, RVA was detected in vomit samples in a high prevalence (41%). RVA was observed mainly in the age group between 1 and 5 years old (75% of the cases), and seasonality with a high detection rate in winter season was observed for the two consecutive years of surveillance. To our knowledge, this study represents the first detection and molecular characterization of RVA in Salto department, Northwestern region of Uruguay; and the first identification of the emerging genotype G12 in the country.

  10. Evaluation of GenoType MTBDRplus for the rapid detection of drug-resistant tuberculosis in Ghana.

    PubMed

    Asante-Poku, Adwoa; Otchere, Isaac Darko; Danso, Emelia; Mensah, David Delali; Bonsu, Frank; Gagneux, Sebastien; Yeboah-Manu, Dorothy

    2015-08-01

    Rapid but simple diagnostic tools for the detection of drug-resistant (DR) tuberculosis (TB) have been acknowledged as being important for its effective management and control. To establish a molecular line-probe assay (GenoType MTBDRplus) for detecting DR-TB in Ghana. We first screened 113 Mycobacterium tuberculosis isolates using the indirect proportion method and MTBDRplus. The rpoB and katG genes and the promoter regions of oxyR-ahpC and inhA were sequenced to identify mutations in isolates found to be resistant on phenotypic drug susceptibility testing and/or MTBDRplus. We then analysed an additional 412 isolates using only MTBDRplus. Respectively 43 (8.2%) and 8 (1.5%) isolates were resistant to isoniazid (INH) and rifampicin (RMP), while 8 (1.5%) were multidrug-resistant. In resistant isolates, mutations in codon 450 of rpoB and codon 315 of katG, conferring resistance to respectively RMP and INH, dominated. We found two RMP-resistant isolates with a S450L substitution, each harbouring an additional mutation at S388L and Q409R. Using phenotypic testing as gold standard, the MTBDRplus assay showed a sensitivity and specificity in the detection of RMP and INH resistance and multidrug resistance of respectively 100% and 100%, 83.3% and 100%, and 100% and 100%. The high sensitivity of MTBDRplus makes it a valuable addition to the conventional TB diagnostic algorithm in Ghana.

  11. Rapid detection of duck hepatitis A virus genotype C using reverse transcription loop-mediated isothermal amplification.

    PubMed

    Li, Chuanfeng; Chen, Zongyan; Meng, Chunchun; Liu, Guangqing

    2014-02-01

    A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was used and optimized to develop a rapid and sensitive detection system for duck hepatitis A virus genotype C (DHAV-C) RNA. A set of four specific primers was designed against highly conserved sequences located within the 3D gene from DHAV (strain GX1201). Under optimal reaction conditions, the sensitivity of DHAV-C-specific RT-LAMP was 100-fold higher than that of reverse transcriptase-polymerase chain reaction (RT-PCR), with a detection limit of 0.3pg (6.59×10(4) copies) per reaction. No cross-reactivity was observed from the samples of other duck viruses, which is in good accordance with RT-PCR. Furthermore, a positive reaction can be visually inspected by observing turbidity or color change after the addition of SYBR green I dye. The DHAV-C-specific RT-LAMP assay was applied to the samples and compared with RT-PCR. The positive-sample ratios were 26.7% (12 of 45) by RT-LAMP and 20% (9 of 45) by RT-PCR. Therefore, the newly developed RT-LAMP assay is a rapid, specific, sensitive, and cost-effective method of DHAV-C detection. This assay has potential applications in both clinical diagnosis and field surveillance of DHAV-C infection.

  12. Use of genotypic selection to detect P53 codon 273 CGT>CTT transversion: application to an occupationally exposed population.

    PubMed

    Carton, T; Tan, X D; Hartemann, P; Joyeux, M

    2007-01-01

    CGT>CTT transversion in codon 273 of the P53 tumor-suppressor gene is one of the major mutations detected in human tumors. Within an epidemiological framework, we investigated the use of a genotypic selection method to measure this point mutation. The allele-specific polymerase chain reaction (AS-PCR) that was developed was able to detect 10 mutant copies of the gene among a total of 5 x 10(5) wild-type copies. We used this assay to detect CGT>CTT transversions in buccal cell DNA of production workers (n=76) from a viscose factory exposed to carbon disulfide (amongst other pollutants) and in the DNA of non-exposed office workers (n=67). The mutation appeared more frequently in the exposed than in the non-exposed worker who were smokers. The results of the study indicate that occupational exposure results in a significant increase in P53 CGT>CTT transversions and more especially identified occupational exposure in combination with smoking as a significant risk factor for the mutation. We conclude that AS-PCR of the P53 273rd codon transversions is a suitable technique for studying the effects of occupational exposure.

  13. Detecting genotyping errors and describing black bear movement in northern Idaho

    Treesearch

    Michael K. Schwartz; Samuel A. Cushman; Kevin S. McKelvey; Jim Hayden; Cory Engkjer

    2006-01-01

    Non-invasive genetic sampling has become a favored tool to enumerate wildlife. Genetic errors, caused by poor quality samples, can lead to substantial biases in numerical estimates of individuals. We demonstrate how the computer program DROPOUT can detect amplification errors (false alleles and allelic dropout) in a black bear (Ursus americanus) dataset collected in...

  14. A new molecular diagnostic tool for quantitatively detecting and genotyping “Candidatus Liberibacter species”

    USDA-ARS?s Scientific Manuscript database

    A new molecular diagnostic method was developed for quantitative detection of “Candidatus Liberibacter” species associated with citrus Huanglongbing (“Ca. Liberibacter asiaticus”, “Ca. Liberibacter africanus” and “Ca. Liberibacter americanus”) and potato zebra chip disorder (“Ca. Liberibacter solana...

  15. Sensitive detection of multiple hepatitis A virus genotypes with a single polony-based assay

    USDA-ARS?s Scientific Manuscript database

    Hepatitis A virus (HAV) is one of the major causes of non-bacterial gastroenteritis in humans worldwide. HAV is mostly transmitted via direct person-to-person contact, or by consumption of contaminated foods and water. Since only a few viral particles may cause disease, detection of low levels of HA...

  16. International Collaborative Study To Compare Reverse Transcriptase PCR Assays for Detection and Genotyping of Noroviruses

    PubMed Central

    Vinjé, Jan; Vennema, Harry; Maunula, Leena; von Bonsdorff, Carl-Henrik; Hoehne, Marina; Schreier, Eckart; Richards, Alison; Green, Jon; Brown, David; Beard, Suzanne S.; Monroe, Stephan S.; de Bruin, Erwin; Svensson, Lennart; Koopmans, Marion P. G.

    2003-01-01

    To allow more rapid and internationally standardized assessment of the spread of noroviruses (previously called Norwalk-like viruses [NLVs]) as important food-borne pathogens, harmonization of methods for their detection is needed. Diagnosis of NLVs in clinical diagnostic laboratories is usually performed by reverse transciptase PCR (RT-PCR) assays. In the present study, the performance of five different RT-PCR assays for the detection of NLVs was evaluated in an international collaborative study by five laboratories in five countries with a coded panel of 91 fecal specimens. The assays were tested for their sensitivity, detection limit, and ease of standardization. In total, NLVs could be detected by at least one RT-PCR assay in 69 (84%) of the samples that originally tested positive. Sensitivity ranged from 52 to 73% overall and from 54 to 100% and 58 to 85% for genogroup I and II viruses, respectively. In all, 64% of the false-negative results were obtained with a set of diluted stools (n = 20) that may have lost quality upon storage. Sensitivity was improved when these samples were excluded from analysis. No one single assay stood out as the best, although the p1 assay demonstrated the most satisfactory overall performance. To promote comparability of data, this assay will be recommended for newly starting groups in future collaborative studies. PMID:12682125

  17. FIRST MOLECULAR DETECTION AND VP7 (G) GENOTYPING OF GROUP A ROTAVIRUS BY SEMI-NESTED RT-PCR FROM SEWAGE IN NIGERIA

    PubMed Central

    MOTAYO, Babatunde Olanrewaju; ADENIJI, Adekunle Johnson; FANEYE, Adedayo Omotayo

    2016-01-01

    SUMMARY Rotavirus is the leading cause of viral gastroenteritis worldwide, and sewage is a major source of the virus dissemination in the environment. Our aim was to detect and genotype rotaviruses from sewages in Nigeria. One hundred and ninety sewage samples were collected between June 2014 and January 2015. The two phase concentration method using PEG 6000 and dextran was used to concentrate sewage samples following WHO protocols. Molecular detection was performed by RT-PCR, and VP7 genotyping by semi-nested multiplex PCR. A total of 14.2% (n = 27) samples tested positive. Monthly distribution showed that June to September had a lower rate (3.7% to 7.4%), while October to January recorded 11% to 26%. Genotype G1 predominated followed by G8, G9, G4 and lastly G2, 7.4% (n = 2) of isolates were nontypeable. This is the first report of rotavirus detection in sewages from Nigeria. Genotype G1 remains the most prevalent genotype. This observation calls for an effort by the governmental authorities to implement a molecular surveillance, both clinical and environmental, in order to provide vital information for the control and the vaccine efficacy not only in Nigeria, but globally. PMID:27828615

  18. A method for rapid detection and genotype identification of hepatitis C virus 1-6 by one-step reverse transcription loop-mediated isothermal amplification.

    PubMed

    Nyan, Dougbeh-Chris; Swinson, Kevin L

    2016-02-01

    Hepatitis C virus (HCV) is probably the leading cause of liver cirrhosis and hepatocellular carcinoma globally. Diagnostic tools conventionally used for the detection and identification of HCV infection are technically demanding, time-consuming, and costly for resource-limited environments. This study reports the development of the first rapid loop-mediated reverse transcription isothermal amplification assay that rapidly detects and identifies HCV genotypes in blood components. RNA extracted from donor plasma and serum specimens was applied to a one-step reverse transcription loop-mediated isothermal amplification reaction performed with HCV-specific oligonucleotides. Reactions were conducted at 63.5 °C for 30-60 min. The diagnostic characteristics of the assay were investigated and validated with clinical specimens. Electrophoretic analysis of amplification revealed detection and identification of HCV genotypes 1-6. Positive amplification revealed unique ladder-like banding patterns that identified each HCV genotype. The assay demonstrated a sensitivity of 91.5% and specificity of 100%. Rapid naked-eye detection of HCV infection was facilitated by observation of an intense fluorescent glow of amplified targets under UV illumination. These diagnostic characteristics highlight the potential utility of this assay for the rapid detection and genotype identification of HCV infection in field and point-of-care settings in endemic regions and resource-limited environments. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  19. Detection and genotyping of canine coronavirus RNA in diarrheic dogs in Japan.

    PubMed

    Soma, Takehisa; Ohinata, Tsuyoshi; Ishii, Hiroshi; Takahashi, Toshikazu; Taharaguchi, Satoshi; Hara, Motonobu

    2011-04-01

    To clarify the prevalence of canine coronavirus (CCoV) infection in Japan, faecal samples from 109 dogs with diarrhoea were examined for CCoV RNA together with canine parvovirus type 2 (CPV-2) DNA. The detection rates of CCoV and CPV-2 for dogs aged less than 1 year were 66.3% and 43.8%, while those for dogs aged 1 year or older were 6.9% and 10.3%, respectively, which were significantly different (p<0.0001 and p=0.0003, respectively), indicating not CPV-2 but CCoV is an important diarrhoea-causing organism in juvenile dogs. Among the CCoV-positive dogs, 65.5% and 72.7% showed to be positive for CCoV types I and II, respectively, and simultaneous detection rate of both types was high at 40.0%. Furthermore, transmissible gastroenteritis virus (TGEV)-like CCoV RNA was detected from 8 dogs. These findings indicate that CCoV type I and TGEV-like CCoV are already circulating in Japan, though no reports have been presented to date. Copyright © 2010 Elsevier Ltd. All rights reserved.

  20. Molecular detection and genotyping of Japanese Encephalitis Virus in mosquitoes during a 2010 outbreak in the Republic of Korea

    USGS Publications Warehouse

    Seo, Hyun-Ji; Kim, Heung Chul; Klein, Terry A.; Ramey, Andrew M.; Lee, Ji-Hyee; Kyung, Soon-Goo; Park, Jee-Yong; Cho, In-Soo; Yeh, Jung-Yong

    2013-01-01

    Japanese encephalitis virus (JEV), a mosquito-borne zoonotic pathogen, is one of the major causes of viral encephalitis. To reduce the impact of Japanese encephalitis among children in the Republic of Korea (ROK), the government established a mandatory vaccination program in 1967. Through the efforts of this program only 0-7 (mean 2.1) cases of Japanese encephalitis were reported annually in the ROK during the period of 1984-2009. However, in 2010 there was an outbreak of 26 confirmed cases of Japanese encephalitis, including 7 deaths. This represented a >12-fold increase in the number of confirmed cases of Japanese encephalitis in the ROK as compared to the mean number reported over the last 26 years and a 3.7-fold increase over the highest annual number of cases during this same period (7 cases). Surveillance of adult mosquitoes was conducted during the 2010 outbreak of Japanese encephalitis in the ROK. A total of 6,328 culicine mosquitoes belonging to 12 species from 5 genera were collected at 6 survey sites from June through October 2010 and assayed by reverse-transcription polymerase chain reaction (RT-PCR) for the presence of JEV. A total of 34/371 pooled samples tested positive for JEV (29/121 Culex tritaeniorhynchus, 4/64 Cx. pipiens, and 1/26 Cx. bitaeniorhynchus) as confirmed by sequencing of the pre-membrane and envelope protein coding genes. The maximum likelihood estimates of JEV positive individuals per 1,000 culicine vectors for Cx. tritaeniorhynchus, Cx. pipiens, and Cx. bitaeniorhynchus were 11.8, 5.6, and 2.8, respectively. Sequences of the JEV pre-membrane and envelope protein coding genes amplified from the culicine mosquitoes by RT-PCR were compared with those of JEV genotypes I-V. Phylogenetic analyses support the detection of a single genotype (I) among samples collected from the ROK in 2010.

  1. Molecular Detection and Genotyping of Japanese Encephalitis Virus in Mosquitoes during a 2010 Outbreak in the Republic of Korea

    PubMed Central

    Klein, Terry A.; Ramey, Andrew M.; Lee, Ji-Hye; Kyung, Soon-Goo; Park, Jee-Yong; Cho, Yun Sang; Cho, In-Soo; Yeh, Jung-Yong

    2013-01-01

    Japanese encephalitis virus (JEV), a mosquito-borne zoonotic pathogen, is one of the major causes of viral encephalitis. To reduce the impact of Japanese encephalitis among children in the Republic of Korea (ROK), the government established a mandatory vaccination program in 1967. Through the efforts of this program only 0–7 (mean 2.1) cases of Japanese encephalitis were reported annually in the ROK during the period of 1984–2009. However, in 2010 there was an outbreak of 26 confirmed cases of Japanese encephalitis, including 7 deaths. This represented a >12-fold increase in the number of confirmed cases of Japanese encephalitis in the ROK as compared to the mean number reported over the last 26 years and a 3.7-fold increase over the highest annual number of cases during this same period (7 cases). Surveillance of adult mosquitoes was conducted during the 2010 outbreak of Japanese encephalitis in the ROK. A total of 6,328 culicine mosquitoes belonging to 12 species from 5 genera were collected at 6 survey sites from June through October 2010 and assayed by reverse-transcription polymerase chain reaction (RT-PCR) for the presence of JEV. A total of 34/371 pooled samples tested positive for JEV (29/121 Culex tritaeniorhynchus, 4/64 Cx. pipiens, and 1/26 Cx. bitaeniorhynchus) as confirmed by sequencing of the pre-membrane and envelope protein coding genes. The maximum likelihood estimates of JEV positive individuals per 1,000 culicine vectors for Cx. tritaeniorhynchus, Cx. pipiens, and Cx. bitaeniorhynchus were 11.8, 5.6, and 2.8, respectively. Sequences of the JEV pre-membrane and envelope protein coding genes amplified from the culicine mosquitoes by RT-PCR were compared with those of JEV genotypes I-V. Phylogenetic analyses support the detection of a single genotype (I) among samples collected from the ROK in 2010. PMID:23390520

  2. Performance evaluation of Anyplex™II HPV28 detection kit in a routine diagnostic setting: comparison with the HPV Sign® Genotyping Test.

    PubMed

    Marcuccilli, Fabbio; Farchi, Francesca; Mirandola, Walter; Ciccozzi, Massimo; Paba, Pierpaolo; Bonanno, Elena; Perno, Carlo Federico; Ciotti, Marco

    2015-06-01

    Anyplex™II HPV28 is a new PCR assay designed for HPV genotyping. It can detect 28 HPV types including 19 high-risk and 9 low-risk types. This study evaluated the performance of Anyplex™II HPV28 on 123 fresh cervical samples screened in parallel with HPV Sign® Genotyping Test. Of the 123 samples screened, 93 were positive, 15 negative, and 15 discordant. The total number of HPV positive samples combined was 108: 38 single infections and 70 multiple infections. The agreement between the two tests was 87.8%, κ=0.592. Genotype specific agreement was strong for HPV 16 (k=0.761), HPV 18 (k=0.674), and HPV 35 (k=0.796). Sensitivity and specificity of Anyplex™II HPV28 assay using HPV Sign® Genotyping Test as reference was 84.8% and 94%; conversely, sensitivity and specificity of HPV Sign® Genotyping Test was 29% and 99.5%. Anyplex™II HPV28 assay is a sensitive and specific assay suitable for HPV genotyping but requires clinical validation.

  3. Agreement for HPV genotyping detection between self-collected specimens on a FTA cartridge and clinician-collected specimens

    PubMed Central

    Guan, YaoYao; Gravitt, Patti E.; Howard, Roslyn; Eby, Yolanda J.; Wang, Shaoming; Li, Belinda; Feng, Changyan; Qiao, You-Lin; Castle, Philip E.

    2016-01-01

    The current method of transporting self-collected cervicovaginal specimen for HPV DNA testing relies on liquid based medium, which is challenging and expensive to transport. A novel, dry storage and transportation device, Whatman indicating FTA™ Elute Cartridge, avoids some of the pitfalls of liquid-based medium. This method has been shown to be comparable to liquid-based collection medium, but relative performance of self-collected (SC) and clinician-collected (CC) samples onto FTA cards has not been reported. The objective of this study is to compare the analytic performance of self- and clinician-collected samples onto FTA cartridges for the detection of carcinogenic HPV using Linear Array. There was a 91% agreement, 69% positive agreement, and kappa of 0.75 between the clinician-collected and self-collected specimens for detection of any carcinogenic HPV genotype. When the HPV results were categorized hierarchically according to cervical cancer risk, there was no difference in the distribution of the HPV results for the clinician- and self-collected specimens (p = 0.7). This study concludes that FTA elute cartridge is a promising method of specimen transport for cervical cancer screening programs considering using self-collected specimen and HPV testing. Larger studies with clinical endpoints are now needed to assess the clinical performance. PMID:23370404

  4. Detection and differentiation of human papillomavirus genotypes HPV-6 and HPV-11 by FRET-based real-time PCR.

    PubMed

    Kocjan, Bostjan J; Seme, Katja; Poljak, Mario

    2008-11-01

    A real-time PCR (RT-PCR) assay was developed based on fluorescence resonance energy transfer (FRET) hybridization probe technology, allowing very sensitive and specific detection of HPV-6 and HPV-11, reliable differentiation of HPV-6 and HPV-11, as well as prototypic and non-prototypic HPV-6 genomic variants, in a single PCR reaction. The primers and probe were designed on the basis of multiple alignments of 74 HPV-6 E2 gene sequences and 20 HPV-11 E2 gene sequences. Testing on defined plasmid standards showed that the RT-PCR allowed simple and reliable identification of HPV-6 and HPV-11 using type specific amplification followed by probe-specific post-amplification dissociation analysis. Sensitivity, assessed by probit analysis at a 95% detection level, was 42.9, 43.4, and 25.3 DNA copies per assay for prototypic and non-prototypic HPV-6 variants and HPV-11, respectively. The results obtained by the developed assay on 51 HPV DNA-negative samples and 149 HPV DNA-positive samples, including 81 HPV-6 positive samples (19 prototypic and 62 non-prototypic HPV-6 variants), 28 HPV-11 positive samples, 10 samples of HPV-44 and HPV-74 (the closest relatives of HPV-6 and HPV-11) and 30 samples of 15 other important alpha HPV, showed complete agreement with those obtained with the INNO-LiPA human papillomavirus (HPV) Genotyping Assay and HPV-6 E2 and E6 gene sequencing.

  5. Phenotypic and genotypic detection of methicillin-resistant Staphylococcus aureus in hunting dogs in Maiduguri metropolitan, Borno State, Nigeria

    PubMed Central

    Mustapha, Muhammad; Bukar-Kolo, Yachilla Maryam; Geidam, Yaqub Ahmed; Gulani, Isa Adamu

    2016-01-01

    Aim: To determine the presence of MRSA in hunting dogs in Maiduguri metropolitan. Materials and Methods: Phenotypic methods used includes microscopic technique, colony morphology study, catalase-coagulase tests, and the use of mannitol salt agar test, oxacillin resistance screening agar base, and antibiotic susceptibility testing methods. Genotypic approach was used for deoxyribonucleic acid extraction, and the presence of nuc and mecA gene was detected using polymerase chain reaction (PCR) techniques. Results: Examination of 416 swab samples from nasal and perineal region of dogs revealed a total of 79.5% of S. aureus, where 62.5% of the isolates were MRSA. Molecular analysis revealed that 7nuc genes specific for S. aureus from 20 presumptive MRSA assay were all mecA PCR negative. The isolates were sensitive to gentamicin and ciprofloxacin but proved resistant to cefoxitin and oxacillin. Conclusion: High isolation rate of MRSA was found in hunting dogs. Significant level (p<0.05) of MRSA was isolated in the nasal cavity of hunting dogs than its perineum. Only nuc genes were detected from the MRSA isolates. PMID:27284227

  6. A finite mixture model for genotype and environment interactions: detecting latent population heterogeneity.

    PubMed

    Gillespie, Nathan A; Neale, Michael C

    2006-06-01

    Approaches such as DeFries-Fulker extremes regression (LaBuda et al., 1986) are commonly used in genetically informative studies to assess whether familial resemblance varies as a function of the scores of pairs of twins. While useful for detecting such effects, formal modeling of differences in variance components as a function of pairs' trait scores is rarely attempted. We therefore present a finite mixture model which specifies that the population consists of latent groups which may differ in (i) their means, and (ii) the relative impact of genetic and environmental factors on within-group variation and covariation. This model may be considered as a special case of a factor mixture model, which combines the features of a latent class model with those of a latent trait model. Various models for the class membership of twin pairs may be employed, including additive genetic, common environment, specific environment or major locus (QTL) factors. Simulation results based on variance components derived from Turkheimer and colleagues (2003), illustrate the impact of factors such as the difference in group means and variance components on the feasibility of correctly estimating the parameters of the mixture model. Model-fitting analyses estimated group heritability as .49, which is significantly greater than heritability for the rest of the population in early childhood. These results suggest that factor mixture modeling is sufficiently robust for detecting heterogeneous populations even when group mean differences are modest.

  7. Application of DNA-DNA colony hybridization to the detection of catabolic genotypes in environmental samples.

    PubMed Central

    Sayler, G S; Shields, M S; Tedford, E T; Breen, A; Hooper, S W; Sirotkin, K M; Davis, J W

    1985-01-01

    The application of preexisting DNA hybridization techniques was investigated for potential in determining populations of specific gene sequences in environmental samples. Cross-hybridizations among two degradative plasmids, TOL and NAH, and two cloning vehicles, pLAFR1 and RSF1010, were determined. The detection limits for the TOL plasmid against a nonhomologous plasmid-bearing bacterial background was ascertained. The colony hybridization technique allowed detection of one colony containing TOL plasmid among 10(6) Escherichia coli colonies of nonhomologous DNA. Comparisons between population estimates derived from growth on selective substrates and from hybridizations were examined. Findings indicated that standard sole carbon source enumeration procedures for degradative populations lead to overestimations due to nonspecific growth of other bacteria on the microcontaminant carbon sources present in the media. Population estimates based on the selective growth of a microcosm population on two aromatic substrates (toluene and naphthalene) and estimates derived from DNA-DNA colony hybridizations, using the TOL or NAH plasmid as a probe, corresponded with estimates of substrate mineralization rates and past exposure to environmental contaminants. The applications of such techniques are hoped to eventually allow enumeration of any specific gene sequences in the environment, including both anabolic and catabolic genes. In addition, this procedure should prove useful in monitoring recombinant DNA clones released into environmental situations. Images PMID:4004244

  8. High-resolution melting analysis for detection of a single-nucleotide polymorphism and the genotype of the myostatin gene in warmblood horses.

    PubMed

    Serpa, Priscila B S; Garbade, Petra; Natalini, Cláudio C; Pires, Ananda R; Tisotti, Tainor M

    2017-01-01

    OBJECTIVE To develop a high-resolution melting (HRM) assay to detect the g.66493737C>T polymorphism in the myostatin gene (MSTN) and determine the frequency of 3 previously defined g.66493737 genotypes (T/T, T/C, and C/C) in warmblood horses. SAMPLES Blood samples from 23 horses. PROCEDURES From each blood sample, DNA was extracted and analyzed by standard PCR methods and an HRM assay to determine the MSTN genotype. Three protocols (standard protocol, protocol in which a high-salt solution was added to the reaction mixture before the first melting cycle, and protocol in which an unlabeled probe was added to the reaction mixture before analysis) for the HRM assay were designed and compared. Genotype results determined by the HRM protocol that generated the most consistent melting curves were compared with those determined by sequencing. RESULTS The HRM protocol in which an unlabeled probe was added to the reaction mixture generated the most consistent melting curves. The genotypes of the g.66493737C>T polymorphism were determined for 22 horses (16 by HRM analysis and 20 by sequencing); 14, 7, and 1 had the T/T, T/C, and C/C genotypes, respectively. The genotype determined by HRM analysis agreed with that determined by sequencing for 14 of 16 horses. The frequency of alleles T and C was 79.5% and 20.5%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that HRM analysis may be a faster and more economical alternative than PCR methods for genotyping. Genotyping results might be useful as predictors of athletic performance for horses.

  9. Development of a Rapid High-Throughput Method for High-Resolution Melting Analysis for Routine Detection and Genotyping of Noroviruses▿

    PubMed Central

    Tajiri-Utagawa, Etsuko; Hara, Masayuki; Takahashi, Kuniaki; Watanabe, Mayumi; Wakita, Takaji

    2009-01-01

    We developed a simple, rapid, high-throughput detection and genotyping method for noroviruses using real-time reverse transcription-PCR (RT-PCR) and high-resolution melting (HRM) analysis to create a difference plot. The capsid gene was amplified by real-time RT-PCR in the presence of ResoLight HRM dye, a saturating DNA dye. Following optimization of the HRM assay conditions, the major norovirus genotypes were selected. Because we had only small quantities of the patient stool samples used in this study, we constructed plasmids for each genotype and used these to optimize the HRM assay. We selected six stool samples, each positive for one of the six dominant subtypes of noroviruses that have been circulating in Japan, namely, genotypes 4, 8, and 9 from genogroup 1 and genotypes 3, 4, and 10 from genogroup 2. The specific high-resolution derivate plot of the HRM assay for each plasmid was constructed by subtracting the melting-curve shape of the plasmid from the reference or base curve. The RNAs extracted from 14 clinical samples positive for small round structured viruses were then directly analyzed using the HRM assay. The HRM data from the clinical RNA samples corresponded with the genotype results obtained by RT-PCR and sequencing of the clinical samples. In addition, the HRM data from the clinical RNA samples corresponded with the HRM data from the six reference plasmid DNAs, indicating that this assay is useful for the direct detection and genotyping of noroviruses in clinical samples. This assay requires no multiplexing or hybridization probes and provides a new approach to the genetic screening of noroviruses in clinical virology laboratories. PMID:19073870

  10. Genotype-specific fluorogenic RT-PCR assays for the detection and quantitation of canine coronavirus type I and type II RNA in faecal samples of dogs.

    PubMed

    Decaro, Nicola; Martella, Vito; Ricci, Dominga; Elia, Gabriella; Desario, Costantina; Campolo, Marco; Cavaliere, Nicola; Di Trani, Livia; Tempesta, Maria; Buonavoglia, Canio

    2005-12-01

    Two genotype-specific fluorogenic RT-PCR assays were developed for the detection and quantitation of canine coronavirus (CCoV) type I and type II RNA in the faeces of dogs with diarrhoea. Both the fluorogenic assays showed high specificity, sensitivity and reproducibility, allowing a precise quantitation of CCoV type I and type II RNA over a linear range of about eight orders of magnitude (from 10(1) to 10(8) copies of standard RNA). Comparison with genotype-specific gel-based RT-PCR assays revealed that the fluorogenic assays were more sensitive and more rapid than conventional amplifications, with a large increase in throughput. The genotype-specific fluorogenic assays were then used to detect and measure viral loads in the faecal samples collected from dogs naturally or experimentally infected with type I, type II, or both genotypes. Of 174 samples collected from naturally infected dogs, 77 were positive for CCoV type I and 46 for CCoV type II. Thirty-eight dogs were found to be infected naturally by both genotypes, with viral RNA titres generally higher for type I in comparison to type II. At the same time, dogs infected experimentally shed type I RNA with higher titres with respect to type II.

  11. Differential Detection of Human Papillomavirus Genotypes and Cervical Intraepithelial Neoplasia by Four Commercial Assays

    PubMed Central

    Bonde, Jesper; Preisler, Sarah; Ejegod, Ditte; Rygaard, Carsten; Lynge, Elsebeth

    2016-01-01

    Laboratories now can choose from >100 human papillomavirus (HPV) assays for cervical screening. Our previous analysis based on the data from the Danish Horizon study, however, showed that four widely used assays, Hybrid Capture 2 (HC2), cobas, CLART, and Aptima, frequently do not detect the same HPV infections. Here, we determined the characteristics of the concordant samples (all four assays returning a positive HPV test result) and discordant samples (all other HPV-positive samples) in primary cervical screening at 30 to 65 years of age (n = 2,859) and in a concurrent referral population from the same catchment area (n = 885). HPV testing followed the manufacturers' protocols. Women with abnormal cytology were managed according to the routine recommendations. Cytology-normal/HPV-positive women were invited for repeated testing in 18 months. Screening history and histologically confirmed cervical intraepithelial neoplasia (CIN) in 2.5 years after the baseline testing were determined from the national pathology register. HPV-positive women undergoing primary screening having concordant samples were more likely to harbor high-risk infections and less likely to harbor only low-risk infections than women with discordant samples. Additionally, assay signal strengths were substantially higher in concordant samples. More than 80% of ≥CIN2 results were found for women with concordant samples, and no ≥CIN2 results were found when the infection was detected by only one assay. These patterns were similar in the referral population despite the younger age and higher number of HPV infections. HPV test result discordance identified a cluster of low-risk HPV infections that were hardly ever associated with high-grade CIN and, almost exclusively, represented false-positive screening findings. PMID:27535689

  12. Cladograms with Path to Event (ClaPTE): A novel algorithm to detect associations between genotypes or phenotypes using phylogenies

    PubMed Central

    Handelman, Samuel K; Aaronson, Jacob M.; Seweryn, Michal; Voronkin, Igor; Kwiek, Jesse J.; Sadee, Wolfgang; Verducci, Joseph S.; Janies, Daniel A.

    2015-01-01

    Background Associations between genotype and phenotype provide insight into the evolution of pathogenesis, drug resistance, and the spread of pathogens between hosts. However, common ancestry can lead to apparent associations between biologically unrelated features. The novel method Cladograms with Path to Event (ClaPTE) detects associations between character-pairs (either a pair of mutations or a mutation paired with a phenotype) while adjusting for common ancestry, using phylogenetic trees. Methods ClaPTE tests for character-pairs changing close together on the phylogenetic tree, consistent with an associated character-pair. ClaPTE is compared to three existing methods (independent contrasts, mixed model, and likelihood ratio) to detect character-pair associations adjusted for common ancestry. Comparisons utilize simulations on gene trees for: HIV Env, HIV promoter, and bacterial DnaJ and GuaB; and case studies for Oseltamavir resistance in Influenza, and for DnaJ and GuaB. Simulated data include both true-positive/associated character-pairs, and true-negative/not-associated character-pairs, used to assess type I (frequency of p-values in true-negatives) and type II (sensitivity to true-positives) error control. Results and conclusions ClaPTE has competitive sensitivity and better type I error control than existing methods. In the Influenza/Oseltamavir case study, ClaPTE reports no new permissive mutations but detects associations between adjacent (in primary sequence) amino acid positions which other methods miss. In the DnaJ and GuaB case study, ClaPTE reports more frequent associations between positions both from the same protein family than between positions from different families, in contrast to other methods. In both case studies, the results from ClaPTE are biologically plausible. PMID:25577610

  13. Detection of Common, Emerging and Uncommon VP4, and VP7 Human Group A Rotavirus Genotypes from Urban Sewage Samples in Uruguay.

    PubMed

    Tort, Luis Fernando Lopez; Victoria, Matías; Lizasoain, Andrés; García, Mariana; Berois, Mabel; Cristina, Juan; Leite, José Paulo Gagliardi; Gómez, Mariela Martínez; Miagostovich, Marize Pereira; Colina, Rodney

    2015-12-01

    Environmental approach has proven to be a useful tool for epidemiological studies demonstrating through environmental studies the diversity of viruses circulating in a given population. The aim of this study was to perform a phylogenetic characterization of the group A rotavirus (RVA) glycoprotein (G)- and protease-sensitive (P)-genotypes obtained from sewage samples (n = 116) collected in six cities of Uruguay during March 2011 to April 2013. A worldwide standardized semi-nested multiplex RT-PCR (SNM RT-PCR) protocol directed against VP4 and VP7 genes were conducted for RVA detection and consensual DNA fragments were submitted to nucleotide sequencing. P and/or G genotype was successfully determined by phylogenetic analysis in 61% (n = 37) of the positive samples obtained by SNM RT-PCR (n = 61). The RVA genotypes were as follow: G1 (n = 2), G2 (n = 14), G3 (n = 5), G12 (n = 2), P[4] (n = 4), P[8] (n = 16), and P[3] (n = 2). Interestingly, through phylogenetic analysis, emerging, and uncommon human genotypes could be detected. Results obtained from the comparison of RVA genotypes detected in the current study and Uruguayan RVA strains previously described for contemporary clinical pediatric cases showed that monitoring sewage may be a good screening option for a rapid and economical overview of the circulating genotypes in the surrounding human population and a useful approximation to study RVA epidemiology in a future vaccine monitoring program. The present study represents the first report in Uruguay that describes the phylogenetic diversity of RVA from urban sewage samples.

  14. [Helicobacter pylori: cagA analysis and vacA genotyping in Chile. Detection of a s2/m1 strain].

    PubMed

    Martínez, A; González, C; Kawaguchi, F; Montoya, R; Corvalán, A; Madariaga, J; Roa, J; García, A; Salgado, F; Solar, H; Palma, M

    2001-10-01

    The genes cagA and vacA encode H pylori virulence factors. To genotype these genes in H pylori strains isolated from patients with upper gastrointestinal symptoms. We studied 50 patients who underwent an upper gastrointestinal endoscopy, with positive culture for H pylori. Detection of cagA and vacA genotyping was done using polymerase chain reactions. The gene cagA was detected in 19 samples (38%). Signal sequences s1 and s2 of vacA gene were detected in 16 samples each (32%). There was simultaneous amplification of s1 and s2 in 6 samples and they were not detected in 9 samples. The middle region of vacA was m1 in 9 samples, m2 in 29 samples and there was simultaneous amplification of m1 and m2 in 12 samples. In 16 samples (32%), more than one type of signal sequence or medial region was detected. Of those patients in whom vacA was the only genotype detected, 15 were s2/m2, 7 were s1/m1, 4 were s1/m2 and 1 was s2/m1. In these patients, the infection with cagA- H pylori strains, predominates, the prevalence of infection with s1 or s2 strains is similar and the predominant medial region is m2.

  15. AN EVALUATION OF CRYPTOSPORIDIUM PARVUM GENOTYPING

    EPA Science Inventory

    We evaluated the specificity and sensitivity of 11 previously described species differentiation and genotyping PCR protocols for detection of Cryptosporidium parasites. Genomic DNA from three species of Crytosporidium parasites (genotype 1 and genotype 2 of C. parvum, C. muris, a...

  16. AN EVALUATION OF CRYPTOSPORIDIUM PARVUM GENOTYPING

    EPA Science Inventory

    We evaluated the specificity and sensitivity of 11 previously described species differentiation and genotyping PCR protocols for detection of Cryptosporidium parasites. Genomic DNA from three species of Crytosporidium parasites (genotype 1 and genotype 2 of C. parvum, C. muris, a...

  17. A novel DNA extraction and duplex polymerase chain reaction assay for the rapid detection of Prototheca zopfii genotype 2 in milk.

    PubMed

    Gao, J; Hou, R-G; Zhang, H-Q; He, J-Z; Li, S-M; Su, J-L; Han, B

    2011-09-01

    To develop a PCR-based assay to detect Prototheca zopfii (P. zopfii) and its mastitis-related subtype (genotype 2) directly from milk samples. The DNA extraction method herein is based on the lysing properties of chemical agents, mechanical grinding and DNA-binding properties of silica particles; this method was developed to rapidly extract DNA directly from P. zopfii in bovine milk. Two pairs of primers specific for P. zopfii and genotype 2 were used in the duplex PCR, and a sensitivity test showed that the detection level was 5 × 10(2) colony-forming units (CFU) ml(-1) for P. zopfii and 5 × 10(3) CFU ml(-1) for genotype 2. Furthermore, a practical survey of 23 milk samples showed that the assay produced results that were in accordance with those obtained by the conventional microbiology method. The DNA extraction method is effective in isolating sufficient quantities of DNA from P. zopfii in milk for PCR analysis. The PCR assay is economical, sensitive and more rapid than the conventional culture method. The assay could be used as an alternative method for the rapid the detection of bovine mastitis resulting from P. zopfii genotype 2. © 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.

  18. Generalist dinoflagellate endosymbionts and host genotype diversity detected from mesophotic (67-100 m depths) coral Leptoseris

    PubMed Central

    Chan, Yvonne L; Pochon, Xavier; Fisher, Marla A; Wagner, Daniel; Concepcion, Gregory T; Kahng, Samuel E; Toonen, Robert J; Gates, Ruth D

    2009-01-01

    Background Mesophotic corals (light-dependent corals in the deepest half of the photic zone at depths of 30 - 150 m) provide a unique opportunity to study the limits of the interactions between corals and endosymbiotic dinoflagellates in the genus Symbiodinium. We sampled Leptoseris spp. in Hawaii via manned submersibles across a depth range of 67 - 100 m. Both the host and Symbiodinium communities were genotyped, using a non-coding region of the mitochondrial ND5 intron (NAD5) and the nuclear ribosomal internal transcribed spacer region 2 (ITS2), respectively. Results Coral colonies harbored endosymbiotic communities dominated by previously identified shallow water Symbiodinium ITS2 types (C1_ AF333515, C1c_ AY239364, C27_ AY239379, and C1b_ AY239363) and exhibited genetic variability at mitochondrial NAD5. Conclusion This is one of the first studies to examine genetic diversity in corals and their endosymbiotic dinoflagellates sampled at the limits of the depth and light gradients for hermatypic corals. The results reveal that these corals associate with generalist endosymbiont types commonly found in shallow water corals and implies that the composition of the Symbiodinium community (based on ITS2) alone is not responsible for the dominance and broad depth distribution of Leptoseris spp. The level of genetic diversity detected in the coral NAD5 suggests that there is undescribed taxonomic diversity in the genus Leptoseris from Hawaii. PMID:19747389

  19. Familial Case of Pelizaeus-Merzbacher Disorder Detected by Oligoarray Comparative Genomic Hybridization: Genotype-to-Phenotype Diagnosis

    PubMed Central

    Kariminejad, Roxana; Hosseini, Kaveh; Moshtagh, Azadeh; Abbassi, Gole Maryam; Sadatian, Neda; Bazrgar, Masood; Kariminejad, Ariana; Kariminejad, Mohamad Hassan

    2017-01-01

    Introduction. Pelizaeus-Merzbacher disease (PMD) is an X-linked recessive hypomyelinating leukodystrophy characterized by nystagmus, spastic quadriplegia, ataxia, and developmental delay. It is caused by mutation in the PLP1 gene. Case Description. We report a 9-year-old boy referred for oligoarray comparative genomic hybridization (OA-CGH) because of intellectual delay, seizures, microcephaly, nystagmus, and spastic paraplegia. Similar clinical findings were reported in his older brother and maternal uncle. Both parents had normal phenotypes. OA-CGH was performed and a 436 Kb duplication was detected and the diagnosis of PMD was made. The mother was carrier of this 436 Kb duplication. Conclusion. Clinical presentation has been accepted as being the mainstay of diagnosis for most conditions. However, recent developments in genetic diagnosis have shown that, in many congenital and sporadic disorders lacking specific phenotypic manifestations, a genotype-to-phenotype approach can be conclusive. In this case, a diagnosis was reached by universal genomic testing, namely, whole genomic array. PMID:28133555

  20. Amplification of the Gp41 gene for detection of mutations conferring resistance to HIV-1 fusion inhibitors on genotypic assay

    NASA Astrophysics Data System (ADS)

    Tanumihardja, J.; Bela, B.

    2017-08-01

    Fusion inhibitors have potential for future use in HIV control programs in Indonesia, so the capacity to test resistance to such drugs needs to be developed. Resistance-detection with a genotypic assay began with amplification of the target gene, gp41. Based on the sequence of the two most common HIV subtypes in Indonesia, AE and B, a primer pair was designed. Plasma samples containing both subtypes were extracted to obtain HIV RNA. Using PCR, the primer pair was used to produce the amplification product, the identity of which was checked based on length under electrophoresis. Eleven plasma samples were included in this study. One-step PCR using the primer pair was able to amplify gp41 from 54.5% of the samples, and an unspecific amplification product was seen in 1.1% of the samples. Amplification failed in 36.4% of the samples, which may be due to an inappropriate primer sequence. It was also found that the optimal annealing temperature for producing the single expected band was 57.2 °C. With one-step PCR, the designed primer pair amplified the HIV-1 gp41 gene from subtypes AE and B. However, further research should be done to determine the conditions that will increase the sensitivity and specificity of the amplification process.

  1. Generalist dinoflagellate endosymbionts and host genotype diversity detected from mesophotic (67-100 m depths) coral Leptoseris.

    PubMed

    Chan, Yvonne L; Pochon, Xavier; Fisher, Marla A; Wagner, Daniel; Concepcion, Gregory T; Kahng, Samuel E; Toonen, Robert J; Gates, Ruth D

    2009-09-11

    Mesophotic corals (light-dependent corals in the deepest half of the photic zone at depths of 30-150 m) provide a unique opportunity to study the limits of the interactions between corals and endosymbiotic dinoflagellates in the genus Symbiodinium. We sampled Leptoseris spp. in Hawaii via manned submersibles across a depth range of 67-100 m. Both the host and Symbiodinium communities were genotyped, using a non-coding region of the mitochondrial ND5 intron (NAD5) and the nuclear ribosomal internal transcribed spacer region 2 (ITS2), respectively. Coral colonies harbored endosymbiotic communities dominated by previously identified shallow water Symbiodinium ITS2 types (C1_ AF333515, C1c_ AY239364, C27_ AY239379, and C1b_ AY239363) and exhibited genetic variability at mitochondrial NAD5. This is one of the first studies to examine genetic diversity in corals and their endosymbiotic dinoflagellates sampled at the limits of the depth and light gradients for hermatypic corals. The results reveal that these corals associate with generalist endosymbiont types commonly found in shallow water corals and implies that the composition of the Symbiodinium community (based on ITS2) alone is not responsible for the dominance and broad depth distribution of Leptoseris spp. The level of genetic diversity detected in the coral NAD5 suggests that there is undescribed taxonomic diversity in the genus Leptoseris from Hawaii.

  2. Development and evaluation of a seminested PCR for detection and differentiation of Babesia gibsoni (Asian genotype) and B. canis DNA in canine blood samples.

    PubMed

    Birkenheuer, Adam J; Levy, Michael G; Breitschwerdt, Edward B

    2003-09-01

    Canine babesiosis has recently been recognized as an emerging infectious disease of dogs in North America. We sought to develop a seminested PCR to detect and differentiate Babesia gibsoni (Asian genotype), B. canis subsp. vogeli, B. canis subsp. canis, and B. canis subsp. rossi DNA in canine blood samples. An outer primer pair was designed to amplify an approximately 340-bp fragment of the 18S rRNA genes from B. gibsoni (Asian genotype), B. canis subsp. vogeli, B. canis subsp. rossi, and B. canis subsp. canis but not mammalian DNA. Forward primers were designed that would specifically amplify a smaller fragment from each organism in a seminested PCR. The practical limit of detection was 50 organisms/ml of mock-infected EDTA anticoagulated whole blood. The primer pair also amplified an approximately 370-bp fragment of the B. gibsoni (USA/California genotype) 18S rRNA gene from the blood of an experimentally infected dog with a high percentage of parasitemia. Amplicons were not detected when DNA extracted from the blood of a dog that was naturally infected with Theileria annae at a low percentage of parasitemia was amplified. Due to limited sensitivity, this test is not recommended for the routine diagnosis of B. gibsoni (USA/California genotype) or T. annae. The PCR test did not amplify Toxoplasma gondii, Neospora caninum, Leishmania infantum, Cryptosporidium parvum, or canine DNA under any of the conditions tested. The seminested PCR test was able to detect and discriminate B. gibsoni (Asian genotype), B. canis subsp. vogeli, B. canis subsp. canis, and B. canis subsp. rossi DNA in blood samples from infected dogs.

  3. Detection, genotyping and quantitation of multiple hpv infections in south African women with cervical squamous cell carcinoma.

    PubMed

    Lebelo, Ramokone L; Bogers, Johannes J; Thys, Sofie; Depuydt, Christophe; Benoy, Ina; Selabe, S Gloria; Bida, Meshack N; Mphahlele, M Jeffrey

    2015-09-01

    In Africa, data is limited on quantitation of human papillomavirus (HPV) types in women with multiple infections. This study applied a real time PCR (qPCR) assay for detection, genotyping and quantitation of multiple HPV infections in 90 tissue blocks of South African women with cervical squamous cell carcinoma. One sample with multiple HPV types was subjected to laser micro-dissection and qPCR. Four samples were negative for β-globin and these were excluded from the analysis. The HPV DNA positivity rate was 93.0% (80/86). All 80 positives showed the presence of HR HPV types; HPV 68 was the only type negative in all the samples. Overall, HPV 16 was positive in most of the samples (88.8%), followed by HPV 56 (28.7%), HPV 18 (20.0%) and HPV 39 (18.7%). More than half of the samples (65.0%) had multiple infections. HPV 16 was present in majority of single (85.7%) and multiple infections (90.4%). HPV 16 showed higher viral loads in 70.3% of the HPV 16 co-infected samples. In one multiple infected sample laser micro-dissection and qPCR identified HPV 18 with higher viral load as the most likely cause of the invasive lesion. There is large number of multiple HPV infections in South African women with cervical squamous cell carcinoma. HPV 16 is the most frequently detected type and often presents with higher viral load, suggesting it could be responsible for pathogenesis of the lesions in the majority of cases.

  4. Detection of parasitizing coccidia and determination of host crane species, sex and genotype by faecal DNA analysis.

    PubMed

    Honma, H; Suyama, Y; Nakai, Y

    2011-11-01

    In Japan, the three main crane species are the endangered red-crowned crane (Grus japonensis) inhabiting Hokkaido, the northernmost island of Japan; the vulnerable hooded crane (Grus monacha); and the vulnerable white-naped crane (Grus vipio). Both the hooded and white-naped cranes migrate in winter to Izumi in Kyushu, the southern island of Japan. In this study, we investigated the cranes and their coccidian parasites, through a targeted molecular approach using faecal DNA to develop a noninvasive method for infectious disease research. To determine the origin of noninvasively collected faecal samples, host species were identified by sequencing a region of approximately 470 bp of the mitochondrial 16S ribosomal RNA gene in the faecal DNA. Furthermore, to avoid sample redundancy, individual determination was performed by fragment analysis using microsatellite and sex-linked markers. For microsatellite genotyping, previously reported markers and markers isolated in this study were examined, and seven loci for red-crowned cranes, eight for hooded cranes and six for white-naped cranes displayed polymorphisms. A low error rate was demonstrated by comparing microsatellite data generated from faecal DNA samples with that generated from feather DNA samples, indicating a high reliability. Polymerase chain reaction-based capillary electrophoresis (PCR-CE), employing genetic markers in the second internal transcribed spacer (ITS2) of nuclear ribosomal DNA, was employed to detect crane coccidia. The sensitivity of detection of PCR-CE using faecal DNA was inferior to that with traditional microscopy; however, our results suggest that PCR-CE can depict crane coccidia diversity with higher resolution and it is a useful tool to characterize community composition of coccidia in detail. © 2011 Blackwell Publishing Ltd.

  5. COLD-PCR and innovative microarray substrates for detecting and genotyping MPL exon 10 W515 substitutions.

    PubMed

    Brisci, Angela; Damin, Francesco; Pietra, Daniela; Galbiati, Silvia; Boggi, Sabrina; Casetti, Ilaria; Rumi, Elisa; Chiari, Marcella; Cazzola, Mario; Ferrari, Maurizio; Cremonesi, Laura

    2012-12-01

    Myeloproliferative neoplasms (MPNs) include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Somatic mutations in exon 10 of the MPL (myeloproliferative leukemia virus oncogene) gene, mainly substitutions encoding W515 variants, have recently been described in a minority of patients with ET or PMF. We optimized analytically sensitive methods for detecting and genotyping MPL variants. We used DNA previously isolated from circulating granulocytes of 60 patients with MPN that had previously been analyzed by high-resolution melting (HRM), direct sequencing, and the TaqMan allelic-discrimination assay. We developed conditions for enriching tumor mutant alleles with COLD-PCR (coamplification at lower denaturation temperature PCR) and coupled it with direct sequencing. Assays were designed for identifying MPL W515 substitutions with full COLD-PCR protocols. In parallel, we used innovative microarray substrates to develop assays for evaluating the mutant burden in granulocyte cells. Mutations that were present at very low levels in patients who had previously been scored as having an MPL variant by HRM and as wild type by direct sequencing were successfully identified in granulocyte DNA. Notably, the microarray approach displayed analytical sensitivities of 0.1% to 5% mutant allele, depending on the particular mutation. This analytical sensitivity is similar to that obtained with COLD-PCR. The assay requires no enrichment strategy and allows both the characterization of each variant allele and the evaluation of its proportion in every patient. These procedures, which are transferable to clinical diagnostic laboratories, can be used for detecting very low proportions of minority mutant alleles that cannot be identified by other, conventional methods. © 2012 American Association for Clinical Chemistry

  6. SYBR Green based real-time RT-PCR assay for detection and genotype prediction of bovine noroviruses and assessment of clinical significance in Norway.

    PubMed

    Jor, Evert; Myrmel, Mette; Jonassen, Christine M

    2010-10-01

    A novel SYBR Green based real-time RT-PCR assay for detection of genogroup III bovine noroviruses (BoNoV) was developed and the assay applied to 419 faecal samples from calves with and without diarrhoea. The samples were obtained from 190 Norwegian dairy and beef herds. BoNoV was detected in 49.6% of the samples from 61.1% of the herds indicating that BoNoV is ubiquitous in Norway. The overall prevalence was not significantly different in diarrhoea and non-diarrhoea samples. Analyses of polymerase gene sequences revealed both genotype III/1 and III/2 with genotype III/2 (Newbury2-like) being the most prevalent. Detected capsid sequences were restricted to Newbury2-like and the chimeric Bo/Thirsk10/00/UK strain. The RNA polymerase genotypes of the circulating BoNoVs in Norway were predicted by melting temperature analysis. Additional data from a challenge experiment suggest that a high proportion of young calves are shedding low levels of BoNoV for a prolonged time after recovering from the associated diarrhoea. The findings may explain some of the discrepancies in detection rates from previous studies and explain why some studies have failed to detect significant prevalence differences between calves with and without diarrhoea. It may also shed new light on some epidemiological aspects of norovirus infections.

  7. Highly multiplex and sensitive SNP genotyping method using a three-color fluorescence-labeled ligase detection reaction coupled with conformation-sensitive CE.

    PubMed

    Choi, Woong; Jung, Gyoo Yeol

    2017-02-01

    For the development of clinically useful genotyping methods for SNPs, accuracy, simplicity, sensitivity, and cost-effectiveness are the most important criteria. Among the methods currently being developed for SNP genotyping technology, the ligation-dependent method is considered the simplest for clinical diagnosis. However, sensitivity is not guaranteed by the ligation reaction alone, and analysis of multiple targets is limited by the detection method. Although CE is an attractive alternative to error-prone hybridization-based detection, the multiplex assay process is complicated because of the size-based DNA separation principle. In this study, we employed the ligase detection reaction coupled with high-resolution CE-SSCP to develop an accurate, sensitive, and simple multiplex genotyping method. Ligase detection reaction could amplify ligated products through recurrence of denaturation and ligation reaction, and SSCP could separate these products according to each different structure conformation without size variation. Thus, simple and sensitive SNP analysis can be performed using this method involving the use of similar-sized probes, without complex probe design steps. We found that this method could not only accurately discriminate base mismatches but also quantitatively detect 37 SNPs of the tp53 gene, which are used as targets in multiplex analysis, using three-color fluorescence-labeled probes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Quantitative detection and genotyping of Helicobacter pylori from stool using droplet digital PCR reveals variation in bacterial loads that correlates with cagA virulence gene carriage

    PubMed Central

    Talarico, Sarah; Safaeian, Mahboobeh; Gonzalez, Paula; Hildesheim, Allan; Herrero, Rolando; Porras, Carolina; Cortes, Bernal; Larson, Ann; Fang, Ferric C.; Salama, Nina R.

    2015-01-01

    Background Epidemiologic studies of the carcinogenic stomach bacterium Helicobacter pylori have been limited by the lack of non-invasive detection and genotyping methods. We developed a new stool-based method for detection, quantification, and partial genotyping of H. pylori using droplet digital PCR (ddPCR), which allows for increased sensitivity and absolute quantification by PCR partitioning. Materials and Methods Stool-based ddPCR assays for H. pylori 16S gene detection and cagA virulence gene typing were tested using a collection of 50 matched stool and serum samples from Costa Rican volunteers and 29 H. pylori stool antigen-tested stool samples collected at a U.S. hospital. Results The stool-based H. pylori 16S ddPCR assay had a sensitivity of 84% and 100% and a specificity of 100% and 71% compared to serology and stool antigen tests, respectively. The stool-based cagA genotyping assay detected cagA in 22 (88%) of 25 stools from CagA antibody-positive individuals and 4 (16%) of 25 stools from CagA antibody-negative individuals from Costa Rica. All 26 of these samples had a Western-type cagA allele. Presence of serum CagA antibodies was correlated with a significantly higher load of H. pylori in the stool. Conclusions The stool-based ddPCR assays are a sensitive, non-invasive method for detection, quantification, and partial genotyping of H. pylori. The quantitative nature of ddPCR-based H. pylori detection revealed significant variation in bacterial load among individuals that correlates with presence of the cagA virulence gene. These stool-based ddPCR assays will facilitate future population-based epidemiologic studies of this important human pathogen. PMID:26667241

  9. Quantitative Detection and Genotyping of Helicobacter pylori from Stool using Droplet Digital PCR Reveals Variation in Bacterial Loads that Correlates with cagA Virulence Gene Carriage.

    PubMed

    Talarico, Sarah; Safaeian, Mahboobeh; Gonzalez, Paula; Hildesheim, Allan; Herrero, Rolando; Porras, Carolina; Cortes, Bernal; Larson, Ann; Fang, Ferric C; Salama, Nina R

    2016-08-01

    Epidemiologic studies of the carcinogenic stomach bacterium Helicobacter pylori have been limited by the lack of noninvasive detection and genotyping methods. We developed a new stool-based method for detection, quantification, and partial genotyping of H. pylori using droplet digital PCR (ddPCR), which allows for increased sensitivity and absolute quantification by PCR partitioning. Stool-based ddPCR assays for H. pylori 16S gene detection and cagA virulence gene typing were tested using a collection of 50 matched stool and serum samples from Costa Rican volunteers and 29 H. pylori stool antigen-tested stool samples collected at a US hospital. The stool-based H. pylori 16S ddPCR assay had a sensitivity of 84% and 100% and a specificity of 100% and 71% compared to serology and stool antigen tests, respectively. The stool-based cagA genotyping assay detected cagA in 22 (88%) of 25 stools from CagA antibody-positive individuals and four (16%) of 25 stools from CagA antibody-negative individuals from Costa Rica. All 26 of these samples had a Western-type cagA allele. Presence of serum CagA antibodies was correlated with a significantly higher load of H. pylori in the stool. The stool-based ddPCR assays are a sensitive, noninvasive method for detection, quantification, and partial genotyping of H. pylori. The quantitative nature of ddPCR-based H. pylori detection revealed significant variation in bacterial load among individuals that correlates with presence of the cagA virulence gene. These stool-based ddPCR assays will facilitate future population-based epidemiologic studies of this important human pathogen. © 2015 John Wiley & Sons Ltd.

  10. Comparison of Detection Rate and Mutational Pattern of Drug-Resistant Mutations Between a Large Cohort of Genotype B and Genotype C Hepatitis B Virus-Infected Patients in North China.

    PubMed

    Li, Xiaodong; Liu, Yan; Xin, Shaojie; Ji, Dong; You, Shaoli; Hu, Jinhua; Zhao, Jun; Wu, Jingjing; Liao, Hao; Zhang, Xin-Xin; Xu, Dongping

    2017-06-01

    The study aimed to investigate the association of prevalent genotypes in China (HBV/C and HBV/B) with HBV drug-resistant mutations. A total of 13,847 nucleos(t)ide analogue (NA)-treated patients with chronic HBV infection from North China were enrolled. HBV genotypes and resistant mutations were determined by direct sequencing and confirmed by clonal sequencing if necessary. HBV/B, HBV/C, and HBV/D occupied 14.3%, 84.9%, and 0.8% across the study population, respectively. NA usage had no significant difference between HBV/B- and HBV/C-infected patients. Lamivudine-resistant mutations were more frequently detected in HBV/C-infected patients, compared with HBV/B-infected patients (31.67% vs. 25.26%, p < 0.01). Adefovir- and entecavir-resistant mutation detection rates were similar, but the mutational pattern was different between the two genotypes. For adefovir-resistant mutations, HBV/C-infected patients had a higher detection rate of rtA181 V (HBV/C 5.29% vs. HBV/B 1.36%, p < 0.01) and a lower detection rate of rtN236T (2.70% vs. 6.54%, p < 0.01). For entecavir-resistant mutations, HBV/C-infected patients had a higher detection rate of rtM204 V/I+T184 substitution or S202G/C (3.66% vs. 2.16%, p < 0.01) and a lower detection rate of rtM204 V/I+M250 V/I/L substitution (0.67% vs. 1.46%, p < 0.01). Multidrug-resistant mutations (defined as coexistence of mutation to nucleoside and nucleotide analogues) were detected in 104 patients. HBV/C-infected patients had a higher detection rate of multidrug-resistant mutation than HBV/B-infected patients (0.83% vs. 0.35%, p < 0.05). The study for the first time clarified that HBV/C-infected patients had a higher risk to develop multidrug-resistant mutations, compared with HBV/B-infected patients; and HBV/C- and HBV/B-infected patients had different inclinations in the ETV-resistant mutational pattern.

  11. [Evaluation of rapid genotype assay for the identification of gram-positive cocci from blood cultures and detection of mecA and van genes].

    PubMed

    Gülhan, Barış; Atmaca, Selahattin; Ozekinci, Tuncer; Suay, Adnan

    2011-10-01

    Rapid and accurate identification of bacterial pathogens grown in blood cultures of patients with sepsis is crucial for prompt initiation of appropriate therapy in order to decrease related morbidity and mortality rates. Although current automated blood culture systems led to a significant improvement in bacterial detection time, more rapid identification systems are still needed to optimise the establishment of treatment. Novel genotype technology which is developed for the rapid diagnosis of sepsis, is a molecular genetic assay based on DNA multiplex amplification with biotinylated primers followed by hybridization to membrane bound probes. The aim of this study was to evaluate the performance of "Genotype® BC gram-positive” test for the identification of gram-positive cocci grown in blood cultures and rapid detection of mecA and van genes. This test uses DNA.STRIP® technology which includes a panel of probes for identification of 17 gram-positive bacterial species and is able to determinate the methicillin and vancomycin resistance mediating genes (mecA and vanA, vanB, vanC1, vanC2/C3) simultaneously, in a single test run. A total of 55 positive blood cultures from BACTECTM Plus/F (Becton Dickinson, USA) aerobic and pediatric blood culture vials were included in the study. The isolates which exhibit gram-positive coccus morphology by Gram staining were identified by Genotype ® BC gram-positive test (Hain Life Science, Germany). All of the samples were also identified with the use of Phoenix PMIC/ID Panel (Becton Dickinson, USA) and antibiotic susceptibilities were determined. Of the 55 blood culture isolates, 17 were identified as Staphylococcus epidermidis [all were methicillin-resistant (MR)], 9 were S.aureus (one was MR), 18 were S.hominis (10 were MR), 4 were E.faecalis, 3 were E. faecium (one was vanconycin-resistant), 2 were S.saprophyticus (one was MR), 1 was S.warneri and 1 was S.haemolyticus, by Phoenix automated system. Genotype® BC gram

  12. Genotyping of Endocervical Chlamydia trachomatis Strains and Detection of Serological Markers of Acute and Chronic Inflammation in Their Host.

    PubMed

    Taheri Beni, Behrouz; Jenab, Anahita; Roghanian, Rasoul; Motamedi, Hossein; Golbang, Naser; Golbang, Pouran; Yazdi, Javad Zaeimi

    2012-07-01

    Chlamydia trachomatis (C. trachomatis) is the most prevalent cause of bacterial sexually transmitted infections (STI) recognized throughout the world. The aim of this study is to determine different genotypes of genital C. trachomatis and the association between the serological markers of inflammation and genotypes of C. trachomatis in sexually active women (n=80) attending Shahid Beheshti Hospital in Isfahan, Iran. In this descriptive study, endocervical swabs were collected from 80 women. There were 17 endocervical samples that showed positivity for C. trachomatis by plasmid polymerase chain reaction (PCR) using KL1 and KL2 primers. The omp1 gene was directly amplified in 17 plasmid PCR positive samples and was used to differentiate the clinical genotypes by omp1 gene PCR-restriction fragment length polymorphism (PCR-RFLP). The levels of IgG and IgA specific to C. trachmatis and C-reactive protein (CRP) were evaluated. Based on restriction-digestion patterns, four genotypes were identified. Genotypes E (35.3%) and F (35.3%) were the most prevalent, followed by D/Da (23.5%) and K (5.9%). There was no significant association between genotypes and the presence of IgG and CRP. Patients infected with genotype E showed a serological marker of chronic inflammation, i.e. IgA seropositivity, significantly more than patients infected with other genotypes (p=0.042). Nested PCR could increase the sensitivity of omp1 amplification. Based on the presence of IgA, chronic C. trachomatis infections were observed more frequently among genotype E-infected patients in our population.

  13. Detection of a new subgenotype of hepatitis B virus genotype A in Cameroon but not in neighbouring Nigeria.

    PubMed

    Hübschen, J M; Mbah, P O; Forbi, J C; Otegbayo, J A; Olinger, C M; Charpentier, E; Muller, C P

    2011-01-01

    To further investigate the genetic diversity of hepatitis B virus (HBV) genotype A in Africa, we analysed 263 HBV strains from Nigeria (n=163) and Cameroon (n=100). Phylogenetic analysis of S fragment sequences attributed 175 strains (66.5%) to genotype E and 88 (33.5%) to genotype A. In Cameroon, genotype A strains were the most prevalent (79/100, 79.0%), whereas, in Nigeria, genotype E was highly dominant (154/163, 94.5%). The genotype A strains grouped with reference strains of subgenotype A3 (n=8), the provisional subgenotype A5 (n=43), a recently reported new variant from Rwanda (n=35), or as outliers (n=2). Ten complete genome sequences obtained from strains that clustered with the new variant from Rwanda formed a separate group supported by a bootstrap value of 96. The between-group distance to other potential or recognized subgenotypes of genotype A was at least 3.81%. Thus, the new group of strains could be considered as a new subgenotype of HBV genotype A, tentatively named 'A7'. Interestingly, the 'A7' strains from Rwanda and Cameroon showed an interspersed clustering, but essentially no other (sub)genotypes were shared between the two countries, suggesting that 'A7' may have evolved in a yet unknown place and may have only relatively recently spread to Rwanda and Western Cameroon. Strains attributed to provisional subgenotype A5 were found for the first time in Cameroon (n=36) and Central Nigeria (n=2), indicating that A5 is more widespread than previously thought.

  14. Evaluation of genotype MTBDRplus VER 2.0 line probe assay for the detection of MDR-TB in smear positive and negative sputum samples.

    PubMed

    Meaza, Abyot; Kebede, Abebaw; Yaregal, Zelalem; Dagne, Zekarias; Moga, Shewki; Yenew, Bazezew; Diriba, Getu; Molalign, Helina; Tadesse, Mengistu; Adisse, Desalegn; Getahun, Muluwork; Desta, Kassu

    2017-04-17

    Multi drug resistant tuberculosis (MDR-TB) poses formidable challenges to TB control due to its complex diagnostic and treatment challenges and often associated with a high rate of mortality. Accurate and rapid detection of MDR-TB is critical for timely initiation of treatment. Line Probe Assay (LPA) is a qualitative in vitro diagnostic test based on DNA-STRIP technology for the identification of the M. tuberculosis complex and its resistance to rifampicin (RMP) and/or isoniazid (INH). Hain Lifescience, GmbH, Germany has improved the sensitivity of Genotype MTBDRplus VER 2.0 LPA for the detection of MDR-TB; with the possibility of applying the tool in smear negative sputum samples. A cross sectional study was conducted on 274 presumptive MDR-TB patients referred to the National TB Reference Laboratory (NTRL), Ethiopian Public Health Institute (EPHI) who submitted sputum samples for laboratory diagnosis of drug resistant-TB testing. Seventy-two smear and culture positive samples processed in smear positive direct LPA category and 197 smear negative sputum samples were processed for direct LPA. Among the smear negative samples 145 (73.6%) were culture negative and 26 (13.2%) were culture positive. All specimens were processed using NALC-NaOH method and ZN smear microscopy done from sediments. Genotype MTBDRplus VER 2.0 done from processed sputum sediments and the result was compared against the reference, BACTEC MGIT 960 culture and DST. Sensitivity, specificity, PPV and NPV of Genotype MTBDRplus VER 2.0 assay was determined and P-value <0.05 was considered as statistically significant. The sensitivity, specificity, PPV and NPV of Genotype MTBDRplus VER 2.0 LPA were 96.4, 100, 100 and 96.9%, respectively for the detection of MDR-TB from direct smear positive sputum samples. The sensitivity, specificity, PPV and NPV of Genotype MTBDR plus VER 2.0 LPA were 77.8, 97.2, 82.4 and 97.2%, respectively, for the detection of M. tuberculosis from direct smear negative sputum

  15. Comparing triage algorithms using HPV DNA genotyping, HPV E7 mRNA detection and cytology in high-risk HPV DNA-positive women.

    PubMed

    Luttmer, Roosmarijn; Berkhof, Johannes; Dijkstra, Maaike G; van Kemenade, Folkert J; Snijders, Peter J F; Heideman, Daniëlle A M; Meijer, Chris J L M

    2015-06-01

    High-risk human papillomavirus (hrHPV) DNA positive women require triage testing to identify those with high-grade cervical intraepithelial neoplasia or cancer (≥CIN2). Comparing three triage algorithms (1) E7 mRNA testing following HPV16/18/31/33/45/52/58 genotyping (E7 mRNA test), (2) HPV16/18 DNA genotyping and (3) cytology, for ≥CIN2 detection in hrHPV DNA-positive women. hrHPV DNA-positive women aged 18-63 years visiting gynecology outpatient clinics were included in a prospective observational cohort study. From these women a cervical scrape and colposcopy-directed biopsies were obtained. Cervical scrapes were evaluated by cytology, HPV DNA genotyping by bead-based multiplex genotyping of GP5+6+-PCR-products, and presence of HPV16/18/31/33/45/52/58 E7 mRNA using nucleic acid sequence-based amplification (NASBA) in DNA positive women for respective HPV types. Sensitivities and specificities for ≥CIN2 were compared between E7 mRNA test and HPV16/18 DNA genotyping in the total group (n=348), and E7 mRNA test and cytology in a subgroup of women referred for non-cervix-related gynecological complaints (n=133). Sensitivity for ≥CIN2 of the E7 mRNA test was slightly higher than that of HPV16/18 DNA genotyping (66.9% versus 60.9%; ratio 1.10, 95% CI: 1.0002-1.21), at similar specificity (54.8% versus 52.3%; ratio 1.05, 95% CI: 0.93-1.18). Neither sensitivity nor specificity of the E7 mRNA test differed significantly from that of cytology (sensitivity: 68.8% versus 75.0%; ratio 0.92, 95% CI: 0.72-1.17; specificity: 59.4% versus 65.3%; ratio 0.91, 95% CI: 0.75-1.10). For detection of ≥CIN2 in hrHPV DNA-positive women, an algorithm including E7 mRNA testing following HPV16/18/31/33/45/52/58 DNA genotyping performs similar to HPV16/18 DNA genotyping or cytology. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Development and evaluation of a real-time RT-qPCR for detection of Crimean-Congo hemorrhagic fever virus representing different genotypes.

    PubMed

    Jääskeläinen, Anne J; Kallio-Kokko, Hannimari; Ozkul, Aykut; Bodur, Hurrem; Korukruoglu, Gulay; Mousavi, Mehrdad; Pranav, Patel; Vaheri, Antti; Mirazimi, Ali; Vapalahti, Olli

    2014-12-01

    Crimean-Congo hemorrhagic fever (CCHF) is a zoonotic disease caused by a nairovirus belonging to family Bunyaviridae. The CCHF virus (CCHFV) can be transmitted to humans by Hyalomma ticks as well as by direct contact with infected body fluids or tissues from viremic livestock or humans. Our aim was to set up a fast RT-qPCR for detection of the different CCHFV genotypes in clinical samples, including an inactivation step to make the sample handling possible in lower biosafety levels (BSL) than BSL-4. This method was evaluated against commercial reference assays and international External Quality Assessment (EQA) samples. The analytical limit of detection for the developed CCHFV-S RT-qPCR was 11 CCHFV genomes per reaction. After exclusion of four dubious samples, we studied 38 CCHFV-positive samples (using reference tests) of which 38 were found positive by CCHFV-S RT-qPCR, suggesting a sensitivity of 100%. CCHFV-S RT q-PCR detected all eight different CCHFV strains representing five different CCHFV genotypes. In conclusion, the CCHFV-S RT-qPCR described in this study was evaluated using various sources of CCHFV samples and shown to be an accurate tool to detect human CCHFV infection caused by different genotypes of the virus.

  17. PennCNV: An integrated hidden Markov model designed for high-resolution copy number variation detection in whole-genome SNP genotyping data

    PubMed Central

    Wang, Kai; Li, Mingyao; Hadley, Dexter; Liu, Rui; Glessner, Joseph; Grant, Struan F.A.; Hakonarson, Hakon; Bucan, Maja

    2007-01-01

    Comprehensive identification and cataloging of copy number variations (CNVs) is required to provide a complete view of human genetic variation. The resolution of CNV detection in previous experimental designs has been limited to tens or hundreds of kilobases. Here we present PennCNV, a hidden Markov model (HMM) based approach, for kilobase-resolution detection of CNVs from Illumina high-density SNP genotyping data. This algorithm incorporates multiple sources of information, including total signal intensity and allelic intensity ratio at each SNP marker, the distance between neighboring SNPs, the allele frequency of SNPs, and the pedigree information where available. We applied PennCNV to genotyping data generated for 112 HapMap individuals; on average, we detected ∼27 CNVs for each individual with a median size of ∼12 kb. Excluding common rearrangements in lymphoblastoid cell lines, the fraction of CNVs in offspring not detected in parents (CNV-NDPs) was 3.3%. Our results demonstrate the feasibility of whole-genome fine-mapping of CNVs via high-density SNP genotyping. PMID:17921354

  18. Construction and applications of DNA probes for detection of polychlorinated biphenyl-degrading genotypes in toxic organic-contaminated soil environments

    SciTech Connect

    Walia, S.; Khan, A.; Rosenthal, N. )

    1990-01-01

    Several DNA probes for polychlorinated biphenyl (PCB)-degrading genotypes were constructed from PCB-degrading bacteria. These laboratory-engineered DNA probes were used for the detection, enumeration, and isolation of specific bacteria degrading PCBs. Dot blot analysis of purified DNA from toxic organic chemical-contaminated soil bacterial communities showed positive DNA-DNA hybridization with a 32P-labeled DNA probe (pAW6194, cbpABCD). Less than 1% of bacterial colonies isolated from garden topsoil and greater than 80% of bacteria isolated from PCB-contaminated soils showed DNA homologies with 32P-labeled DNA probes. Some of the PCB-degrading bacterial isolates detected by the DNA probe method did not show biphenyl clearance. The DNA probe method was found to detect additional organisms with greater genetic potential to degrade PCBs than the biphenyl clearance method did. Results from this study demonstrate the usefulness of DNA probes in detecting specific PCB-degrading bacteria, abundance of PCB-degrading genotypes, and genotypic diversity among PCB-degrading bacteria in toxic chemical-polluted soil environments. We suggest that the DNA probe should be used with caution for accurate assessment of PCB-degradative capacity within soils and further recommend that a combination of DNA probe and biodegradation assay be used to determine the abundance of PCB-degrading bacteria in the soil bacterial community.

  19. Molecular Detection and Genotyping of Coxiella-Like Endosymbionts in Ticks that Infest Horses in South Korea

    PubMed Central

    Seo, Min-Goo; Lee, Seung-Hun; Ouh, In-Ohk; Lee, Gwang Hyeop; Goo, Youn-Kyoung; Kim, Seungjoon; Kwon, Oh-Deog

    2016-01-01

    Members of the genus Coxiella can be transmitted from ticks to humans during contact with animals; Coxiella may thus spread from the infected horses or ticks to humans. In this study, the presence of Coxiella burnetii and Coxiella-like endosymbionts (CLE) in ticks found on infested horses was determined using PCR and genotyping. A total of 213 ticks were randomly collected from 51 horses (4–5 ticks per horse) raised on Jeju Island, Korea, between 2009 and 2013. All ticks were morphologically identified as adult Haemaphysalis longicornis, a predominant tick species widespread in Korea. Based on the results of nested PCR and 16S rRNA sequencing, CLE were detected in 121 (52.4%, 95% CI: 45.9–58.8) ticks. CLE 16S rRNA sequences from 9 randomly selected ticks were 100% identical. Phylogenetic analysis showed that these 9 sequences were highly similar (97.9–100%) to the sequences of clade B species, like the CLE previously described to be found in Haemaphysalis spp. This study showed that CLE are prevalent in ticks that infest horses reared on Jeju Island, and this is, to the best of our knowledge, the first study to describe CLE occurrence in ticks infesting animals reared in Korea. Because of the high prevalence of CLE in ticks found on horses, CLE transmission from ticks to other animals and humans remains a possibility. This warrants a detailed study of other hosts and regions. Considering the zoonotic potential of Coxiella, further strategic surveillance of Coxiella transmission is necessary. PMID:27792764

  20. Hepatitis C virus long-term persistence in peripheral blood mononuclear cells in patients with haemophilia. Detection of occult genotype 1.

    PubMed

    Parodi, C; García, G; Monzani, M C; Culasso, A; Aloisi, N; Corti, M; Campos, R; de E de Bracco, M M; Baré, P

    2015-07-01

    Peripheral blood mononuclear cells (PBMC) from chronic hepatitis C virus-infected persons can harbour viral variants that are not detected in plasma samples. We explored the presence and persistence of HCV genotypes in plasma and PBMC cultures from 25 HCV-monoinfected and 25 HIV/HCV-coinfected patients with haemophilia. Cell cultures were performed at different time points between 1993 and 2010-2011, and the HCV genome was examined in culture supernatants. Sequential plasma samples were studied during the same time period. Analysing sequential plasma samples, 21% of patients had mixed-genotype infections, while 50% had mixed infections determined from PBMC culture supernatants. HIV coinfection was significantly associated with the presence of mixed infections (OR = 4.57, P = 0.02; 95% CI = 1.38-15.1). In our previous study, genotype 1 was found in 72% of 288 patients of this cohort. Similar results were obtained with the sequential plasma samples included in this study, 69% had genotype 1. However, when taking into account plasma samples and the results from PBMC supernatants, genotype 1 was identified in 94% of the population. The PBMC-associated variants persisted for 10 years in some subjects, emphasizing their role as long-term reservoirs. The presence of genotype 1 in PBMC may be associated with therapeutic failure and should not be disregarded when treating haemophilic patients who have been infected by contaminated factor concentrates. The clinical implications of persistent lymphotropic HCV variants deserve further examination among multiple exposed groups of HCV-infected patients.

  1. Development of a Reverse Transcription-Quantitative PCR System for Detection and Genotyping of Aichi Viruses in Clinical and Environmental Samples

    PubMed Central

    Hata, Akihiko; Yamashita, Teruo; Haramoto, Eiji; Minagawa, Hiroko; Katayama, Hiroyuki

    2013-01-01

    Aichi viruses (AiVs) have been proposed as a causative agent of human gastroenteritis potentially transmitted by fecal-oral routes through contaminated food or water. In the present study, we developed a TaqMan minor groove binder (MGB)-based reverse transcription-quantitative PCR (RT-qPCR) system that is able to quantify AiVs and differentiate between genotypes A and B. This system consists of two assays, an AiV universal assay utilizing a universal primer pair and a universal probe and a duplex genotype-specific assay utilizing the same primer pair and two genotype-specific probes. The primers and probes were designed based on multiple alignments of the 21 available AiV genome sequences containing the capsid gene. Using a 10-fold dilution of plasmid DNA containing the target sequences, it was confirmed that both assays allow detection and quantification of AiVs with a quantitative range of 1.0 × 101 to 1.0 × 107 copies/reaction, and the genotype-specific assay reacts specifically to each genotype. To validate the newly developed assays, 30 clinical stool specimens were subsequently examined with the assays, and the AiV RNA loads were determined to be 1.4 × 104 to 6.6 × 109 copies/g stool. We also examined 12 influent and 12 effluent wastewater samples collected monthly for a 1-year period to validate the applicability of the assays for detection of AiVs in environmental samples. The AiV RNA concentrations in influent and effluent wastewater were determined to be up to 2.2 × 107 and 1.8 × 104 copies/liter, respectively. Our RT-qPCR system is useful for routine diagnosis of AiVs in clinical stool specimens and environmental samples. PMID:23603673

  2. A simple and rapid genotyping assay for simultaneous detection of two ADRB2 allelic variants using fluorescence resonance energy transfer probes and melting curve analysis.

    PubMed

    Sábato, M Fernanda; Irani, Anne-Marie; Bukaveckas, Bonny L; Schwartz, Lawrence B; Wilkinson, David S; Ferreira-Gonzalez, Andrea

    2008-05-01

    Allelic variants at codons 16 and 27 of the beta(2)-adrenergic receptor gene (ADRB2) have shown clinical and pharmacological implications in asthma, hypertension, ischemic heart failure, diabetes, obesity, and cystic fibrosis. We have developed a simultaneous genotyping assay for the c.46A>G and c.79C>G allelic variants using hybridization probes and melting curve analysis. The assay was optimized on a panel of 30 DNA samples of known ADRB2 genotype as determined by sequencing with 100% concordance between the two techniques. Melting temperature (Tm) ranges for the different genotypes were obtained using data from three independent experiments. Single peaks for p.Arg16Arg (Tm = 57.76 degrees C +/- 0.10 degrees C) and p.Gly16Gly (Tm = 66.73 degrees C +/- 0.18 degrees C) and two melting peaks for p.Arg16Gly were obtained. Similarly, single peaks for p.Gln27Gln (Tm = 53.98 degrees C +/- 0.19 degrees C) and p.Glu27Glu (Tm = 64.93 degrees C +/- 0.16 degrees C) and two peaks for p.Gln27Glu were detected. Independent operators easily assigned genotypes in a sample set of 385 asthmatic patients. Haplotype and allele frequencies were in concordance with previously published data: Arg allele frequencies in children/adults were 0.34/0.30 in Caucasians and 0.45/0.52 in African Americans, and Gln allele frequencies were 0.58/0.52 in Caucasians and 0.82/0.84 in African Americans. Thus, the ADRB2 genotyping assay represents a highly reliable and rapid technique for routine clinical use in the simultaneous detection of ADRB2 variants.

  3. Detection of a New Community Genotype Methicillin-Resistant Staphylococcus aureus Clone That Is Unrelated to the USA300 Clone and That Causes Pediatric Infections in Colombia

    PubMed Central

    Marquez-Ortiz, Ricaurte Alejandro; Alvarez-Olmos, Martha I.; Leal, Aura Lucia; Castro, Betsy Esperanza; Vanegas, Natasha

    2013-01-01

    The dissemination of a clone of community genotype methicillin-resistant Staphylococcus aureus (CG-MRSA) that is related to USA300 has been reported in Latin America. We recently detected isolates of a new clone of CG-MRSA (spa type t1635 and ACME-negative) that was genetically unrelated to the USA300 clone and that causes infections in children in Colombia. This finding indicates the appearance of a new clone of CG-MRSA in our region. PMID:23241375

  4. Simultaneous detection and genotyping of porcine reproductive and respiratory syndrome virus (PRRSV) by real-time RT-PCR and amplicon melting curve analysis using SYBR Green.

    PubMed

    Martínez, E; Riera, P; Sitjà, M; Fang, Y; Oliveira, S; Maldonado, J

    2008-08-01

    The feasibility of using a SYBR Green-based real-time RT-PCR assay (SYBR Green ReTi RT-PCR) followed by melting curve analysis (MCA) for detecting and genotyping porcine reproductive and respiratory syndrome virus (PRRSV) was assessed. The SYBR Green ReTi RT-PCR and a previously reported two-step, non-nested RT-PCR assays were simultaneously tested on selected European (EU) and North American (US) PRRSV strains and isolates collected from diverse clinical, temporal, and geographical origins. The validation experiments showed that the optimised SYBR Green ReTi RT-PCR can sensitively and specifically detect PRRSV, consistently detecting as little as 0.03TCID(50)/sample of each virus genotype, with no type-bias and no amplification signal for other swine pathogens. After MCA, two well-differentiated melting temperature (T(m)) profiles for each virus genotype were obtained, as sequencing confirmed it. High repeatability was obtained for the T(m) values, with intra-run coefficients of variation (CoVs) of 0.25 and 0.32 and inter-run CoVs of 0.42 and 0.52 for EU and US genotypes, respectively. The sensitivity of the SYBR Green ReTi RT-PCR (100%) was higher than that of the RT-PCR (95.7%) when testing field isolates. This greater sensitivity of the SYBR Green ReTi RT-PCR was further confirmed by the detection of a higher proportion of PRRSV-positive diagnostic specimens (29.7%) than by the RT-PCR (28.5%). The SYBR Green ReTi RT-PCR test detected infection as early as 2 dpi in the sera of experimentally infected pigs regardless of virus genotype, and discriminated negative (non-inoculated), EU- and US-infected pigs. In conclusion, the reported SYBR Green ReTi RT-PCR assay coupled with MCA can detect and type PRRSV and may be useful as an alternative diagnostic assay in diverse PRRSV epidemiological circumstances.

  5. Feline blood genotyping versus phenotyping, and detection of non-AB blood type incompatibilities in UK cats.

    PubMed

    Tasker, S; Barker, E N; Day, M J; Helps, C R

    2014-04-01

    The aim of this study was to determine the agreement between AB blood phenotyping and genotyping and determine whether non-AB blood type incompatibilities exist in UK cats. Blood samples underwent phenotyping (A, B or AB) using microplate agglutination, and genotyping (AA, Ab or bb) using pyrosequencing of a fragment of the cytidine monophospho-N-acetylneuraminic acid hydroxylase gene. Non-AB blood type incompatibilities were investigated by cross-matching against reference blood of the same phenotype. Of 112 cats tested, 86 (77%) were blood phenotype A, 19 (17%) type B and 7 (6%) type AB. Genotype and initial phenotype agreed in 96% (107 of 112) of cats, but 5 were discordant; these were all B phenotype with either AA (n=2) or Ab (n=3) genotype. Two of the five cats had repeat blood samples tested: one was reclassified as phenotype A; the other remained phenotype B. Two cats had incompatibilities on minor cross-match, but these were attributed to phenotyping errors. Unknown mutation(s) associated with phenotype B, resulting in false AA or Ab genotyping, were evident in a small number of cases in this study. No conclusive evidence for non-AB blood type incompatibilities was found.

  6. Biomedical Information Extraction: Mining Disease Associated Genes from Literature

    ERIC Educational Resources Information Center

    Huang, Zhong

    2014-01-01

    Disease associated gene discovery is a critical step to realize the future of personalized medicine. However empirical and clinical validation of disease associated genes are time consuming and expensive. In silico discovery of disease associated genes from literature is therefore becoming the first essential step for biomarker discovery to…

  7. Biomedical Information Extraction: Mining Disease Associated Genes from Literature

    ERIC Educational Resources Information Center

    Huang, Zhong

    2014-01-01

    Disease associated gene discovery is a critical step to realize the future of personalized medicine. However empirical and clinical validation of disease associated genes are time consuming and expensive. In silico discovery of disease associated genes from literature is therefore becoming the first essential step for biomarker discovery to…

  8. Direct detection and genotyping of Chlamydia trachomatis in cervical scrapes by using polymerase chain reaction and restriction fragment length polymorphism analysis.

    PubMed Central

    Lan, J; Walboomers, J M; Roosendaal, R; van Doornum, G J; MacLaren, D M; Meijer, C J; van den Brule, A J

    1993-01-01

    Detection and genotyping of Chlamydia trachomatis were optimized by using a polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis performed directly with crude cells of cervical scrapes. Different PCR pretreatment methods were evaluated on samples which were positive for C. trachomatis by cell culture. In comparison with DNA extraction and different proteolytic digestion methods, a simple pretreatment of 10 min of boiling appeared to be optimal for PCR amplification. Crude samples (n = 209) were first screened for C. trachomatis by both cell culture and plasmid PCR. Subsequently, positive samples found by plasmid PCR were subjected to a direct omp1 PCR-based RFLP analysis to differentiate C. trachomatis serovars A to K, Ba, Da, and L1 to L3 and serovariant D-. All cervical scrapes that were found positive for C. trachomatis by cell culture (n = 30) were also positive by plasmid PCR and omp1 PCR and could be easily genotyped. In addition, of the culture-negative group, eight samples were found positive by plasmid PCR. Five of these eight samples were also positive by omp1 PCR; of these five, two were positive by a nested omp1 PCR. Genotyping by RFLP analysis of the 35 omp1 PCR-positive samples showed that serovars D, E, and F are the most prevalent types found in cervical scrapes, while serovariant D- was also detected. This study shows that direct PCR and PCR-based RFLP analysis are feasible for detection and genotyping of C. trachomatis in cervical scrapes and are more sensitive than culture-based serotyping. Images PMID:8099080

  9. Identification of Four Distinct Genotypes of Candida dubliniensis and Detection of Microevolution In Vitro and In Vivo

    PubMed Central

    Gee, Sarah F.; Joly, Sophie; Soll, David R.; Meis, Jacques F. G. M.; Verweij, Paul E.; Polacheck, Itzhack; Sullivan, Derek J.; Coleman, David C.

    2002-01-01

    The present study investigates further the population structure of Candida dubliniensis and its ability to exhibit microevolution. Using 98 isolates (including 80 oral isolates) from 94 patients in 15 countries, we confirmed the existence of two distinct populations within the species C. dubliniensis, designated Cd25 group I and Cd25 group II, respectively, on the basis of DNA fingerprints generated with the C. dubliniensis-specific probe Cd25. The majority of Cd25 group I isolates (48 of 71, 67.6%) were from human immunodeficiency virus (HIV)-infected individuals, whereas the majority of Cd25 group II isolates (19 of 27, 70.4%) were from HIV-negative individuals (P ≤ 0.001). Nucleotide sequence analysis of the internal transcribed spacer (ITS) regions of the rRNA genes from 19 representative isolates revealed the presence of four separate genotypes. All of the Cd25 group I isolates tested belonged to genotype 1, while the Cd25 group II population was comprised of three distinct genotypes (genotypes 2 to 4), which corresponded to distinct clades within the Cd25 group II population. These findings were confirmed using genotype-specific PCR primers with 70 isolates. We also showed that C. dubliniensis can exhibit microevolution in vivo and in vitro as occurs in other yeast species. DNA fingerprinting using the C. dubliniensis probes Cd25, Cd24, and Cd1 and karyotype analysis of multiple oral isolates recovered from the same specimen from each of eight separate patients revealed microevolution in six of eight of the clonal populations. Similarly, sequential clonal isolates from various anatomical sites in two separate patients exhibited microevolution. Microevolution was also shown to occur when two clinical isolates susceptible to fluconazole were exposed to the drug in vitro. The epidemiological significance of the four C. dubliniensis genotypes and the ability of C. dubliniensis to undergo microevolution has yet to be established. PMID:11825972

  10. Oligoclonal bands in the cerebrospinal fluid of amyotrophic lateral sclerosis patients with disease-associated mutations.

    PubMed

    Ticozzi, Nicola; Tiloca, Cinzia; Mencacci, Niccolò E; Morelli, Claudia; Doretti, Alberto; Rusconi, Daniela; Colombrita, Claudia; Sangalli, Davide; Verde, Federico; Finelli, Palma; Messina, Stefano; Ratti, Antonia; Silani, Vincenzo

    2013-01-01

    In amyotrophic lateral sclerosis (ALS) cerebrospinal fluid (CSF) analysis is usually performed to exclude inflammatory processes of the central nervous system. Although in a small subset of patients an intrathecal synthesis of IgG is detectable, usually there is no clear explanation for this evidence. This study investigates the occurrence of oligoclonal bands (OCBs) in the CSF of a large series of ALS patients, attempting a correlation with genotype data. CSF was collected from 259 ALS patients. CSF parameters were measured according to standard procedures, and detection of OCBs performed by isoelectric focusing. The patients were screened for mutations in SOD1, FUS, TARDBP, ANG, OPTN, and C9ORF72. We observed the presence of OCBs in the CSF of 9/259 ALS patients (3.5 %), and of disease-associated mutations in 12 cases. OCBs were significantly more frequent in mutation carriers compared to the remaining cohort (3/12 vs 6/247; p < 0.01). Among patients with OCBs, two patients had the TARDBP p.A382T mutation (one of which in homozygous state), and one the ANG p.P-4S variant. Both patients carrying the p.A382T mutation had an atypical phenotype, one of them manifesting signs suggestive of a cerebellar involvement, and the other presenting neuroradiological findings suggestive of an inflammatory disorder of the central nervous system. Our results suggest that ALS patients with OCBs may harbor mutations in disease-causing genes. We speculate that mutations in both TARDBP and ANG genes may disrupt the blood-brain barrier (BBB), promoting local immune responses and neuroinflammation. The role of mutant TARDBP and ANG genes on BBB integrity of ALS patients warrants further investigation.

  11. Detection of a Knockdown Resistance Mutation Associated with Permethrin Resistance in the Body Louse Pediculus humanus corporis by Use of Melting Curve Analysis Genotyping

    PubMed Central

    Drali, Rezak; Benkouiten, Samir; Badiaga, Sékéné; Bitam, Idir

    2012-01-01

    Louse-borne diseases are prevalent in the homeless, and body louse eradication has thus far been unsuccessful in this population. We aim to develop a rapid and robust genotyping method usable in large field-based clinical studies to monitor permethrin resistance in the human body louse Pediculus humanus corporis. We assessed a melting curve analysis genotyping method based on real-time PCR using hybridization probes to detect the M815I-T917I-L920F knockdown resistance (kdr) mutation in the paraorthologous voltage-sensitive sodium channel (VSSC) α subunit gene, which is associated with permethrin resistance. The 908-bp DNA fragment of the VSSC gene, encoding the α subunit of the sodium channel and encompassing the three mutation sites, was PCR sequenced from 65 lice collected from a homeless population. We noted a high prevalence of the 3 indicated mutations in the body lice collected from homeless people (100% for the M815I and L920F mutations and 56.73% for the T917I mutation). These results were confirmed by melting curve analysis genotyping, which had a calculated sensitivity of 100% for the M815I and T917I mutations and of 98% for the L920F mutation. The specificity was 100% for M815I and L920F and 96% for T917I. Melting curve analysis genotyping is a fast, sensitive, and specific tool that is fully compatible with the analysis of a large number of samples in epidemiological surveys, allowing the simultaneous genotyping of 96 samples in just over an hour (75 min). Thus, it is perfectly suited for the epidemiological monitoring of permethrin resistance in human body lice in large-scale clinical studies. PMID:22573588

  12. Using RNA-Seq for gene identification, polymorphism detection and transcript profiling in two alfalfa genotypes with divergent cell wall composition in stems

    PubMed Central

    2011-01-01

    Background Alfalfa, [Medicago sativa (L.) sativa], a widely-grown perennial forage has potential for development as a cellulosic ethanol feedstock. However, the genomics of alfalfa, a non-model species, is still in its infancy. The recent advent of RNA-Seq, a massively parallel sequencing method for transcriptome analysis, provides an opportunity to expand the identification of alfalfa genes and polymorphisms, and conduct in-depth transcript profiling. Results Cell walls in stems of alfalfa genotype 708 have higher cellulose and lower lignin concentrations compared to cell walls in stems of genotype 773. Using the Illumina GA-II platform, a total of 198,861,304 expression sequence tags (ESTs, 76 bp in length) were generated from cDNA libraries derived from elongating stem (ES) and post-elongation stem (PES) internodes of 708 and 773. In addition, 341,984 ESTs were generated from ES and PES internodes of genotype 773 using the GS FLX Titanium platform. The first alfalfa (Medicago sativa) gene index (MSGI 1.0) was assembled using the Sanger ESTs available from GenBank, the GS FLX Titanium EST sequences, and the de novo assembled Illumina sequences. MSGI 1.0 contains 124,025 unique sequences including 22,729 tentative consensus sequences (TCs), 22,315 singletons and 78,981 pseudo-singletons. We identified a total of 1,294 simple sequence repeats (SSR) among the sequences in MSGI 1.0. In addition, a total of 10,826 single nucleotide polymorphisms (SNPs) were predicted between the two genotypes. Out of 55 SNPs randomly selected for experimental validation, 47 (85%) were polymorphic between the two genotypes. We also identified numerous allelic variations within each genotype. Digital gene expression analysis identified numerous candidate genes that may play a role in stem development as well as candidate genes that may contribute to the differences in cell wall composition in stems of the two genotypes. Conclusions Our results demonstrate that RNA-Seq can be

  13. A comparison of human papillomavirus genotype-specific DNA and E6/E7 mRNA detection to identify anal precancer among HIV-infected men who have sex with men

    PubMed Central

    Castle, Philip E.; Follansbee, Stephen; Borgonovo, Sylvia; Tokugawa, Diane; Schwartz, Lauren M.; Lorey, Thomas S.; LaMere, Brandon; Gage, Julia C.; Fetterman, Barbara; Darragh, Teresa M.; Rodriguez, Ana Cecilia; Wentzensen, Nicolas

    2012-01-01

    Background Human papillomavirus (HPV) RNA detection is reportedly more specific for the detection of anogenital precancer than HPV DNA but it is unknown whether this is due to detection of RNA or due to HPV genotype restriction. Materials and Methods 363 human immunodeficiency virus (HIV)-positive men who have sex with men had two anal cytology samples taken and were evaluated using high-resolution anoscopy and biopsies of visible lesions. Anal specimens were tested for E6/E7 RNA for 5 carcinogenic HPV genotypes (HPV16, 18, 31, 33, and 45) and tested for the DNA of 13 carcinogenic HPV genotypes. Results DNA testing was more likely to be positive than RNA testing (53% vs. 48%, p = 0.02) for the same 5 HPV genotypes in aggregate. When restricted to 5 HPV genotypes targeted by the RNA test, the sensitivity to detect anal precancer was the same for DNA and RNA (81%) while RNA was more specific than DNA (65% vs. 58%, p = 0.007). By comparison, DNA detection of all 13 carcinogenic HPV genotypes was more sensitive (96% vs. 81%, p = 0.001) but much less specific (65% vs. 33%, p < 0.001) compared to RNA detection of the 5 HPV genotypes. Conclusion After controlling for HPV genotypes, RNA was only slightly more specific than DNA detection for anal precancer. Impact DNA or RNA testing for a subset of the most carcinogenic HPV genotypes may be useful for distinguishing between those HPV-positive men at higher and lower risk of anal precancer and cancer. PMID:23155136

  14. A novel species within the Fusarium graminearum complex from Ethiopia detected by a multilocus genotyping assay and molecular phylogenetics

    USDA-ARS?s Scientific Manuscript database

    Twenty isolates resembling members of the Fusarium graminearum species complex (Fg complex; O’Donnell et al., Fungal Genet. Biol. 41:600-623, 2004) were isolated from ground wheat samples collected in two different geographic areas in Ethiopia. Results of a multilocus genotyping (MLGT) assay (Ward ...

  15. Development of an efficient genotyping method to detect obese mutation in the mouse leptin gene for use in SPF barrier facilities.

    PubMed

    Ayabe, Hiroaki; Ikeda, Shogo; Maruyama, Shigeri; Shioyama, Seiji; Kikuchi, Mayumi; Kawaguchi, Ayako; Yamada, Tetsuo; Ikeda, Takuya

    2013-01-01

    We have developed a rapid and efficient genotyping method for detection of the mouse leptin obese mutation (Lep(ob)) using tetra-primer amplification refractory mutation system-polymerase chain reaction (tetra-primer ARMS-PCR). In this method, whole blood collected onto gamma-ray sterilized Flinders Technology Associates (FTA) filter paper is used as PCR template without a DNA purification step. Three genotypes (Lep(ob)/Lep(ob), Lep(ob)/+ and +/+) differentiated by single-tube PCR and electrophoresis were perfectly consistent with those determined by PCR-restriction fragment length polymorphism (PCR-RFLP). This method can save material costs and operation time, because it does not require restriction enzyme digestion and could be set up in most specific pathogen-free (SPF) barrier facilities.

  16. Detection of the emerging rotavirus G12P[8] genotype at high frequency in brazil in 2014: Successive replacement of predominant strains after vaccine introduction.

    PubMed

    Luchs, Adriana; Cilli, Audrey; Morillo, Simone Guadagnucci; Gregório, Debora de Souza; de Souza, Karen Aparecida Farias; Vieira, Heloísa Rosa; Fernandes, Adeline de Mira; Carmona, Rita de Cássia Compagnoli; Timenetsky, Maria do Carmo Sampaio Tavares

    2016-04-01

    The continuum characterization of rotavirus (RVA) genotypes is essential to understand how vaccine introduction could impact virus epidemiology. In the present study, an unexpected rapid changing pattern of RVA genotypes distribution in Brazilian population during three followed seasons is described. From January/2012 to December/2014, a total of 3441 fecal specimens were collected from collaborating centers across Southern, Southeastern and Midwest of Brazil. All specimens were screened for RVA using ELISA, and genotyped by RT-PCR. Differences in proportions were tested using Chi-Squares. A p-value of less than 0.05 was considered statistically significant. RVA was detected in 19.7% (677/3441). Among RVA positive cases (n=677), a total of 652 (96.3%) samples were successfully amplified by RT-PCR. G3P[8] remained prevalent in 2012 (37.6%, 69/185) and 2013 (40.1%, 74/186) (χ(2)=0.107, p=0.743), but declined markedly in 2014 (3.5%, 10/281) (χ(2)=71.770, p=0.000). G12P[8] was second highest strain in 2012 (22.7%, 42/185), decrease rapidly in 2013 (2.7%, 5/186) (χ(2)=26.224, p=0.000) and re-emerged as the predominant genotype in 2014 (86.6%, 243/281) (χ(2)=118.299, p=0.000). From July/2014, G12P[8] was the single genotype detected in all regions studied. The sudden emergence, spread and predominance of G12P[8] strain in Brazil, raised the hypothesis of a possible G12 outbreak being in progress. Nationally, the long term decline in gastroenteritis hospitalization observed in the country after RVA vaccine introduction was confirmed. Nevertheless, the sharp increase in diarrhea hospitalization prevalence from 2013 to 2014 observed in Southern and Southeastern regions is consistent with what appears to be an outbreak of G12P[8]. Continued surveillance is needed to verify the effectiveness of the RotarixTM vaccine in Brazil together with potential emergence of unusual genotypes. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Toward fully automated genotyping: allele assignment, pedigree construction, phase determination, and recombination detection in Duchenne muscular dystrophy.

    PubMed Central

    Perlin, M. W.; Burks, M. B.; Hoop, R. C.; Hoffman, E. P.

    1994-01-01

    Human genetic maps have made quantum leaps in the past few years, because of the characterization of > 2,000 CA dinucleotide repeat loci: these PCR-based markers offer extraordinarily high PIC, and within the next year their density is expected to reach intervals of a few centimorgans per marker. These new genetic maps open new avenues for disease gene research, including large-scale genotyping for both simple and complex disease loci. However, the allele patterns of many dinucleotide repeat loci can be complex and difficult to interpret, with genotyping errors a recognized problem. Furthermore, the possibility of genotyping individuals at hundreds or thousands of polymorphic loci requires improvements in data handling and analysis. The automation of genotyping and analysis of computer-derived haplotypes would remove many of the barriers preventing optimal use of dense and informative dinucleotide genetic maps. Toward this end, we have automated the allele identification, genotyping, phase determinations, and inheritance consistency checks generated by four CA repeats within the 2.5-Mbp, 10-cM X-linked dystrophin gene, using fluorescein-labeled multiplexed PCR products analyzed on automated sequencers. The described algorithms can deconvolute and resolve closely spaced alleles, despite interfering stutter noise; set phase in females; propagate the phase through the family; and identify recombination events. We show the implementation of these algorithms for the completely automated interpretation of allele data and risk assessment for five Duchenne/Becker muscular dystrophy families. The described approach can be scaled up to perform genome-based analyses with hundreds or thousands of CA-repeat loci, using multiple fluorophors on automated sequencers. PMID:7942856

  18. Toward fully automated genotyping: Allele assignment, pedigree construction, phase determination, and recombination detection in Duchenne muscular dystrophy

    SciTech Connect

    Perlin, M.W.; Burks, M.B.; Hoop, R.C.; Hoffman, E.P.

    1994-10-01

    Human genetic maps have made quantum leaps in the past few years, because of the characterization of >2,000 CA dinucleotide repeat loci: these PCR-based markers offer extraordinarily high PIC, and within the next year their density is expected to reach intervals of a few centimorgans per marker. These new genetic maps open new avenues for disease gene research, including large-scale genotyping for both simple and complex disease loci. However, the allele patterns of many dinucleotide repeat loci can be complex and difficult to interpret, with genotyping errors a recognized problem. Furthermore, the possibility of genotyping individuals at hundreds or thousands of polymorphic loci requires improvements in data handling and analysis. The automation of genotyping and analysis of computer-derived haplotypes would remove many of the barriers preventing optimal use of dense and informative dinucleotide genetic maps. Toward this end, we have automated the allele identification, genotyping, phase determinations, and inheritance consistency checks generated by four CA repeats within the 2.5-Mbp, 10-cM X-linked dystrophin gene, using fluorescein-labeled multiplexed PCR products analyzed on automated sequencers. The described algorithms can deconvolute and resolve closely spaced alleles, despite interfering stutter noise; set phase in females; propagate the phase through the family; and identify recombination events. We show the implementation of these algorithms for the completely automated interpretation of allele data and risk assessment for five Duchenne/Becker muscular dystrophy families. The described approach can be scaled up to perform genome-based analyses with hundreds or thousands of CA-repeat loci, using multiple fluorophors on automated sequencers. 16 refs., 5 figs., 1 tab.

  19. Lack of detection of host associated differences in Newcastle disease viruses of genotype VIId isolated from chickens and geese.

    PubMed

    Wang, Yuyang; Duan, Zhiqiang; Hu, Shunlin; Kai, Yan; Wang, Xiaobo; Song, Qingqing; Zhong, Lei; Sun, Qing; Wang, Xiaoquan; Wu, Yantao; Liu, Xiufan

    2012-09-13

    The goose is usually considered to be resistant even to strains of Newcastle disease virus (NDV) that are markedly virulent for chickens. However, ND outbreaks have been frequently reported in goose flocks in China since the late 1990s with the concurrent emergence of genotype VIId NDV in chickens. Although the NDVs isolated from both chickens and geese in the past 15 years have been predominantly VIId viruses, published data comparing goose- and chicken-originated ND viruses are scarce and controversial. In this paper, we compared genotype VIId NDVs originated from geese and chickens genetically and pathologically. Ten entire genomic sequences and 329 complete coding sequences of individual genes from genotype VIId NDVs of both goose- and chicken-origin were analyzed. We then randomly selected two goose-originated and two chicken-originated VIId NDVs and compared their pathobiology in both geese and chickens in vivo and in vitro with genotype IV virus Herts/33 as a reference. The results showed that all the VIId NDVs either from geese or from chickens shared high sequence homology and characteristic amino acid substitutions and clustered together in phylogenetic trees. In addition, geese and chickens infected by goose or chicken VIId viruses manifested very similar pathological features distinct from those of birds infected with Herts/33. There is no genetic or phenotypic difference between genotype VIId NDVs originated from geese and chickens. Therefore, no species-preference exists for either goose or chicken viruses and more attention should be paid to the trans-species transmission of VIId NDVs between geese and chickens for the control and eradication of ND.

  20. Lack of detection of host associated differences in Newcastle disease viruses of genotype VIId isolated from chickens and geese

    PubMed Central

    2012-01-01

    Background The goose is usually considered to be resistant even to strains of Newcastle disease virus (NDV) that are markedly virulent for chickens. However, ND outbreaks have been frequently reported in goose flocks in China since the late 1990s with the concurrent emergence of genotype VIId NDV in chickens. Although the NDVs isolated from both chickens and geese in the past 15 years have been predominantly VIId viruses, published data comparing goose- and chicken-originated ND viruses are scarce and controversial. Results In this paper, we compared genotype VIId NDVs originated from geese and chickens genetically and pathologically. Ten entire genomic sequences and 329 complete coding sequences of individual genes from genotype VIId NDVs of both goose- and chicken-origin were analyzed. We then randomly selected two goose-originated and two chicken-originated VIId NDVs and compared their pathobiology in both geese and chickens in vivo and in vitro with genotype IV virus Herts/33 as a reference. The results showed that all the VIId NDVs either from geese or from chickens shared high sequence homology and characteristic amino acid substitutions and clustered together in phylogenetic trees. In addition, geese and chickens infected by goose or chicken VIId viruses manifested very similar pathological features distinct from those of birds infected with Herts/33. Conclusions There is no genetic or phenotypic difference between genotype VIId NDVs originated from geese and chickens. Therefore, no species-preference exists for either goose or chicken viruses and more attention should be paid to the trans-species transmission of VIId NDVs between geese and chickens for the control and eradication of ND. PMID:22971647

  1. Evaluation of an array-based method for human papillomavirus detection and genotyping in comparison with conventional methods used in cervical cancer screening.

    PubMed

    García-Sierra, Nerea; Martró, Elisa; Castellà, Eva; Llatjós, Mariona; Tarrats, Antoni; Bascuñana, Elisabet; Díaz, Rosana; Carrasco, María; Sirera, Guillem; Matas, Lurdes; Ausina, Vicente

    2009-07-01

    Cervical cancer is the second-most prevalent cancer in young women around the world. Infection with human papillomavirus (HPV), especially high-risk HPV types (HR-HPV), is necessary for the development of this cancer. HPV-DNA detection is increasingly being used in cervical cancer screening programs, together with the Papanicolau smear test. We evaluated the usefulness of introducing this new array-based HPV genotyping method (i.e., Clinical Arrays Papillomavirus Humano) in the cervical cancer screening algorithm in our center. The results obtained using this method were compared to those obtained by the hybrid capture II high-risk HPV DNA test (HC-II) and Papanicolau in a selected group of 408 women. The array-based assay was performed in women that were HC-II positive or presented cytological alterations. Among 246 array-positive patients, 123 (50%) presented infection with >or=2 types, and HR-HPV types were detected in 206 (83.7%), mainly HPV-16 (24.0%). Up to 132 (33.2%) specimens were classified as ASCUS (for atypical squamous cells of undetermined significance), and only 48 (36.4%) of them were HPV-DNA positive by either assay; however, 78.7% of these cases were caused by HR-HPV types. The agreement between both HPV-DNA detection techniques was fairly good (n = 367). Screening with Papanicolau smear and HC-II tests, followed by HPV detection and genotyping, provided an optimal identification of women at risk for the development of cervical cancer. Furthermore, with the identification of specific genotypes, either in single or multiple infections, a better prediction of disease progression was achieved. The array method also made allowed us to determine the possible contribution of the available vaccines in our setting.

  2. Evaluation of an Array-Based Method for Human Papillomavirus Detection and Genotyping in Comparison with Conventional Methods Used in Cervical Cancer Screening▿

    PubMed Central

    García-Sierra, Nerea; Martró, Elisa; Castellà, Eva; Llatjós, Mariona; Tarrats, Antoni; Bascuñana, Elisabet; Díaz, Rosana; Carrasco, María; Sirera, Guillem; Matas, Lurdes; Ausina, Vicente

    2009-01-01

    Cervical cancer is the second-most prevalent cancer in young women around the world. Infection with human papillomavirus (HPV), especially high-risk HPV types (HR-HPV), is necessary for the development of this cancer. HPV-DNA detection is increasingly being used in cervical cancer screening programs, together with the Papanicolau smear test. We evaluated the usefulness of introducing this new array-based HPV genotyping method (i.e., Clinical Arrays Papillomavirus Humano) in the cervical cancer screening algorithm in our center. The results obtained using this method were compared to those obtained by the hybrid capture II high-risk HPV DNA test (HC-II) and Papanicolau in a selected group of 408 women. The array-based assay was performed in women that were HC-II positive or presented cytological alterations. Among 246 array-positive patients, 123 (50%) presented infection with ≥2 types, and HR-HPV types were detected in 206 (83.7%), mainly HPV-16 (24.0%). Up to 132 (33.2%) specimens were classified as ASCUS (for atypical squamous cells of undetermined significance), and only 48 (36.4%) of them were HPV-DNA positive by either assay; however, 78.7% of these cases were caused by HR-HPV types. The agreement between both HPV-DNA detection techniques was fairly good (n = 367). Screening with Papanicolau smear and HC-II tests, followed by HPV detection and genotyping, provided an optimal identification of women at risk for the development of cervical cancer. Furthermore, with the identification of specific genotypes, either in single or multiple infections, a better prediction of disease progression was achieved. The array method also made allowed us to determine the possible contribution of the available vaccines in our setting. PMID:19439534

  3. An Improved PCR-RFLP Assay for Detection and Genotyping of Asymptomatic Giardia lamblia Infection in a Resource-Poor Setting.

    PubMed

    Hawash, Yoursry; Ghonaim, M M; Al-Shehri, S S

    2016-02-01

    Laboratory workers, in resource-poor countries, still consider PCR detection of Giardia lamblia more costly and more time-consuming than the classical parasitological techniques. Based on 2 published primers, an in-house one-round touchdown PCR-RFLP assay was developed. The assay was validated with an internal amplification control included in reactions. Performance of the assay was assessed with DNA samples of various purities, 91 control fecal samples with various parasite load, and 472 samples of unknown results. Two cysts per reaction were enough for PCR detection by the assay with exhibited specificity (Sp) and sensitivity (Se) of 100% and 93%, respectively. Taking a published small subunit rRNA reference PCR test results (6%; 29/472) as a nominated gold standard, G. lamblia was identified in 5.9% (28/472), 5.2%, (25/472), and 3.6% (17/472) by PCR assay, RIDA(®) Quick Giardia antigen detection test (R-Biopharm, Darmstadt, Germany), and iodine-stained smear microscopy, respectively. The percent agreements (kappa values) of 99.7% (0.745), 98.9% (0.900), and 97.7% (0.981) were exhibited between the assay results and that of the reference PCR, immunoassay, and microscopy, respectively. Restriction digestion of the 28 Giardia-positive samples revealed genotype A pattern in 12 and genotype B profile in 16 samples. The PCR assay with the described format and exhibited performance has a great potential to be adopted in basic clinical laboratories as a detection tool for G. lamblia especially in asymptomatic infections. This potential is increased more in particular situations where identification of the parasite genotype represents a major requirement as in epidemiological studies and infection outbreaks.

  4. An Improved PCR-RFLP Assay for Detection and Genotyping of Asymptomatic Giardia lamblia Infection in a Resource-Poor Setting

    PubMed Central

    Hawash, Yoursry; Ghonaim, M. M.; Al-Shehri, S. S.

    2016-01-01

    Laboratory workers, in resource-poor countries, still consider PCR detection of Giardia lamblia more costly and more time-consuming than the classical parasitological techniques. Based on 2 published primers, an in-house one-round touchdown PCR-RFLP assay was developed. The assay was validated with an internal amplification control included in reactions. Performance of the assay was assessed with DNA samples of various purities, 91 control fecal samples with various parasite load, and 472 samples of unknown results. Two cysts per reaction were enough for PCR detection by the assay with exhibited specificity (Sp) and sensitivity (Se) of 100% and 93%, respectively. Taking a published small subunit rRNA reference PCR test results (6%; 29/472) as a nominated gold standard, G. lamblia was identified in 5.9% (28/472), 5.2%, (25/472), and 3.6% (17/472) by PCR assay, RIDA® Quick Giardia antigen detection test (R-Biopharm, Darmstadt, Germany), and iodine-stained smear microscopy, respectively. The percent agreements (kappa values) of 99.7% (0.745), 98.9% (0.900), and 97.7% (0.981) were exhibited between the assay results and that of the reference PCR, immunoassay, and microscopy, respectively. Restriction digestion of the 28 Giardia-positive samples revealed genotype A pattern in 12 and genotype B profile in 16 samples. The PCR assay with the described format and exhibited performance has a great potential to be adopted in basic clinical laboratories as a detection tool for G. lamblia especially in asymptomatic infections. This potential is increased more in particular situations where identification of the parasite genotype represents a major requirement as in epidemiological studies and infection outbreaks. PMID:26951972

  5. Detection and differentiation of genotype I and III Japanese encephalitis virus in mosquitoes by multiplex reverse transcriptase-polymerase chain reaction.

    PubMed

    Chen, Y Y; Lin, J W; Fan, Y C; Chiou, S S

    2014-02-01

    Japanese encephalitis (JE) is a disease that threatens both human and animal populations in Asian countries, and the causative agent of JE, Japanese encephalitis virus (JEV), has recently changed from genotype III (GIII) to genotype I (GI). However, a test for the rapid differentiation of GI and GIII JEV is still unavailable, especially one that can be used for mosquito-based surveillance. We have designed GI- and GIII-specific primer sets for the rapid detection and differentiation of GI and GIII JEV by multiplex reverse transcriptase-polymerase chain reaction (multiplex RT-PCR). The GI-specific and GIII-specific primer sets were able to specifically amplify the target gene from GI and GIII JEV, respectively. The limitations of detection were 0.00225 and 0.225 pfu for the GI-specific and GIII-specific primers, respectively. Using a mixture of GI-specific and GIII-specific primers, the multiplex RT-PCR was able to specifically detect and differentiate GI and GIII JEV. The multiplex RT-PCR was able to successfully differentiate GI and GIII virus in JEV-infected mosquitoes. Thus, a sensitive and specific multiplex RT-PCR system for the rapid detection and differentiation of GI and GIII JEV has been developed, and this test is likely to be valuable when carrying out mosquito-based JEV surveillance. © 2012 Blackwell Verlag GmbH.

  6. The Decay of Disease Association with Declining Linkage Disequilibrium: A Fine Mapping Theorem.

    PubMed

    Maadooliat, Mehdi; Bansal, Naveen K; Upadhya, Jiblal; Farazi, Manzur R; Li, Xiang; He, Max M; Hebbring, Scott J; Ye, Zhan; Schrodi, Steven J

    2016-01-01

    Several important and fundamental aspects of disease genetics models have yet to be described. One such property is the relationship of disease association statistics at a marker site closely linked to a disease causing site. A complete description of this two-locus system is of particular importance to experimental efforts to fine map association signals for complex diseases. Here, we present a simple relationship between disease association statistics and the decline of linkage disequilibrium from a causal site. Specifically, the ratio of Chi-square disease association statistics at a marker site and causal site is equivalent to the standard measure of pairwise linkage disequilibrium, r(2). A complete derivation of this relationship from a general disease model is shown. Quite interestingly, this relationship holds across all modes of inheritance. Extensive Monte Carlo simulations using a disease genetics model applied to chromosomes subjected to a standard model of recombination are employed to better understand the variation around this fine mapping theorem due to sampling effects. We also use this relationship to provide a framework for estimating properties of a non-interrogated causal site using data at closely linked markers. Lastly, we apply this way of examining association data from high-density genotyping in a large, publicly-available data set investigating extreme BMI. We anticipate that understanding the patterns of disease association decay with declining linkage disequilibrium from a causal site will enable more powerful fine mapping methods and provide new avenues for identifying causal sites/genes from fine-mapping studies.

  7. Collective judgment predicts disease-associated single nucleotide variants

    PubMed Central

    2013-01-01

    Background In recent years the number of human genetic variants deposited into the publicly available databases has been increasing exponentially. The latest version of dbSNP, for example, contains ~50 million validated Single Nucleotide Variants (SNVs). SNVs make up most of human variation and are often the primary causes of disease. The non-synonymous SNVs (nsSNVs) result in single amino acid substitutions and may affect protein function, often causing disease. Although several methods for the detection of nsSNV effects have already been developed, the consistent increase in annotated data is offering the opportunity to improve prediction accuracy. Results Here we present a new approach for the detection of disease-associated nsSNVs (Meta-SNP) that integrates four existing methods: PANTHER, PhD-SNP, SIFT and SNAP. We first tested the accuracy of each method using a dataset of 35,766 disease-annotated mutations from 8,667 proteins extracted from the SwissVar database. The four methods reached overall accuracies of 64%-76% with a Matthew's correlation coefficient (MCC) of 0.38-0.53. We then used the outputs of these methods to develop a machine learning based approach that discriminates between disease-associated and polymorphic variants (Meta-SNP). In testing, the combined method reached 79% overall accuracy and 0.59 MCC, ~3% higher accuracy and ~0.05 higher correlation with respect to the best-performing method. Moreover, for the hardest-to-define subset of nsSNVs, i.e. variants for which half of the predictors disagreed with the other half, Meta-SNP attained 8% higher accuracy than the best predictor. Conclusions Here we find that the Meta-SNP algorithm achieves better performance than the best single predictor. This result suggests that the methods used for the prediction of variant-disease associations are orthogonal, encoding different biologically relevant relationships. Careful combination of predictions from various resources is therefore a good strategy

  8. Phenotypic and genotypic detection of Candida albicans and Candida dubliniensis strains isolated from oral mucosa of AIDS pediatric patients.

    PubMed

    Livério, Harisson Oliveira; Ruiz, Luciana da Silva; Freitas, Roseli Santos de; Nishikaku, Angela; Souza, Ana Clara de; Paula, Claudete Rodrigues; Domaneschi, Carina

    2017-04-13

    The aim of this study was to assess a collection of yeasts to verify the presence of Candida dubliniensis among strains isolated from the oral mucosa of AIDS pediatric patients which were initially characterized as Candida albicans by the traditional phenotypic method, as well as to evaluate the main phenotypic methods used in the discrimination between the two species and confirm the identification through genotypic techniques, i.e., DNA sequencing. Twenty-nine samples of C. albicans isolated from this population and kept in a fungi collection were evaluated and re-characterized. In order to differentiate the two species, phenotypic tests (Thermotolerance tests, Chromogenic medium, Staib agar, Tobacco agar, Hypertonic medium) were performed and genotypic techniques using DNA sequencing were employed for confirmation of isolated species. Susceptibility and specificity were calculated for each test. No phenotypic test alone was sufficient to provide definitive identification of C. dubliniensis or C. albicans, as opposed to results of molecular tests. After amplification and sequencing of specific regions of the 29 studied strains, 93.1% of the isolates were identified as C. albicans and 6.9% as C. dubliniensis. The Staib agar assay showed a higher susceptibility (96.3%) in comparison with other phenotypic techniques. Therefore, genotypic methods are indispensable for the conclusive identification and differentiation between these species.

  9. Development of a Novel Genus-Specific Real-Time PCR Assay for Detection and Differentiation of Bartonella Species and Genotypes

    PubMed Central

    Bai, Ying; Malania, Lile; Winchell, Jonas M.; Kosoy, Michael Y.

    2012-01-01

    The genus Bartonella includes numerous species with varied host associations, including several that infect humans. Development of a molecular diagnostic method capable of detecting the diverse repertoire of Bartonella species while maintaining genus specificity has been a challenge. We developed a novel real-time PCR assay targeting a 301-bp region of the ssrA gene of Bartonella and demonstrated specific amplification in over 30 Bartonella species, subspecies, and strains. Subsequent analysis of ssrA sequences was sufficient to discriminate Bartonella species and provided phylogenetic data consistent with that of gltA, a commonly used gene for differentiating Bartonella genotypes. Using this assay, we identified Bartonella DNA in 29% and 47% of blood specimens from elk in Wyoming and cattle in the Republic of Georgia, respectively. Sequence analysis of a subset of genotypes from elk specimens revealed a cluster most closely related to Bartonella capreoli, and genotypes from cattle were identified as Bartonella bovis, both Bartonella species commonly found in wild and domestic ruminants. Considering the widespread geographic distribution and infectivity potential to a variety of hosts, this assay may be an effective diagnostic method for identification of Bartonella infections in humans and have utility in Bartonella surveillance studies. PMID:22378904

  10. Detection of hepatitis B virus infection in wild-born chimpanzees (Pan troglodytes verus): phylogenetic relationships with human and other primate genotypes.

    PubMed

    MacDonald, D M; Holmes, E C; Lewis, J C; Simmonds, P

    2000-05-01

    Infection with hepatitis B virus (HBV) was detected by serological testing for HBV surface antigen and by PCR assay for HBV DNA in serum samples from two common chimpanzees (Pan troglodytes subsp. verus) born in West Africa. The complete genome sequences obtained by nucleotide sequencing of overlapping DNA fragments amplified by PCR were compared with HBV variants recovered from other primates and with human genotypes A to F. Both chimpanzee sequences were 3, 182 nucleotides in length, and the surface gene sequence predicted the existence of a, d, and w serological determinants. Neither sequence contained stop codons in the precore region. On phylogenetic analysis, the HBV variants infecting the chimpanzees clustered together with a third chimpanzee HBV isolate independently obtained from an infected captive animal (A. J. Zuckerman, A. Thornton, C. R. Howard, K. N. Tsiquaye, D. M. Jones, and M. R. Brambell, Lancet ii:652-654, 1978), with an overall sequence similarity of >94%. This provides strong evidence for a chimpanzee-specific genotype of HBV which circulates in nature. These findings add to the recent evidence for infection in the wild of other Old and New World primates (gibbon, orangutan, and woolly monkey) with species-specific variants of HBV. There is no evidence for close phylogenetic clustering of variants found so far in primates with any of the established HBV genotypes from humans. With the new evidence for the widespread distribution of HBV in primates, hypotheses for the origins of human infection are reviewed.

  11. Human papillomavirus genotyping, human papillomavirus mRNA expression, and p16/Ki-67 cytology to detect anal cancer precursors in HIV-infected MSM.

    PubMed

    Wentzensen, Nicolas; Follansbee, Stephen; Borgonovo, Sylvia; Tokugawa, Diane; Schwartz, Lauren; Lorey, Thomas S; Sahasrabuddhe, Vikrant V; Lamere, Brandon; Gage, Julia C; Fetterman, Barbara; Darragh, Teresa M; Castle, Philip E

    2012-11-13

    Anal cancer incidence is high in HIV-infected MSM. Screening for anal intraepithelial lesions and cancers is performed at specialized clinics and relies on high-resolution anoscopy (HRA) and anal cytology. Both approaches have limited reproducibility and sensitivity for detecting anal cancer precursors. We evaluated biomarkers for human papillomavirus (HPV)-related disease in a population of HIV-infected MSM. A cross-sectional screening study with passive follow-up included 363 MSM followed at a HIV/AIDS clinic. All men had anal cytology samples taken and were evaluated using HRA and anal biopsies. Using a composite endpoint of biopsy results and cytology, we compared the performance of HPV16/18 genotyping, HPVE6/E7 mRNA expression, and p16/Ki-67 cytology to detect high-grade anal intraepithelial neoplasias (AINs). For all biomarkers analyzed, there was a significant trend of increasing percentage of men testing positive with increasing severity of disease (P < 0.001). HPV DNA testing had the highest sensitivity for anal intraepithelial neoplasia grade 2 and anal intraepithelial neoplasia grade 3 (AIN3), followed by p16/Ki-67, HPVE6/E7 mRNA testing, and HPV16/18 genotyping. The highest Youden's index was observed for HPVE6/E7 mRNA testing, followed by HPV16/18 genotyping, p16/Ki-67 cytology, and HPV DNA testing. Increasing the threshold for positivity of p16/Ki-67 to five or more positive cells led to significantly higher specificity, but unchanged sensitivity for detecting AIN3. Molecular features of anal disease categories are similar to those of corresponding cervical lesions. Biomarkers evaluated for cervical cancer screening may be used for primary anal cancer screening or to decide who should require immediate treatment vs. expectant management.

  12. Comparison of real-time multiplex human papillomavirus (HPV) PCR assays with the linear array HPV genotyping PCR assay and influence of DNA extraction method on HPV detection.

    PubMed

    Roberts, Christine C; Swoyer, Ryan; Bryan, Janine T; Taddeo, Frank J

    2011-05-01

    Real-time human papillomavirus (HPV) type-specific multiplex PCR assays were developed to detect HPV DNA in specimens collected for the efficacy determination of the quadrivalent HPV (type 6, 11, 16, and 18) L1 virus-like particle (VLP) vaccine (Gardasil). We evaluated the concordance between type-specific multiplex HPV PCR and the widely used, commercially available Roche Linear Array genotyping PCR assay. Female genital swab specimens were tested for the presence of L1, E6, and E7 sequences of HPV type 6 (HPV6), HPV11, HPV16, HPV18, HPV31, HPV45, HPV52, and HPV58 and E6 and E7 sequences of HPV33, HPV35, HPV39, HPV51, HPV56, and HPV59 in type- and gene-specific real-time multiplex PCR assays. Specimens were also tested for the presence of L1 sequences using two versions of the Roche Linear Array genotyping assay. Measures of concordance of a modified version of the Linear Array and the standard Linear Array PCR assay were evaluated. With specimen DNA extraction using the Qiagen Spin blood kit held as the constant, multiplex PCR assays detect more HPV-positive specimens for the 14 HPV types common to both than either version of the Linear Array HPV genotyping assay. Type-specific agreements between the assays were good, at least 0.838, but were often driven by negative agreement in HPV types with low prevalence, as evidenced by reduced proportions of positive agreement. Overall HPV status agreements ranged from 0.615 for multiplex PCR and standard Linear Array to 0.881 for multiplex PCR and modified Linear Array. An alternate DNA extraction technique, that used by the Qiagen MinElute kit, impacted subsequent HPV detection in both the multiplex PCR and Linear Array assays.

  13. Complement genetics, deficiencies, and disease associations.

    PubMed

    Mayilyan, Karine R

    2012-07-01

    The complement system is a key component of innate immunity. More than 45 genes encoding the proteins of complement components or their isotypes and subunits, receptors, and regulators have been discovered. These genes are distributed throughout different chromosomes, with 19 genes comprising three significant complement gene clusters in the human genome. Genetic deficiency of any early component of the classical pathway (C1q, C1r/s, C2, C4, and C3) is associated with autoimmune diseases due to the failure of clearance of immune complexes (IC) and apoptotic materials, and the impairment of normal humoral response. Deficiencies of mannan-binding lectin (MBL) and the early components of the alternative (factor D, properdin) and terminal pathways (from C3 onward components: C5, C6, C7, C8, C9) increase susceptibility to infections and their recurrence. While the association of MBL deficiency with a number of autoimmune and infectious disorders has been well established, the effects of the deficiency of other lectin pathway components (ficolins, MASPs) have been less extensively investigated due to our incomplete knowledge of the genetic background of such deficiencies and the functional activity of those components. For complement regulators and receptors, the consequences of their genetic deficiency vary depending on their specific involvement in the regulatory or signalling steps within the complement cascade and beyond. This article reviews current knowledge and concepts about the genetic load of complement component deficiencies and their association with diseases. An integrative presentation of genetic data with the latest updates provides a background to further investigations of the disease association investigations of the complement system from the perspective of systems biology and systems genetics.

  14. Sequence-specific label-free nucleic acid biosensor for the detection of the hepatitis C virus genotype 1a using a disposable pencil graphite electrode.

    PubMed

    Donmez, Soner; Arslan, Fatma; Arslan, Halit

    2016-05-01

    In this paper, we demonstrate a simple, sensitive, inexpensive, disposable and label-free electrochemical nucleic acid biosensor for the detection of the hepatitis C virus genotype 1a (HCV1a). The nucleic acid biosensor was designed with the amino-linked inosine-substituted 20-mer probes, which were immobilized onto a disposable pencil graphite electrode (PGE) by covalent linking. The proposed nucleic acid biosensor was linear in the range of 0.05 and 0.75 μM, exhibiting a limit of detection of 54.9 nM. The single-stranded synthetic PCR product analogs of HCV1a were also detected with satisfactory results under optimal conditions, showing the potential application of this biosensor.

  15. Comparison of the f-HPV typing™ and Hybrid Capture II® assays for detection of high-risk HPV genotypes in cervical samples.

    PubMed

    Cañadas, María-Paz; Cirigliano, Vincenzo; Darwich, Laila; Sirera, Guillermo; Coll, Josep; Clotet, Bonaventura; Videla, Sebastian

    2012-07-01

    Human papillomavirus genotyping is being considered in cervical screening programs and for monitoring the effectiveness of HPV vaccination. Both approaches require access to fast, easy and high-throughput technology. The aim of this study was to compare a new commercial assay (f-HPV typing™) with the Hybrid Capture II® (HC2) to detect HPV infection. The F-HPV typing is a multiplex fluorescent PCR method recognizing E6 and E7 regions of 13 high-risk (HR) HPV types, the same set of HR-types targeted HC2 test. A subset of 157 cervical samples was tested with both assays. The percentage of positive HR-HPV DNA samples was 24% (37/155) by HC2 and 33% (49/155) by f-HPV typing. Concordant results were found in 133/155 (overall agreement, 85.8%; Cohen's kappa=0.65). The analytical sensitivity and specificity of f-HPV were 97.6 and 93, respectively. In conclusion, this study shows that the f-HPV assay provides a good alternative to HC2 to detect HPV infection, allowing simple and rapid HPV genotyping and detecting multiple infections.

  16. GenoType MTBDRsl for Molecular Detection of Second-Line-Drug and Ethambutol Resistance in Mycobacterium tuberculosis Strains and Clinical Samples

    PubMed Central

    Lacoma, A.; García-Sierra, N.; Prat, C.; Maldonado, J.; Ruiz-Manzano, J.; Haba, L.; Gavin, P.; Samper, S.; Ausina, V.

    2012-01-01

    The purpose of this study was to evaluate the GenoType MTBDRsl assay (Hain Lifescience GmbH, Nehren, Germany) for its ability to detect resistance to fluoroquinolones (FLQ), injectable second-line antibiotics [kanamycin (KM) and capreomycin (CM)], and ethambutol (EMB) in Mycobacterium tuberculosis clinical strains and directly in clinical samples. A total of 34 clinical strains were characterized with the Bactec 460 TB system. Fifty-four clinical samples from 16 patients (5 were smear negative and 49 were smear positive) were also tested directly. The corresponding isolates of the clinical specimens were also analyzed with the Bactec 460TB. When there was a discrepancy between assays, pyrosequencing was performed. The overall rates of concordance of the MTBDRsl and the Bactec 460TB for the detection of FLQ, KM/CM, and EMB susceptibility in clinical strains were 72.4% (21/29), 88.8% (24/27), and 67.6% (23/34), whereas for clinical samples, rates were 86.5% (45/52), 92.3% (48/52), and 56% (28/50), respectively. In conclusion, the GenoType MTBDRsl assay may be a useful tool for making early decisions regarding KM/CM susceptibility and to a lesser extent regarding FLQ and EMB susceptibility. The test is able to detect mutations in both clinical strains and samples with a short turnaround time. However, for correct management of patients with extensively drug-resistant tuberculosis, results must be confirmed by a phenotypical method. PMID:22075597

  17. GenoType MTBDRsl for molecular detection of second-line-drug and ethambutol resistance in Mycobacterium tuberculosis strains and clinical samples.

    PubMed

    Lacoma, A; García-Sierra, N; Prat, C; Maldonado, J; Ruiz-Manzano, J; Haba, L; Gavin, P; Samper, S; Ausina, V; Domínguez, J

    2012-01-01

    The purpose of this study was to evaluate the GenoType MTBDRsl assay (Hain Lifescience GmbH, Nehren, Germany) for its ability to detect resistance to fluoroquinolones (FLQ), injectable second-line antibiotics [kanamycin (KM) and capreomycin (CM)], and ethambutol (EMB) in Mycobacterium tuberculosis clinical strains and directly in clinical samples. A total of 34 clinical strains were characterized with the Bactec 460 TB system. Fifty-four clinical samples from 16 patients (5 were smear negative and 49 were smear positive) were also tested directly. The corresponding isolates of the clinical specimens were also analyzed with the Bactec 460TB. When there was a discrepancy between assays, pyrosequencing was performed. The overall rates of concordance of the MTBDRsl and the Bactec 460TB for the detection of FLQ, KM/CM, and EMB susceptibility in clinical strains were 72.4% (21/29), 88.8% (24/27), and 67.6% (23/34), whereas for clinical samples, rates were 86.5% (45/52), 92.3% (48/52), and 56% (28/50), respectively. In conclusion, the GenoType MTBDRsl assay may be a useful tool for making early decisions regarding KM/CM susceptibility and to a lesser extent regarding FLQ and EMB susceptibility. The test is able to detect mutations in both clinical strains and samples with a short turnaround time. However, for correct management of patients with extensively drug-resistant tuberculosis, results must be confirmed by a phenotypical method.

  18. In Vitro vs In Silico Detected SNPs for the Development of a Genotyping Array: What Can We Learn from a Non-Model Species?

    PubMed Central

    Lepoittevin, Camille; Frigerio, Jean-Marc; Garnier-Géré, Pauline; Salin, Franck; Cervera, María-Teresa; Vornam, Barbara; Harvengt, Luc; Plomion, Christophe

    2010-01-01

    Background There is considerable interest in the high-throughput discovery and genotyping of single nucleotide polymorphisms (SNPs) to accelerate genetic mapping and enable association studies. This study provides an assessment of EST-derived and resequencing-derived SNP quality in maritime pine (Pinus pinaster Ait.), a conifer characterized by a huge genome size (∼23.8 Gb/C). Methodology/Principal Findings A 384-SNPs GoldenGate genotyping array was built from i/ 184 SNPs originally detected in a set of 40 re-sequenced candidate genes (in vitro SNPs), chosen on the basis of functionality scores, presence of neighboring polymorphisms, minor allele frequencies and linkage disequilibrium and ii/ 200 SNPs screened from ESTs (in silico SNPs) selected based on the number of ESTs used for SNP detection, the SNP minor allele frequency and the quality of SNP flanking sequences. The global success rate of the assay was 66.9%, and a conversion rate (considering only polymorphic SNPs) of 51% was achieved. In vitro SNPs showed significantly higher genotyping-success and conversion rates than in silico SNPs (+11.5% and +18.5%, respectively). The reproducibility was 100%, and the genotyping error rate very low (0.54%, dropping down to 0.06% when removing four SNPs showing elevated error rates). Conclusions/Significance This study demonstrates that ESTs provide a resource for SNP identification in non-model species, which do not require any additional bench work and little bio-informatics analysis. However, the time and cost benefits of in silico SNPs are counterbalanced by a lower conversion rate than in vitro SNPs. This drawback is acceptable for population-based experiments, but could be dramatic in experiments involving samples from narrow genetic backgrounds. In addition, we showed that both the visual inspection of genotyping clusters and the estimation of a per SNP error rate should help identify markers that are not suitable to the GoldenGate technology in species

  19. European Multicenter Evaluation of High-Density DNA Probe Arrays for Detection of Hepatitis B Virus Resistance Mutations and Identification of Genotypes

    PubMed Central

    Tran, N.; Berne, R.; Chann, R.; Gauthier, M.; Martin, D.; Armand, M.-A.; Ollivet, A.; Teo, C. G.; Ijaz, S.; Flichman, D.; Brunetto, M.; Bielawski, K. P.; Pichoud, C.; Zoulim, F.; Vernet, G.

    2006-01-01

    Polymorphisms along the hepatitis B virus (HBV) genome have an impact on disease outcome, sensitivity to antiviral treatment, escape from vaccination, and laboratory diagnosis. We have designed a diagnostic tool based on duplex amplification of the whole HBV genome and a high-density DNA chip designed to detect 245 mutations, 20 deletions, and 2 insertions at 151 positions and to determine the genotype of the virus in serum. Assay performances were evaluated with 170 samples, characterized by determination of viral load and sequencing of the Pol, S, and precore genes and the basal core promoter. One hundred fifty-three samples (90%) could be amplified and analyzed by the chip. Only two samples with more than 103 genome copies/ml could not be analyzed. Genotype had no impact on analytical sensitivity. Reproducibility studies showed no difference between repeats for codon and genotype determination. Genotype determination by sequencing and the chip were concordant in 148 of 151 samples. Twelve thousand one hundred sixty-one codons were analyzed by both techniques. Only 89.4% could be determined by sequencing, and among the remaining 11,335 codons, 92.8% were identical by sequencing and the chip. Failures to identify an amino acid by the chip were mainly due to reduced hybridization efficiency attributed to unexpected polymorphisms. Optimization of the chip-based reagent for the analysis of the HBV genome is ongoing. This first evaluation showed that DNA chip technology can provide important information in relation to the clinical management of chronic hepatitis B. PMID:16891494

  20. Real-time PCR assays for detection of Brucella spp. and the identification of genotype ST27 in bottlenose dolphins (Tursiops truncatus).

    PubMed

    Wu, Qingzhong; McFee, Wayne E; Goldstein, Tracey; Tiller, Rebekah V; Schwacke, Lori

    2014-05-01

    Rapid detection of Brucella spp. in marine mammals is challenging. Microbiologic culture is used for definitive diagnosis of brucellosis, but is time consuming, has low sensitivity and can be hazardous to laboratory personnel. Serological methods can aid in diagnosis, but may not differentiate prior exposure versus current active infection and may cross-react with unrelated Gram-negative bacteria. This study reports a real-time PCR assay for the detection of Brucella spp. and application to screen clinical samples from bottlenose dolphins stranded along the coast of South Carolina, USA. The assay was found to be 100% sensitive for the Brucella strains tested, and the limit of detection was 0.27fg of genomic DNA from Brucella ceti B1/94 per PCR volume. No amplification was detected for the non-Brucella pathogens tested. Brucella DNA was detected in 31% (55/178) of clinical samples tested. These studies indicate that the real-time PCR assay is highly sensitive and specific for the detection of Brucella spp. in bottlenose dolphins. We also developed a second real-time PCR assay for rapid identification of Brucella ST27, a genotype that is associated with human zoonotic infection. Positive results were obtained for Brucella strains which had been identified as ST27 by multilocus sequence typing. No amplification was found for other Brucella strains included in this study. ST27 was identified in 33% (18/54) of Brucella spp. DNA-positive clinical samples. To our knowledge, this is the first report on the use of a real-time PCR assay for identification of Brucella genotype ST27 in marine mammals.

  1. Disease-associated repeat instability and mismatch repair.

    PubMed

    Schmidt, Monika H M; Pearson, Christopher E

    2016-02-01

    Expanded tandem repeat sequences in DNA are associated with at least 40 human genetic neurological, neurodegenerative, and neuromuscular diseases. Repeat expansion can occur during parent-to-offspring transmission, and arise at variable rates in specific tissues throughout the life of an affected individual. Since the ongoing somatic repeat expansions can affect disease age-of-onset, severity, and progression, targeting somatic expansion holds potential as a therapeutic target. Thus, understanding the factors that regulate this mutation is crucial. DNA repair, in particular mismatch repair (MMR), is the major driving force of disease-associated repeat expansions. In contrast to its anti-mutagenic roles, mammalian MMR curiously drives the expansion mutations of disease-associated (CAG)·(CTG) repeats. Recent advances have broadened our knowledge of both the MMR proteins involved in disease repeat expansions, including: MSH2, MSH3, MSH6, MLH1, PMS2, and MLH3, as well as the types of repeats affected by MMR, now including: (CAG)·(CTG), (CGG)·(CCG), and (GAA)·(TTC) repeats. Mutagenic slipped-DNA structures have been detected in patient tissues, and the size of the slip-out and their junction conformation can determine the involvement of MMR. Furthermore, the formation of other unusual DNA and R-loop structures is proposed to play a key role in MMR-mediated instability. A complex correlation is emerging between tissues showing varying amounts of repeat instability and MMR expression levels. Notably, naturally occurring polymorphic variants of DNA repair genes can have dramatic effects upon the levels of repeat instability, which may explain the variation in disease age-of-onset, progression and severity. An increasing grasp of these factors holds prognostic and therapeutic potential.

  2. Neurological diseases associated with viral and Mycoplasma pneumoniae infections

    PubMed Central

    Assaad, F.; Gispen, R.; Kleemola, M.; Syrůček, L.; Esteves, K.

    1980-01-01

    In 1963 the World Health Organization established a system for the collection and dissemination of information on viral infections and by 1976, laboratories in 49 countries were participating in this scheme. The present study is in two parts: part 1 is an analysis of almost 60 000 reports on neurological disease associated with viral and Mycoplasma pneumoniae infections reported during the 10-year period 1967-76. This analysis showed a steady increase in the yearly number of reports of viral neurological diseases, which closely followed the general increase in the overall reporting of virus diseases. Likewise, the seasonal pattern was similar to that seen in general for any given virus. Over 75% of the cases were in children. Over half of all viral neurological diseases were associated with enteroviruses, while the myxoviruses accounted for almost 30%. Among the myxoviruses, mumps virus was by far the most frequently reported. The polioviruses were the agents most commonly detected in cases of paralytic disease. The other enteroviruses, mumps virus, and the herpesviruses were the most frequently reported viruses in cases of aseptic meningitis or encephalitis. On the other hand, one-third to over one-half of the reports on the myxoviruses (excluding mumps and measles) related to ill-defined clinical conditions. Part 2 of the study deals in particular with viruses whose role in neurological disease is less well documented. One laboratory reported an outbreak of adenoviral aseptic meningitis in Czechoslovakia, while another described neurological disease associated with M. pneumoniae infection in Finland. Part 2 also includes a detailed appraisal of viral infections diagnosed in the Netherlands during the period 1973-76. The results are very similar to those routinely reported. PMID:6249511

  3. Detection of rotavirus species A, B and C in domestic mammalian animals with diarrhoea and genotyping of bovine species A rotavirus strains.

    PubMed

    Otto, Peter H; Rosenhain, Stefanie; Elschner, Mandy C; Hotzel, Helmut; Machnowska, Patrycja; Trojnar, Eva; Hoffmann, Kathrin; Johne, Reimar

    2015-09-30

    Rotaviruses (RVs) are a major cause of neonatal diarrhoea in humans and animals worldwide. In this study, 425 faecal samples were collected between 1999 and 2013 from diarrhoeic livestock and companion animals at different locations in Germany and tested for RVs. A previously published real-time RT-PCR assay was optimized for detection of a larger variety of RV species A (RVA) strains, and real-time RT-PCR assays for detection of RV species B (RVB) and C (RVC) were newly developed. The detection limits of the assays were 1.54×10(2), 3.95×10(2) and 3.60×10(3) genome copies for RVA, RVB and RVC, respectively. RVA was identified in 85.2% of bovine samples, 51.2% of porcine samples, 50.0% of feline samples, 43.2% of equine samples and 39.7% of canine samples. RVB was found in 3.0% of bovine samples, 2.7% of equine samples and 1.6% of porcine samples. RVC was detected in 31.0% of porcine samples, 21.7% of feline samples, 9.0% of canine samples and 6.0% of bovine samples. For genotyping, 101 RVA-positive bovine samples were further analysed by semi-nested RT-PCR. Genotype combination G6P[5] was most frequently detected (67.3% of samples), followed by G6P[11] (13.9%), G10P[5] (4.0%), G8P[11] (3.0%), G6P[1] (1.0%), and G10P[11] (1.0%). Mixed RVA infections were detected in 5.9% of samples; no or incomplete typing was possible in 4.0% of the samples. This first overview on RV species and RVA genotypes in diarrhoeic livestock and companion animals from Germany indicates a broad circulation of a large variety of RVs. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. A new pyrosequencing assay for rapid detection and genotyping of Shiga toxin, intimin and O157-specific rfbE genes of Escherichia coli.

    PubMed

    Goji, Noriko; Mathews, Amit; Huszczynski, George; Laing, Chad R; Gannon, Victor P J; Graham, Morag R; Amoako, Kingsley K

    2015-02-01

    Shiga toxin (stx)-producing Escherichia coli (STEC) contamination in food and water is one of the most recognized concerns and a major financial burden in human hygiene control worldwide. Rapid and highly reliable methods of detecting and identifying STEC causing gastroenteric illnesses are crucial to prevent foodborne outbreaks. A number of tests have been developed and commercialized to detect STEC using molecular microbiology techniques. Most of these are designed to identify virulence factors such as Shiga toxin and intimin as well as E. coli O and H antigen serotype specific genes. In order to screen pathogenic STEC without relying on O:H serotyping, we developed a rapid detection and genotyping assay for STEC virulence genes using a PCR-pyrosequencing application. We adapted the PyroMark Q24 Pyrosequencing platform for subtyping 4 major virulence genes, Shiga toxin 1 and 2 (stx1 and stx2), intimin (eae) and O157-antigen gene cluster target rfbE, using Single Nucleotide Polymorphism (SNP) analysis. A total of 224 E. coli strains including isolates from Canadian environment, food and clinical cases were examined. Based on the multiple alignment analysis of 30-80 base nucleotide pyrogram reads, three alleles of the Shiga toxin 1a gene (stx1a) (stx1a-I, stx1a-II, stx1a-III) were identified. Results of the stx1, stx2, eae and rfbE genotyping revealed that each group of O:H serotype shares distinctive characteristics that could be associated with the virulence of each genotype. O157:H7/NM carries stx1a-II (94%), stx2a (82%), λ/γ1-eae (100%) and rfbE type-H7/NM (100%). Whereas isolates of the "Top-6" serotypes (O26, O45, O103, O111, O121, O145) had a high incidence of stx1a-I (90%) and stx2a (100%). stx1a-III (60%) was only observed in non Top-7 (Top-6 plus O157) STEC and Shigella spp. The entire assay, from extracting DNA from colonies on a plate to the generation of sequence information, can be completed in 5h. The method of profiling these 4 STEC pathogenic

  5. Detection and genotyping of torque teno virus (TTV) in healthy blood donors and patients infected with HBV or HCV in Qatar.

    PubMed

    AbuOdeh, Raed; Al-Mawlawi, Naema; Al-Qahtani, Ahmed A; Bohol, Marie Fe F; Al-Ahdal, Mohammed N; Hasan, Haydar A; AbuOdeh, Lamees; Nasrallah, Gheyath K

    2015-07-01

    Torque Teno virus (TTV) has been associated with non A-G hepatitis. The goal of this study was to estimate the infection rates and genotypic characteristics of TTV in the State of Qatar. A total of 644 blood samples representing different nationalities: (i) Qatari (118) and (ii) non-Qatari (526) nationals (mostly from Arab and South Eeast Asia countries) were tested for the presence of TTV DNA by nested PCR. The majority (573) of the blood samples belonged to healthy blood donors, whereas 54 and 53 of the blood samples belonged to patients infected with hepatitis B virus (HBV) and hepatitis C virus (HCV), respectively. The results obtained showed that the TTV infection rates in the healthy blood donors, and those infected with HBV or HCV patients were 81.4, 90.75 and 84.9%, respectively. Significant association between TTV viremia and age, or nationality was observed. Sequence analysis of PCR fragments amplified from the 5'-untranslated region (5'-UTR) of all (531) TTV positive samples showed that 65.5% (348/531) of the PCR fragment sequences were classified into main genogroup 3, followed by main genogroups 5 (24%), 2 (5.8%), and 1 (4.7%). Genogroup 4 was not detected among the our studied subjects. Phylogenetic and pairwise analyses using sequences from TTV viremic samples also showed an overall close similarity to the main genogroup 3. In conclusion, there was no significant difference in the rates of TTV detection among Qataris and non-Qataris and several genotypes, mainly genotype 3, were isolated.

  6. Voluntary intravenous self-administration of alcohol detects an interaction between GABAergic manipulation and GABRG1 polymorphism genotype: a pilot study.

    PubMed

    Plawecki, Martin H; Wetherill, Leah; Vitvitskiy, Victor; Kosobud, Ann; Zimmermann, Ulrich S; Edenberg, Howard J; O'Connor, Sean

    2013-01-01

    Operant responding paradigms quantify a subject's motivation for reward, but such studies employing ingested alcohol cannot assure the same incremental increase in brain exposure to alcohol across subjects because of substantial variability in absorption kinetics. We developed a human progressive ratio (PR) paradigm using the computer-assisted self-infusion of ethanol (CASE) system that overcomes such variability and conducted a pilot study to assess its utility for detecting an interaction of subjects' GABRA2 or GABRG1 genotype and pretreatment with 1 mg of lorazepam (LZ) vs. placebo on their willingness to work for alcohol rewards. Twenty healthy, nondependent drinkers, aged 21 to 27, were balanced on rs279871 and rs2350439 single nucleotide polymorphisms in the GABRA2 and GABRG1 genes, respectively. Subjects worked for alcohol, with water as an alternative reinforcer (AR), using a progressive schedule of a task that required constant attention and adapted to both fatigue and drug effects. Testing began 1 hour after pretreatment with 1 mg LZ or placebo in a crossover design. The CASE system performed well, and the constant attention task was perceived as work by all subjects. GABRA2 homozygosity did not significantly predict either breakpoint or cumulative work, whereas a significant GABRG1 genotype by LZ pretreatment interaction for cumulative work was detected (p = 0.04). Breakpoint revealed a weak trend toward pretreatment drug effects (p = 0.11), and a somewhat stronger interaction of LZ pretreatment with GABRG1 genotype (p = 0.06). GABRG1 status revealed a more complex relationship with respect to motivation for alcohol with and without LZ pretreatment; AG and GG individuals worked more for alcohol under both pretreatment conditions, while AA individuals worked more for the AR. The CASE PR paradigm shows promise as a laboratory method for use in drug development and phenotyping studies. Copyright © 2012 by the Research Society on Alcoholism.

  7. [Detection of a clonal complex with Brucella abortus biovar 2 genotype as founder in B. abortus isolates from Argentina].

    PubMed

    Hollender, Daiana; Conde, Sandra B; Salustio, Eduardo; Samartino, Luis E

    2013-01-01

    Brucella abortus is the causative agent of bovine brucellosis, a worldwide zoonosis. Up to date, eight biovars of B. abortus have been described. In Argentina, biovar 1 is the most frequently isolated. However, biovar 2, which is more pathogenic than biovar 1, is also found. Molecular methods for subtyping isolates are necessary for allowing epidemiological surveillance and control of eradication programs. Due to the genetic homogeneity of the genus Brucella, the development of molecular typing tools has been difficult. The publication of microorganism genomes facilitates the design of this approach. The aim of this work was to employ a Multiple Locus VNTR Analysis (MLVA) scheme for strains from Argentina isolated in our laboratory. From the 56 isolates analyzed, 47 different genotypic profiles were obtained. All the strains typed as biovar 2 showed the same profile. This scheme allowed assigning each isolate to the biovar it belongs to. All the genotypes were related using the goeBURST analysis and biovar 2 was proposed as founder.

  8. Detection and description of a particular Ostreid herpesvirus 1 genotype associated with massive mortality outbreaks of Pacific oysters, Crassostrea gigas, in France in 2008.

    PubMed

    Segarra, Amélie; Pépin, Jean François; Arzul, Isabelle; Morga, Benjamin; Faury, Nicole; Renault, Tristan

    2010-10-01

    Ostreid herpesvirus 1 (OsHV-1) infections have been reported around the world and are associated with high mortalities of the Pacific oyster (Crassostrea gigas). In the summer 2008, abnormal mortality rates ranging from 80% to 100% were reported in France and affected only Pacific oysters. Analyses of oyster samples collected during mortality outbreaks demonstrated a significant detection of OsHV-1 (75% of analysed batches), which appeared stronger than previous years. DNA sequencing based on C and IA regions was carried out on 28 batches of OsHV-1 infected Pacific oysters collected in 2008. Polymorphisms were described in both the C and IA regions and characterized a genotype of OsHV-1 not already reported and termed OsHV-1 microVar. A microsatellite zone present in the C region showed several deletions. Additionally, 44 isolates collected in France and in the USA, from 1995 to 2007 were sequenced and compared to the 2008 sequences. The analyses of 76 sequences showed OsHV-1 microVar detection only in 2008 isolates. These data suggest that OsHV-1 microVar can be assumed as an emergent genotype.

  9. Optimization of a genotyping screening based on hydrolysis probes to detect the main mutations related to Leber hereditary optic neuropathy (LHON)

    PubMed Central

    Miranda, Paulo Maurício do Amor Divino; Fernandes, Marcela Scabello Amaral; Maciel-Guerra, Andréa Trevas; Sartorato, Edi Lúcia

    2017-01-01

    Purpose Leber hereditary optic neuropathy (LHON) is a mitochondrial inherited disease characterized by bilateral vision problems, such as reduced visual acuity, dyschromatopsia, and central or centrocecal scotoma. Of these cases, 95% are caused by three mutations in mitochondrial DNA (mtDNA): m.G11778A, followed by m.T14484C and m.G3460A. The remaining 5% of cases of LHON are caused by rare mutations also present in mtDNA. Although conventional molecular tools for molecular screening of LHON are becoming popular, in most cases these tools are still expensive and time-consuming and are difficult to reproduce. Therefore, to meet the need for more accurate, faster, and cheaper techniques for molecular screening, as well as make it more accessible, we used the high-throughput method TaqMan® OpenArray™ Genotyping platform for developing a customized high-throughput assay for the three main mutations related to LHON. Methods The assay was performed for 87 individuals diagnosed with LHON or acquired optic neuropathy of unknown origin. The three main mutations were screened using the customized assay with the TaqMan® OpenArray™ Genotyping platform, and all reactions were performed in triplicate. The positive and negative results were revalidated with restriction fragment length polymorphism PCR (RFLP-PCR) and Sanger sequencing. Results The main mutations related to LHON were detected in 34 patients with genotyping reactions, of which 27 cases had the m.G11778A mutation, and seven had the m.T14484C mutation. Conclusions The TaqMan® OpenArray™ Genotyping platform was shown to be an effective tool for molecular screening of the most common mutations related to LHON without presenting false positive or negative results for the analyzed mutations. In addition, this tool can be considered a cheaper, faster, and more accurate alternative for molecular screening of LHON mutations than PCR and Sanger sequencing, as 94 genotyping reactions can be performed within 6 h

  10. Optimization of a genotyping screening based on hydrolysis probes to detect the main mutations related to Leber hereditary optic neuropathy (LHON).

    PubMed

    Martins, Fábio Tadeu Arrojo; Miranda, Paulo Maurício do Amor Divino; Fernandes, Marcela Scabello Amaral; Maciel-Guerra, Andréa Trevas; Sartorato, Edi Lúcia

    2017-01-01

    Leber hereditary optic neuropathy (LHON) is a mitochondrial inherited disease characterized by bilateral vision problems, such as reduced visual acuity, dyschromatopsia, and central or centrocecal scotoma. Of these cases, 95% are caused by three mutations in mitochondrial DNA (mtDNA): m.G11778A, followed by m.T14484C and m.G3460A. The remaining 5% of cases of LHON are caused by rare mutations also present in mtDNA. Although conventional molecular tools for molecular screening of LHON are becoming popular, in most cases these tools are still expensive and time-consuming and are difficult to reproduce. Therefore, to meet the need for more accurate, faster, and cheaper techniques for molecular screening, as well as make it more accessible, we used the high-throughput method TaqMan(®) OpenArray(™) Genotyping platform for developing a customized high-throughput assay for the three main mutations related to LHON. The assay was performed for 87 individuals diagnosed with LHON or acquired optic neuropathy of unknown origin. The three main mutations were screened using the customized assay with the TaqMan(®) OpenArray(™) Genotyping platform, and all reactions were performed in triplicate. The positive and negative results were revalidated with restriction fragment length polymorphism PCR (RFLP-PCR) and Sanger sequencing. The main mutations related to LHON were detected in 34 patients with genotyping reactions, of which 27 cases had the m.G11778A mutation, and seven had the m.T14484C mutation. The TaqMan(®) OpenArray(™) Genotyping platform was shown to be an effective tool for molecular screening of the most common mutations related to LHON without presenting false positive or negative results for the analyzed mutations. In addition, this tool can be considered a cheaper, faster, and more accurate alternative for molecular screening of LHON mutations than PCR and Sanger sequencing, as 94 genotyping reactions can be performed within 6 h and specific Taq

  11. Frequency, diagnostic performance of coproantigen detection and genotyping of the Giardia among patients referred to a multi-level teaching hospital in northern India.

    PubMed

    Ghoshal, Ujjala; Shukla, Ratnakar; Pant, Priyannk; Ghoshal, Uday C

    Giardiasis, a common gastrointestinal parasitic infection in tropics, is diagnosed on stool microscopy (gold standard); however, its sensitivity is low due to intermittent fecal shedding. Coproantigen detection (ELISA) is useful but requires further evaluation. We aimed to study: (a) detection of Giardia by stool microscopy and/or coproantigen, (b) diagnostic performance of fecal antigen detection and microscopy, and c) genotypic characterization of G. lamblia using PCR specific for triose phosphate isomerase (tpi) gene. Stool samples from 2992 patients were examined by microscopy from March 2013 to March 2015 in a multi level teaching hospital in northern India. Giardia coproantigen detection was performed by ELISA in a subset of patients. Genetic characterization of G. lamblia was performed by PCR targeting tpi gene in a subset of microscopy positive stool samples. Of 2992 patients, 132 (4.4%) had Giardia by microscopy (cyst/trophozoite) and/or ELISA. ELISA was performed in 264 patients; of them, 127 were positive by microscopy. Sensitivity, specificity, positive and negative predictive values of ELISA were 91, 91, 94, and 91%, respectively, using microscopy as a gold standard. PCR was performed in 116 randomly selected samples having Giardia using tpi gene. Assemblages A and B were found among 44 (38%) and 72 (62%) patients, respectively. Assemblage B was more often associated with malnutrition and loss of appetite than A (48/72 [67%] vs. 21/44 [48%], P = 0.044 and 17/72 [24%] vs. 14/44 [32%], P = 0.019). We conclude that 4.4% of studied population had giardiasis. Fecal antigen is a useful method for diagnosis and assemblage B is the most common genotype.

  12. Detection of hepatitis C virus in patients with terminal renal disease undergoing dialysis in southern Brazil: prevalence, risk factors, genotypes, and viral load dynamics in hemodialysis patients.

    PubMed

    Vidales-Braz, Beatris Maria; da Silva, Naylê Maria Oliveira; Lobato, Rubens; Germano, Fabiana Nunes; da Mota, Luiza Dias; Barros, Elvino J G; de Martinez, Ana Maria Barral

    2015-02-03

    Hepatitis C (HCV) is a serious public health issue, and it is estimated that 3% of the world's population is infected. Patients in hemodialysis units have an increased risk for contracting HCV, and high prevalence rates have been found in hemodialysis units around the world. This study is aimed at determining the prevalence of HCV in patients with terminal chronic renal disease (tCRD) who have been submitted to hemodialysis and peritoneal dialysis in southern Brazil to characterize the most prevalent genotypes, the viral load, and possible risk factors and to assess the validity between the ELISA and RT-PCR detection methods. Of 320 patients from three dialysis units, 318 participated in this study. According to the medical records, 55 patients were reactive to HCV, as determined via ELISA. All 318 samples were submitted to RT-PCR and genotyped using an Abbott Realtime m2000 system. Data obtained through a questionnaire and chemical variables were associated with the HCV. The prevalence of HCV was 18.24% (58), and the concordance between the HCV serology and the RT-PCR was 94%. Three patients were diagnosed to be negative for HCV using the ELISA assay but positive when using RT-PCR. Genotype 1 was the most prevalent (46.7%) genotype, within which subtype 1a was the most frequent (74.1%). One of the risk factors associated with HCV infection was the length of time that the patient had been undergoing hemodialysis treatments (p < 0.001). Additionally, the viral load was found to vary when tested before and after hemodialysis (p < 0.001). The prevalence of HCV in dialysis units continues to remain high, indicating nosocomial contamination. RT-PCR detected the presence of the hepatitis C virus in patients with a non-reactive serology, which highlights the importance of performing molecular tests on dialysis patients. The variation in the viral load in patients submitted to hemodialysis indicates a possible destruction or gripping of viral particles to the

  13. Performance assessment of the GenoType MTBDRsl test and DNA sequencing for detection of second-line and ethambutol drug resistance among patients infected with multidrug-resistant Mycobacterium tuberculosis.

    PubMed

    Huang, Wei-Lun; Chi, Ting-Lin; Wu, Mei-Hua; Jou, Ruwen

    2011-07-01

    The GenoType MTBDRsl test and DNA sequencing were used to rapidly detect second-line drug- and ethambutol (EMB)-resistant Mycobacterium tuberculosis. The ability of these two assays to detect the presence of mutations associated with resistance to fluoroquinolones (FLQ), aminoglycosides/cyclic peptide (AG/CP), and EMB in the gyrA, rrs, and embB genes (for the GenoType MTBDRsl test) and gyrA, gyrB, rrs, eis, embC, embA, embB, and embR genes (for DNA sequencing) was compared to that of conventional agar proportion drug susceptibility testing (DST). We evaluated 234 multidrug-resistant (MDR) M. tuberculosis isolates. The two molecular methods had high levels of specificity (95.8 to 100%). The sensitivities for FLQ resistance detection for both methods were 85.1%. For AG (kanamycin [KM] and amikacin [AM]) and CP (capreomycin CAP]), the sensitivities of resistance detection using the GenoType MTBDRsl test were 43.2%, 84.2%, and 71.4%, respectively, while with the inclusion of an extra gene, eis, in sequencing, the sensitivity reached 70.3% for detection of KM resistance. The sensitivities of EMB resistance detection were 56.2% and 90.7% with the GenoType MTBDRsl test and sequencing, respectively. We found that the GenoType MTBDRsl test can rapidly detect resistance to FLQ, CAP, and AM. The accuracy of the GenoType MTBDRsl test for the detection of FLQ and AM resistance was comparable to that of conventional DST; however, the test was less accurate for the detection of KM and EMB resistance and demonstrated a poor predictive value for CAP resistance. We recommend including new alleles consisting of the eis promoter and embB genes in molecular analysis. However, conventional DST is necessary to rule out false-negative results from molecular assays.

  14. Major human Hepatitis A virus genotype in Hong Kong marine waters and detection by real-time PCR.

    PubMed

    Yang, Ning; Chu, Daniel Ling Ho; Wong, Minnie Man Lai; Qi, Huizhou; Wu, Rudolf Shiu Sun; Kong, Richard Yuen Chong

    2011-12-01

    Marine waters from seven sites around Hong Kong with varying levels of sewage pollution were analyzed for Hepatitis A virus (HAV) by PCR cloning and DNA sequencing of the highly variable VP1/2A junction of the HAV genome. Phylogenetic analysis of 10 PCR clones from each of the HAV-positive marine sites indicated that human HAV genotype IB is the most widely distributed type in Hong Kong waters. A sensitive and quantitative TaqMan-based PCR method targeting the 5'-noncoding region (5'-NCR) of HAV was used to quantify HAV particles in marine water samples along with the total Escherichia coli counts being enumerated on TBX medium for comparison. Our results showed that no correlation of any significance between HAV and E. coli counts was observed which underscores the inadequacy in using E. coli as a sanitary standard to predict the levels of HAV in marine waters. Copyright © 2011 Elsevier Ltd. All rights reserved.

  15. Quick survey for detection, identification and characterization of Acanthamoeba genotypes from some selected soil and water samples across Pakistan.

    PubMed

    Tanveer, Tania; Hameed, Abdul; Gul, Asma; Matin, Abdul

    2015-01-01

    Acanthamoeba is an opportunistic protozoan pathogen which is widely distributed in nature and plays a pivotal role in ecosystem. Acanthamoeba species may cause blinding keratitis and fatal granulomatous encephalitis involving central nervous system. In this study, we investigated the presence of Acanthamoeba in soil and water resources of Pakistan. Here, Acanthamoeba were recovered on non-nutrient agar plate lawn with E. coli and identified by morphological characteristics of the cyst. Furthermore PCR was performed with genus-specific primers followed by direct sequencing of the PCR product for molecular identification. Overall our PCR and sequencing results confirmed pathogenic genotypes including T4 and T15 from both soil and water samples. This is our first report of Acanthamoeba isolation from both soil and water resources of Pakistan which may serve as a potential treat to human health across the country.

  16. Detection and phylogenetic characterization of hepatitis E virus genotype 3 in a captive wild boar in Thailand.

    PubMed

    Wiratsudakul, Anuwat; Sariya, Ladawan; Prompiram, Phirom; Tantawet, Siriporn; Suraruangchai, Duangkhamol; Sedwisai, Poonyapat; Sangkachai, Nareerat; Suksai, Parut; Ratanakorn, Parntep

    2012-09-01

    Hepatitis E virus (HEV) was studied in different types of wild boar captive settings in Thailand, including a wildlife breeding research station, zoo, and commercial wild boar farm, which were located in different locations of Thailand. Fifty-one fecal samples were collected and screened for HEV RNA and then analyzed. One sample obtained from a wildlife breeding research station in Ratchaburi province was HEV positive. Phylogenetic characterization revealed that the virus was HEV genotype 3 and belongs to subgroup 3e, which is closely related to HEV recently isolated from domestic pigs and humans in the country. It was hypothesized that HEV is shared among wild boars, domestic pigs, and humans in Thailand.

  17. Genotyping-By-Sequencing (GBS) Detects Genetic Structure and Confirms Behavioral QTL in Tame and Aggressive Foxes (Vulpes vulpes).

    PubMed

    Johnson, Jennifer L; Wittgenstein, Helena; Mitchell, Sharon E; Hyma, Katie E; Temnykh, Svetlana V; Kharlamova, Anastasiya V; Gulevich, Rimma G; Vladimirova, Anastasiya V; Fong, Hiu Wa Flora; Acland, Gregory M; Trut, Lyudmila N; Kukekova, Anna V

    2015-01-01

    The silver fox (Vulpes vulpes) offers a novel model for studying the genetics of social behavior and animal domestication. Selection of foxes, separately, for tame and for aggressive behavior has yielded two strains with markedly different, genetically determined, behavioral phenotypes. Tame strain foxes are eager to establish human contact while foxes from the aggressive strain are aggressive and difficult to handle. These strains have been maintained as separate outbred lines for over 40 generations but their genetic structure has not been previously investigated. We applied a genotyping-by-sequencing (GBS) approach to provide insights into the genetic composition of these fox populations. Sequence analysis of EcoT22I genomic libraries of tame and aggressive foxes identified 48,294 high quality SNPs. Population structure analysis revealed genetic divergence between the two strains and more diversity in the aggressive strain than in the tame one. Significant differences in allele frequency between the strains were identified for 68 SNPs. Three of these SNPs were located on fox chromosome 14 within an interval of a previously identified behavioral QTL, further supporting the importance of this region for behavior. The GBS SNP data confirmed that significant genetic diversity has been preserved in both fox populations despite many years of selective breeding. Analysis of SNP allele frequencies in the two populations identified several regions of genetic divergence between the tame and aggressive foxes, some of which may represent targets of selection for behavior. The GBS protocol used in this study significantly expanded genomic resources for the fox, and can be adapted for SNP discovery and genotyping in other canid species.

  18. Genotyping-By-Sequencing (GBS) Detects Genetic Structure and Confirms Behavioral QTL in Tame and Aggressive Foxes (Vulpes vulpes)

    PubMed Central

    Johnson, Jennifer L.; Wittgenstein, Helena; Mitchell, Sharon E.; Hyma, Katie E.; Temnykh, Svetlana V.; Kharlamova, Anastasiya V.; Gulevich, Rimma G.; Vladimirova, Anastasiya V.; Fong, Hiu Wa Flora; Acland, Gregory M.; Trut, Lyudmila N.; Kukekova, Anna V.

    2015-01-01

    The silver fox (Vulpes vulpes) offers a novel model for studying the genetics of social behavior and animal domestication. Selection of foxes, separately, for tame and for aggressive behavior has yielded two strains with markedly different, genetically determined, behavioral phenotypes. Tame strain foxes are eager to establish human contact while foxes from the aggressive strain are aggressive and difficult to handle. These strains have been maintained as separate outbred lines for over 40 generations but their genetic structure has not been previously investigated. We applied a genotyping-by-sequencing (GBS) approach to provide insights into the genetic composition of these fox populations. Sequence analysis of EcoT22I genomic libraries of tame and aggressive foxes identified 48,294 high quality SNPs. Population structure analysis revealed genetic divergence between the two strains and more diversity in the aggressive strain than in the tame one. Significant differences in allele frequency between the strains were identified for 68 SNPs. Three of these SNPs were located on fox chromosome 14 within an interval of a previously identified behavioral QTL, further supporting the importance of this region for behavior. The GBS SNP data confirmed that significant genetic diversity has been preserved in both fox populations despite many years of selective breeding. Analysis of SNP allele frequencies in the two populations identified several regions of genetic divergence between the tame and aggressive foxes, some of which may represent targets of selection for behavior. The GBS protocol used in this study significantly expanded genomic resources for the fox, and can be adapted for SNP discovery and genotyping in other canid species. PMID:26061395

  19. Power of multifactor dimensionality reduction for detecting gene-gene interactions in the presence of genotyping error, missing data, phenocopy, and genetic heterogeneity.

    PubMed

    Ritchie, Marylyn D; Hahn, Lance W; Moore, Jason H

    2003-02-01

    The identification and characterization of genes that influence the risk of common, complex multifactorial diseases, primarily through interactions with other genes and other environmental factors, remains a statistical and computational challenge in genetic epidemiology. This challenge is partly due to the limitations of parametric statistical methods for detecting genetic effects that are dependent solely or partially on interactions with other genes and environmental exposures. We previously introduced multifactor dimensionality reduction (MDR) as a method for reducing the dimensionality of multilocus genotype information to improve the identification of polymorphism combinations associated with disease risk. The MDR approach is nonparametric (i.e., no hypothesis about the value of a statistical parameter is made), is model-free (i.e., assumes no particular inheritance model), and is directly applicable to case-control and discordant sib-pair study designs. Both empirical and theoretical studies suggest that MDR has excellent power for identifying high-order gene-gene interactions. However, the power of MDR for identifying gene-gene interactions in the presence of common sources of noise is not currently known. The goal of this study was to evaluate the power of MDR for identifying gene-gene interactions in the presence of noise due to genotyping error, missing data, phenocopy, and genetic or locus heterogeneity. Using simulated data, we show that MDR has high power to identify gene-gene interactions in the presence of 5% genotyping error, 5% missing data, or a combination of both. However, MDR has reduced power for some models in the presence of 50% phenocopy, and very limited power in the presence of 50% genetic heterogeneity. Extending MDR to address genetic heterogeneity should be a priority for the continued methodological development of this new approach. Copyright 2003 Wiley-Liss, Inc.

  20. Robust methods to detect disease-genotype association in genetic association studies: calculate p-values using exact conditional enumeration instead of simulated permutations or asymptotic approximations.

    PubMed

    Langaas, Mette; Bakke, Øyvind

    2014-12-01

    In genetic association studies, detecting disease-genotype association is a primary goal. We study seven robust test statistics for such association when the underlying genetic model is unknown, for data on disease status (case or control) and genotype (three genotypes of a biallelic genetic marker). In such studies, p-values have predominantly been calculated by asymptotic approximations or by simulated permutations. We consider an exact method, conditional enumeration. When the number of simulated permutations tends to infinity, the permutation p-value approaches the conditional enumeration p-value, but calculating the latter is much more efficient than performing simulated permutations. We have studied case-control sample sizes with 500-5000 cases and 500-15,000 controls, and significance levels from 5 × 10(-8) to 0.05, thus our results are applicable to genetic association studies with only a few genetic markers under study, intermediate follow-up studies, and genome-wide association studies. Our main findings are: (i) If all monotone genetic models are of interest, the best performance in the situations under study is achieved for the robust test statistics based on the maximum over a range of Cochran-Armitage trend tests with different scores and for the constrained likelihood ratio test. (ii) For significance levels below 0.05, for the test statistics under study, asymptotic approximations may give a test size up to 20 times the nominal level, and should therefore be used with caution. (iii) Calculating p-values based on exact conditional enumeration is a powerful, valid and computationally feasible approach, and we advocate its use in genetic association studies.

  1. Molecular epidemiology of rotavirus and norovirus in Ile-Ife, Nigeria: high prevalence of G12P[8] rotavirus strains and detection of a rare norovirus genotype.

    PubMed

    Oluwatoyin Japhet, Margaret; Adeyemi Adesina, Olufisayo; Famurewa, Oladiran; Svensson, Lennart; Nordgren, Johan

    2012-09-01

    Rotavirus (RV) and norovirus (NoV) are considered the most common causes of viral gastroenteritis in children. In this study, the prevalence of RV and NoV infection in 55 children with diarrhea from the rural community Akinlalu in Southwestern Nigeria was investigated using real-time PCR assays. The RV and NoV strains were genotyped by PCR and/or sequencing. RV and NoV infections occurred with a prevalence of 34.5% and 25.5% respectively, with predominance in children <1 year. Most infections occurred during the dry season with increasing prevalence of RV as the dry season progressed (October-January). Infections with RV VP6 subgroup (SG) II were more prevalent (27.3%) than SGI (7.3%). Similarly, NoV genogroup II infections were more common (23.6%) than genogroup I (1.8%). Five children out of 55 (9.1%) were co-infected with both RV and NoV. Notably, G12P[8] was the predominant RV strain (36.8%, n = 7), observed for the first time in Nigeria. The VP7 gene of the G12 strains clustered within lineage III, sharing high nucleotide identity with each other (>99%) indicating introduction in Nigeria from a single donor. Furthermore, a putative novel genotype within genogroup I NoV was detected, which till date has only been reported once in humans. To conclude, a high prevalence of the emerging G12P[8] RV strain was observed for the first time in Nigeria, as well as a putative novel NoV genotype in humans. These results provide new information which can be important for future vaccine evaluations and possible introduction in Nigeria.

  2. Evaluation of the detection of 14 high-risk human papillomaviruses with HPV 16 and HPV 18 genotyping for cervical cancer screening

    PubMed Central

    BIAN, MEI-LU; CHENG, JIAO-YING; MA, LI; CONG, XIAO; LIU, JUN; CHEN, YING; CHEN, XI

    2013-01-01

    The American Society for Colposcopy and Cervical Pathology (ASCCP) suggests that women ≥30 years old, with a negative cytopathological test but a positive high-risk (HR) human papillomavirus (HPV) test should undergo HPV 16 and HPV 18 genotyping. If this test is positive, immediate cervical pathology is required. Therefore, the aim of this study was to evaluate the effectiveness and clinical value of testing for 14 HR HPVs with HPV 16 and HPV 18 genotyping for cervical cancer (CC) screening. A total of 424 females from the China-Japan Friendship Hospital were selected and randomly divided into two groups (A and B). All participants underwent two different testing methods: the liquid-based cytology test (LCT) and a HPV DNA test. For the HPV DNA test, participants in group A underwent the hybrid capture II (HC-II) testing method while participants in group B were tested using the quantitative polymerase chain reaction (qPCR; HBRT-H14) method. The sensitivity, specificity, positive predictive value and negative predictive value for the detection of cervical intraepithelial neoplasia (CIN) grade II or greater using HBRT-H14 were 96.30, 78.17, 23.21 and 99.68%, respectively. In Group B, compared with other HR HPV types, HPV 16 and HPV 18 infection led to the increased possibility of cervical lesions graded CIN II or higher (8.11 and 51.28%, respectively). A significant difference in the rates of CC and CIN II or higher was observed among women who were i) infected with HPV 16 and/or HPV 18, ii) infected with other HR HPV types and iii) diagnosed as negative for HR HPV infection (χ2=93.976, P=0.0001). In conclusion, HBRT-H14 is applicable for CC screening with the advantage of genotyping for HPV 16 and HPV 18, which may help to improve triage management for women with negative cytology. PMID:24223668

  3. High sensitivity, loop-mediated isothermal amplification combined with colorimetric gold-nanoparticle probes for visual detection of high risk human papillomavirus genotypes 16 and 18.

    PubMed

    Kumvongpin, Ratchanida; Jearanaikool, Patcharee; Wilailuckana, Chotechana; Sae-Ung, Nattaya; Prasongdee, Prinya; Daduang, Sakda; Wongsena, Metee; Boonsiri, Patcharee; Kiatpathomchai, Wansika; Swangvaree, Sukumarn Sanersak; Sandee, Alisa; Daduang, Jureerut

    2016-08-01

    High-risk human papillomavirus (HR-HPV) causes cervical cancer. HPV16 and HPV18 are the most prevalent strains of the virus reported in women worldwide. Loop-mediated isothermal amplification (LAMP) is an alternative method for DNA detection under isothermal conditions. However, it results in a turbid amplified product which is not easily detected by the naked eye. This study aimed to develop an improved technique by using gold nanoparticles (AuNPs) attached to a single-stranded DNA probe for the detection of HPV16 and HPV18. Detection of the LAMP product by AuNP color change was compared with detection by visual turbidity. The optimal conditions for this new LAMP-AuNP assay were an incubation time of 20min and a temperature of 65°C. After LAMP amplification was complete, its products were hybridized with the AuNP probe for 5min and then detected by the addition of magnesium salt. The color changed from red to blue as a result of aggregation of the AuNP probe under high ionic strength conditions produced by the addition of the salt. The sensitivity of the LAMP-AuNP assay was greater than the LAMP turbidity assay by up to 10-fold for both HPV genotypes. The LAMP-AuNP assay showed higher sensitivity and ease of visualization than did the LAMP turbidity for the detection of HPV16 and HPV18. Additionally, AuNP-HPV16 and AuNP-HPV18 probes were stable for over 1year. The combination of LAMP and the AuNP-probe colorimetric assay offers a simple, rapid and highly sensitive alternative diagnostic tool for the detection of HPV16 and HPV18 in district hospitals or field studies.

  4. Development of a Multiplex PCR Test with Automated Genotyping Targeting E7 for Detection of Six High-Risk Human Papillomaviruses.

    PubMed

    Paes, Eliana Ferreira; de Assis, Angela Maria; Teixeira, Cirbia S Campos; Aoki, Francisco Hideo; Teixeira, Julio Cesar

    2015-01-01

    Cervical cancer is caused by high-risk human papillomaviruses (HPV) and viral detection tests aid in the diagnosis of precursor lesions. In the present study, a molecular test for detection of high-risk HPV DNA, called E7-HPV, was standardized and assessed in samples from women with pre-cancerous lesions. The development of the E7-HPV test for detection and genotyping of six high-risk HPV (types 16, 18, 31, 33, 45 and 52), consisted of evaluating primer quality and adjusting the multiplex PCR conditions. Primer design was based on the E7 region of each HPV, and the fluorochrome 6-FAM was added to PCR primers. Viral detection was performed by capillary electrophoresis in automated sequencer in samples obtained from 60 women (55 with ASC-H/HSIL cytology) from August to September 2013. A non-inferiority analysis was conducted with the cobas HPV test as a reference and following international guidelines for the development of new tests. The two tests had a high concordance rate in HPV16 detection (kappa=0.972), with only one discordant case (cervical intraepithelial neoplasia grade 3, negative with cobas and positive for HPV16 by E7-HPV) and complete agreement in HPV18 detection. When comparing detection of all high-risk HPV, three cases were positive with cobas but negative with E7-HPV, and another three cases were negative with cobas but positive with E7-HPV (HPV16, 31 and 52). When we evaluate the cases initially suspected by cytology, the two tests had the same sensitivity in detection CIN2 or worse. In conclusion, the E7-HPV test has satisfactory initial results, and its development can be continued.

  5. Evaluation of the GenoType MTBDRsl Version 2.0 Assay for Second-Line Drug Resistance Detection of Mycobacterium tuberculosis Isolates in South Africa.

    PubMed

    Gardee, Y; Dreyer, A W; Koornhof, H J; Omar, S V; da Silva, P; Bhyat, Z; Ismail, N A

    2017-03-01

    Early detection of resistance to second-line antituberculosis drugs is important for the management of multidrug-resistant tuberculosis (MDR-TB). The GenoType MTBDRsl version 2.0 (VER 2.0) line probe assay has been redesigned for molecular detection of resistance-conferring mutations of fluoroquinolones (FLQ) (gyrA and gyrB genes) and second-line injectable drugs (SLID) (rrs and eis genes). The study evaluated the diagnostic performance of the GenoType MTBDRsl VER 2.0 assay for the detection of second-line drug resistance compared with phenotypic drug susceptibility testing (DST), using the Bactec MGIT 960 system on Mycobacterium tuberculosis complex isolates from South Africa. A total of 268 repository isolates collected between 2012 and 2014, which were rifampin monoresistant or MDR based on DST, were selected. MTBDRsl VER 2.0 testing was performed on these isolates and the results analyzed. The MTBDRsl VER 2.0 sensitivity and specificity indices for culture isolates were the following: FLQ, 100% (95% confidence interval [CI] 95.8 to 100%) and 98.9% (95% CI, 96.1 to 99.9%); SLID, 89.2% (95% CI, 79.1 to 95.6%) and 98.5% (95% CI, 95.7 to 99.7%). The sensitivity and specificity observed for individual SLID were the following: amikacin, 93.8% (95% CI, 79.2 to 99.2%) and 98.5% (95% CI, 95.5 to 99.7%); kanamycin, 89.2% (95% CI, 79.1 to 95.6%) and 98.5% (95% CI, 95.5 to 99.7%); and capreomycin, 86.2% (95% CI, 68.3 to 96.1%) and 95.9% (95% CI, 92.2 to 98.2%). An interoperator reproducibility of 100% and an overall interlaboratory performance of 93% to 96% were found. The overall improvement in sensitivity and specificity with excellent reproducibility makes the GenoType MTBDRsl VER 2.0 a highly suitable tool for rapid screening of clinical isolates for second-line drug resistance for use in high-burden TB/HIV settings. Copyright © 2017 American Society for Microbiology.

  6. Comparison of Xpert MTB/RIF Assay and GenoType MTBDRplus DNA Probes for Detection of Mutations Associated with Rifampicin Resistance in Mycobacterium tuberculosis.

    PubMed

    Rahman, Arfatur; Sahrin, Mahfuza; Afrin, Sadia; Earley, Keith; Ahmed, Shahriar; Rahman, S M Mazidur; Banu, Sayera

    2016-01-01

    GeneXpert MTB/RIF (Xpert) and Genotype MTBDRplus (DRplus) are two World Health Organization (WHO) endorsed probe based molecular drug susceptibility testing (DST) methods for rapid diagnosis of drug resistant tuberculosis. Both methods target the same 81 bp Rifampicin Resistance Determining Region (RRDR) of bacterial RNA polymerase β subunit (rpoB) for detection of Rifampicin (RIF) resistance associated mutations using DNA probes. So there is a correspondence of the probes of each other and expected similarity of probe binding. We analyzed 92 sputum specimens by Xpert, DRplus and LJ proportion method (LJ-DST). We compared molecular DSTs with gold standard LJ-DST. We wanted to see the agreement level of two molecular methods for detection of RIF resistance associated mutations. The 81bp RRDR region of rpoB gene of discrepant cases between the two molecular methods was sequenced by Sanger sequencing. The agreement of Xpert and DRplus with LJ-DST for detection of RIF susceptibility was found to be 93.5% and 92.4%, respectively. We also found 92.4% overall agreement of two molecular methods for the detection of RIF susceptibility. A total of 84 out of 92 samples (91.3%) had agreement on the molecular locus of RRDR mutation by DRplus and Xpert. Sanger sequencing of 81bp RRDR revealed that Xpert probes detected seven of eight discrepant cases correctly and DRplus was erroneous in all the eight cases. Although the overall concordance with LJ-DST was similar for both Xpert and DRplus assay, Xpert demonstrated more accuracy in the detection of RIF susceptibility for discrepant isolates compared with DRplus. This observation would be helpful for the improvement of probe based detection of drug resistance associated mutations especially rpoB mutation in M. tuberculosis.

  7. Selecting tagging SNPs for association studies using power calculations from genotype data.

    PubMed

    Hu, Xiaolan; Schrodi, Steven J; Ross, David A; Cargill, Michele

    2004-01-01

    Recent studies have indicated that linkage disequilibrium (LD) between single nucleotide polymorphism (SNP) markers can be used to derive a reduced set of tagging SNPs (tSNPs) for genetic association studies. Previous strategies for identifying tSNPs have focused on LD measures or haplotype diversity, but the statistical power to detect disease-associated variants using tSNPs in genetic studies has not been fully characterized. We propose a new approach of selecting tSNPs based on determining the set of SNPs with the highest power to detect association. Two-locus genotype frequencies are used in the power calculations. To show utility, we applied this power method to a large number of SNPs that had been genotyped in Caucasian samples. We demonstrate that a significant reduction in genotyping efforts can be achieved although the reduction depends on genotypic relative risk, inheritance mode and the prevalence of disease in the human population. The tSNP sets identified by our method are remarkably robust to changes in the disease model when small relative risk and additive mode of inheritance are employed. We have also evaluated the ability of the method to detect unidentified SNPs. Our findings have important implications in applying tSNPs from different data sources in association studies. Copyright 2004 S. Karger AG, Basel

  8. Genotype-based association models of complex diseases to detect gene-gene and gene-environment interactions.

    PubMed

    Lobach, Iryna; Fan, Ruzong; Manga, Prashiela

    A central problem in genetic epidemiology is to identify and rank genetic markers involved in a disease. Complex diseases, such as cancer, hypertension, diabetes, are thought to be caused by an interaction of a panel of genetic factors, that can be identified by markers, which modulate environmental factors. Moreover, the effect of each genetic marker may be small. Hence, the association signal may be missed unless a large sample is considered, or a priori biomedical data are used. Recent advances generated a vast variety of a priori information, including linkage maps and information about gene regulatory dependence assembled into curated pathway databases. We propose a genotype-based approach that takes into account linkage disequilibrium (LD) information between genetic markers that are in moderate LD while modeling gene-gene and gene-environment interactions. A major advantage of our method is that the observed genetic information enters a model directly thus eliminating the need to estimate haplotype-phase. Our approach results in an algorithm that is inexpensive computationally and does not suffer from bias induced by haplotype-phase ambiguity. We investigated our model in a series of simulation experiments and demonstrated that the proposed approach results in estimates that are nearly unbiased and have small variability. We applied our method to the analysis of data from a melanoma case-control study and investigated interaction between a set of pigmentation genes and environmental factors defined by age and gender. Furthermore, an application of our method is demonstrated using a study of Alcohol Dependence.

  9. Simultaneous and Sensitive Detection of Human Immunodeficiency Virus Type 1 (HIV) Drug Resistant Genotypes by Multiplex Oligonucleotide Ligation Assay

    PubMed Central

    Ellis, Giovanina M.; Marushchak, Tatyana A.; Koth, Andrew; Vaz, Louise E.; Dross, Sandra E.; Beck, Ingrid A.; Frenkel, Lisa M.

    2013-01-01

    Oligonucleotide ligation assay (OLA) is a highly specific and relatively simple method to detect point mutations encoding HIV-1 drug-resistance, which can detect mutants comprising ≥2–5% of the viral population. Nevirapine (NVP), tenofovir (TDF) and lamivudine (3TC) are antiretroviral drugs (ARV) used worldwide for treatment of HIV infection and prevention of mother-to-child-transmission. Adapting the OLA to detect multiple mutations associated with HIV resistance to these ARV simultaneously would provide an efficient tool to monitor drug resistance in resource-limited settings. Known proportions of mutant and wild-type plasmids were used to optimize a multiplex OLA for detection of K103N, Y181C, K65R, and M184V in HIV subtypes B and C, and V106M and G190A in subtype C. Simultaneous detection of two mutations was impaired if probes annealed to overlapping regions of the viral template, but was sensitive to ≥2–5% when testing codons using non-overlapping probes. PCR products from HIV-subtype B and C-infected individuals were tested by multiplex-OLA and compared to results of single-codon OLA. Multiplex-OLA detected mutations at codon pairs 103/181, 106/190 and 65/184 reliably when compared to singleplex-OLA in clinical specimens. The multiplex-OLA is sensitive and specific and reduces the cost of screening for NVP, TDF and/or 3TC resistance. PMID:23660583

  10. Detection of hepatitis B virus genotypic resistance mutations by coamplification at lower denaturation temperature-PCR coupled with sanger sequencing.

    PubMed

    Liu, Can; Lin, Jinpiao; Chen, Huijuan; Shang, Hongyan; Jiang, Ling; Chen, Jing; Ye, Yang; Yang, Bin; Ou, Qishui

    2014-08-01

    Mutations in the reverse transcriptase (rt) region of the DNA polymerase gene are the primary cause of hepatitis B virus (HBV) drug resistance. In this study, we established a novel method that couples coamplification at lower denaturation temperature (COLD)-PCR and Sanger sequencing, and we applied it to the detection of known and unknown HBV mutations. Primers were designed based on the common mutations in the HBV rt sequence at positions 180 to 215. The critical denaturation temperature (Tc) was established as a denaturing temperature for both fast and full COLD-PCR procedures. For single mutations, when a melting temperature (Tm)-reducing mutation occurred (e.g., C-G → T-A), the sensitivities of fast and full COLD-PCR for mutant detection were 1% and 2%, respectively; when the mutation caused no change in Tm (e.g., C-G → G-C) or raised Tm (e.g., T-A → C-G), only full COLD-PCR improved the sensitivity for mutant detection (2%). For combination mutations, the sensitivities of both full and fast COLD-PCR were increased to 0.5%. The limits of detection for fast and full COLD-PCR were 50 IU/ml and 100 IU/ml, respectively. In 30 chronic hepatitis B (CHB) cases, no mutations were detected by conventional PCR, whereas 18 mutations were successfully detected by COLD-PCR, including low-prevalence mutations (<10%), as confirmed by ultradeep pyrosequencing. In conclusion, COLD-PCR provides a highly sensitive, simple, inexpensive, and practical tool for significantly improving amplification efficacy and detecting low-level mutations in clinical CHB cases. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  11. Detection of norovirus genotype I.3b and II.4 in bioaccumulated blue mussels using different virus recovery methods.

    PubMed

    Comelli, Heidi Lange; Rimstad, Espen; Larsen, Stig; Myrmel, Mette

    2008-09-30

    Noroviruses (NoVs) are the most common non bacterial human pathogens associated with shellfish borne gastroenteritis. Norovirus detection is based on molecular procedures such as reverse transcriptase (RT)-PCR. A variety of methods have been developed to extract viral RNA from complex shellfish matrixes and to reduce the level of RT-PCR inhibitors. The present study had three objectives: 1) Determine the most appropriate sample treatment protocol for detection of NoVs in mussels, 2) Examine whether there is a variation of the binding affinity between a NoV GI and a GII strain to mussel digestive tissue and how this influences the detection sensitivity, 3) Establish an internal control for sample processing and virus detection. Three RNA extraction methods were evaluated on extracts from blue mussels (Mytilus edulis) spiked with NoV GII.4. The most efficient RNA extraction method was subsequently used for evaluation of three virus recovery methods of blue mussels bio accumulated with NoV GI.3b and GII.4. Mengovirus was evaluated as an internal process control and TaqMan RT-PCRs were used for virus detection. Elution of the two viruses from shellfish tissue differed, indicating a difference in binding affinities. Only a method based upon Proteinase K digestion followed by NucliSenseasyMAG was able to detect both NoV GI.3b and GII.4 (3.0% and 3.5% recovery respectively). The results show that the processing method influences the possibility to detect different variants of NoV.

  12. Detection of Hepatitis B Virus Genotypic Resistance Mutations by Coamplification at Lower Denaturation Temperature-PCR Coupled with Sanger Sequencing

    PubMed Central

    Liu, Can; Lin, Jinpiao; Chen, Huijuan; Shang, Hongyan; Jiang, Ling; Chen, Jing; Ye, Yang

    2014-01-01

    Mutations in the reverse transcriptase (rt) region of the DNA polymerase gene are the primary cause of hepatitis B virus (HBV) drug resistance. In this study, we established a novel method that couples coamplification at lower denaturation temperature (COLD)-PCR and Sanger sequencing, and we applied it to the detection of known and unknown HBV mutations. Primers were designed based on the common mutations in the HBV rt sequence at positions 180 to 215. The critical denaturation temperature (Tc) was established as a denaturing temperature for both fast and full COLD-PCR procedures. For single mutations, when a melting temperature (Tm)-reducing mutation occurred (e.g., C-G→T-A), the sensitivities of fast and full COLD-PCR for mutant detection were 1% and 2%, respectively; when the mutation caused no change in Tm (e.g., C-G→G-C) or raised Tm (e.g., T-A→C-G), only full COLD-PCR improved the sensitivity for mutant detection (2%). For combination mutations, the sensitivities of both full and fast COLD-PCR were increased to 0.5%. The limits of detection for fast and full COLD-PCR were 50 IU/ml and 100 IU/ml, respectively. In 30 chronic hepatitis B (CHB) cases, no mutations were detected by conventional PCR, whereas 18 mutations were successfully detected by COLD-PCR, including low-prevalence mutations (<10%), as confirmed by ultradeep pyrosequencing. In conclusion, COLD-PCR provides a highly sensitive, simple, inexpensive, and practical tool for significantly improving amplification efficacy and detecting low-level mutations in clinical CHB cases. PMID:24899029

  13. The Decay of Disease Association with Declining Linkage Disequilibrium: A Fine Mapping Theorem

    PubMed Central

    Maadooliat, Mehdi; Bansal, Naveen K.; Upadhya, Jiblal; Farazi, Manzur R.; Li, Xiang; He, Max M.; Hebbring, Scott J.; Ye, Zhan; Schrodi, Steven J.

    2016-01-01

    Several important and fundamental aspects of disease genetics models have yet to be described. One such property is the relationship of disease association statistics at a marker site closely linked to a disease causing site. A complete description of this two-locus system is of particular importance to experimental efforts to fine map association signals for complex diseases. Here, we present a simple relationship between disease association statistics and the decline of linkage disequilibrium from a causal site. Specifically, the ratio of Chi-square disease association statistics at a marker site and causal site is equivalent to the standard measure of pairwise linkage disequilibrium, r2. A complete derivation of this relationship from a general disease model is shown. Quite interestingly, this relationship holds across all modes of inheritance. Extensive Monte Carlo simulations using a disease genetics model applied to chromosomes subjected to a standard model of recombination are employed to better understand the variation around this fine mapping theorem due to sampling effects. We also use this relationship to provide a framework for estimating properties of a non-interrogated causal site using data at closely linked markers. Lastly, we apply this way of examining association data from high-density genotyping in a large, publicly-available data set investigating extreme BMI. We anticipate that understanding the patterns of disease association decay with declining linkage disequilibrium from a causal site will enable more powerful fine mapping methods and provide new avenues for identifying causal sites/genes from fine-mapping studies. PMID:28018425

  14. Development of a new real-time quantitative PCR assay for the detection of Staphylococcus aureus genotype B in cow milk, targeting the new gene adlb.

    PubMed

    Sartori, C; Boss, R; Ivanovic, I; Graber, H U

    2017-10-01

    The specific and reliable diagnosis of mastitis pathogens is essential for successful sanitation programs. The aim of the present study was to develop and evaluate a new real-time quantitative PCR (qPCR) assay for the very sensitive and specific detection of Staphylococcus aureus genotype B in cow milk samples. This mastitis pathogen is contagious and particularly prevalent in Switzerland and other central European countries. The new test is based on a rapid preparation of bacteria, followed by DNA isolation and qPCR for a unique target gene coding for the adhesion-like bovine protein (adlb). The inclusivity of the new target gene was 97% and the exclusivity 98%, meaning that other genotypes and bacterial species could be excluded with high reliability. The limit of detection of the new assay was 235 staphylococcal cell equivalents/mL of culture. The new test shows high intra- and interassay repeatability. Results are available within 2 d after sampling, allowing farmers and veterinarians to apply sanitation measures immediately. Based on the results of a preliminary field study, the diagnostic sensitivity and specificity of the new qPCR assay are 99 and 100%, respectively. The new analytical procedure is straightforward and can be applied for routine diagnostics. The Authors. Published by the Federation of Animal Science Societies and Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

  15. A sensitive one-step TaqMan amplification approach for detection of rubella virus clade I and II genotypes in clinical samples.

    PubMed

    Claus, C; Bergs, S; Emmrich, N C; Hübschen, J M; Mankertz, A; Liebert, U G

    2017-02-01

    Although teratogenic rubella virus (RV) causes a vaccine-preventable disease, it is still endemic in several countries worldwide. Thus, there is a constant risk of RV importation into non-endemic areas. RV monitoring, especially during measles and Zika virus outbreaks, requires reliable diagnostic tools. For this study, a TaqMan-based one-step reverse transcription-quantitative PCR (RT-qPCR) assay, with the p90 gene as a novel and so far unexplored target for detection of clade I and II genotypes, was developed and evaluated. Automated nucleic acid extraction was carried out. Performance characteristics of the TaqMan RT-qPCR assay were determined for a RV plasmid standard and RNA extracted from virus-infected cell culture supernatants representing clade I and II genotypes. Diagnostic specificity and sensitivity were validated against other RNA and DNA viruses, relevant for RV diagnostic approaches and for RV-positive clinical samples, respectively. The assay is specific and highly sensitive with a limit of detection as low as five to one copies per reaction or 200 infectious virus particles per ml. The coefficients of variation (CV) were specified as intra- (within one run) and inter- (between different runs) assay variation, and calculated based on the standard deviations for the obtained Ct values of the respective samples. Intra- and inter-assay CV values were low, with a maximum of 3.4% and 2.4%, respectively. The assay was shown to be suitable and specific for the analysis of clinical samples. With p90 as a novel target, the highly sensitive and specific TaqMan assay outlined in this study is suitable for RV diagnosis worldwide.

  16. Low prevalence of HPV detection and genotyping in non-muscle invasive bladder cancer using single-step PCR followed by reverse line blot.

    PubMed

    Pichler, Renate; Borena, Wegene; Schäfer, Georg; Manzl, Claudia; Culig, Zoran; List, Sebastian; Neururer, Sabrina; Von Laer, Dorothee; Heidegger, Isabel; Klocker, Helmut; Horninger, Wolfgang; Steiner, Hannes; Brunner, Andrea

    2015-12-01

    To clarify the role of human papillomavirus (HPV) in non-muscle invasive bladder cancer, HPV-DNA was scrutinized in formalin-fixed, paraffin-embedded (FFPE) bladder cancer tissue using single-step PCR (HPV L1) for HPV detection, followed by reverse line blot (RLB) for genotyping. A total of 186 patients who underwent transurethral resection of the bladder due to primary, non-muscle invasive bladder cancer from 2006 to 2009 were reviewed. A positive control group of 22 cervical tissues with cervical carcinoma was included. Histology confirmed urothelial carcinoma in all patients: primary CIS, pTa, pT1 and pTa + pT1 in 14 (7.5 %), 134 (72 %), 36 (19.4 %) and two (1.1 %) patients, respectively. A total of 119 (63.9 %) of them were classified as low-risk, while 67 (36.1 %) were high-risk cancers. Tumor recurrence and progression (≥pT2) were seen in 79 and 11 patients (mean follow-up 45 months). The presence of HPV-DNA by single-step PCR was detected in four (2.2 %) patients. HPV 16 and HPV 6 were positive in two (1.1 %) and one (0.6 %) patient, respectively In one case, no HPV genotype listed on the RLB assay could be identified. In the control group, the HPV infection rate was 100 %: HPV 16 in 12 (54.6 %) patients, HPV 16/18 in four (18.3 %) patients, HPV 18 in two (9.1 %) patients, HPV 16/45 in one patient (4.5 %), HPV 18/33 in one (4.5 %) patient, HPV 16/33 in one (4.5 %) patient and HPV 33 in one (4.5 %) patient. Our study demonstrates low prevalence of HPV infection in FFPE bladder cancer tissue, arguing against the etiological role of HPV in non-muscle urothelial carcinogenesis.

  17. Comparison of the performance in detection of HPV infections between the high-risk HPV genotyping real time PCR and the PCR-reverse dot blot assays.

    PubMed

    Zhang, Lahong; Dai, Yibei; Chen, Jiahuan; Hong, Liquan; Liu, Yuhua; Ke, Qiang; Chen, Yiwen; Cai, Chengsong; Liu, Xia; Chen, Zhaojun

    2017-08-29

    A new multiplex real-time PCR assay, the high-risk HPV genotyping real time PCR assay (HR HPV RT-PCR), has been developed to detect 15 high-risk HPV types with respective viral loads. In this report, a total of 684 cervical specimens from women diagnosed with vaginitis were assessed by the HR HPV RT-PCR and the PCR reaction and reverse dot blot (PCR-RDB) assays, using a PCR-sequencing method as a reference standard. A total coincidence of 97.7% between the HR HPV RT PCR and the PCR-RDB assays was determined with a Kappa value of 0.953. The HR HPV RT PCR assay had sensitivity, specificity, and concordance rates (accuracy) of 99.7%, 99.7%, and 99.7%, respectively, as confirmed by PCR-sequencing, while the PCR-RDB assay had respective rates of 98.8%, 97.1%, and 98.0%. The overall rate of HPV infection, determined by PCR-sequencing, in women diagnosed with vaginitis was 49.85%, including 36.26% of single infection and 13.6% of multiple infections. The most common infections among the 15 high-risk HPV types in women diagnosed with vaginitis were HPV-52, HPV-16, and HPV-58, with a total detection rate of 10.23%, 7.75%, and 5.85%, respectively. We conclude that the HR HPV RT PCR assay exhibits better clinical performance than the PCR-RDB assay, and is an ideal alternative method for HPV genotyping. In addition, the HR HPV RT PCR assay provides HPV DNA viral loads, and could serve as a quantitative marker in the diagnosis and treatment of single and multiple HPV infections. © 2017 Wiley Periodicals, Inc.

  18. Primary Screening for Cervical Cancer Based on High-Risk Human Papillomavirus (HPV) Detection and HPV 16 and HPV 18 Genotyping, in Comparison to Cytology

    PubMed Central

    Constantinidis, Theocharis; Constantinidis, Theodoros C.

    2015-01-01

    Objectives The objective of the present study is to assess the performance of a high-risk human papillomavirus (HR-HPV) DNA test with individual HPV-16/HPV-18 genotyping as a method for primary cervical cancer screening compared with liquid-based cytology (LBC) in a population of Greek women taking part in routine cervical cancer screening. Methods The study, conducted by the “HEllenic Real life Multicentric cErvical Screening” (HERMES) study group, involved the recruitment of 4,009 women, aged 25–55, who took part in routine cervical screening at nine Gynecology Departments in Greece. At first visit cervical specimens were collected for LBC and HPV testing using the Roche Cobas 4800 system. Women found positive for either cytology or HPV were referred for colposcopy, whereas women negative for both tests will be retested after three years. The study is ongoing and the results of the first screening round are reported herein. Results Valid results for cytology and HPV testing were obtained for 3,993 women. The overall prevalence of HR-HPV was 12.7%, of HPV-16 2.7% and of HPV-18 1.4%. Of those referred for colposcopy, cervical intraepithelial neoplasia grade 2 or worse (CIN2+) was detected in 41 women (1.07%). At the threshold of CIN2+, cytology [atypical squamous cells of undetermined significance (ASC-US) or worse] and HPV testing showed a sensitivity of 53.7% and 100% respectively, without change between age groups. Cytology and HPV testing showed specificity of 96.8% and 90.3% respectively, which was increased in older women (≥30) in comparison to younger ones (25–29). Genotyping for HPV16/18 had similar accuracy to cytology for the detection of CIN2+ (sensitivity: 58.5%; specificity 97.5%) as well as for triage to colposcopy (sensitivity: 58.5% vs 53.7% for cytology). Conclusion HPV testing has much better sensitivity than cytology to identify high-grade cervical lesions with slightly lower specificity. HPV testing with individual HPV-16/HPV-18

  19. Detection of Antibiotic Resistance in Leprosy Using GenoType LepraeDR, a Novel Ready-To-Use Molecular Test

    PubMed Central

    Cambau, Emmanuelle; Chauffour-Nevejans, Aurélie; Tejmar-Kolar, Liana; Matsuoka, Masanori; Jarlier, Vincent

    2012-01-01

    Background Although leprosy is efficiently treated by multidrug therapy, resistance to first-line (dapsone, rifampin) and to second-line drugs (fluoroquinolones) was described worldwide. Since Mycobacterium leprae is not growing in vitro, phenotypic susceptibility testing requires a one year experiment in the mouse model and this is rarely performed. Genetics on antibiotic resistance provide the basis for molecular tests able to detect for antibiotic resistance in leprosy. Methodology/Principal Findings A reverse hybridization DNA strip test was developed as the GenoType LepraeDR test. It includes DNA probes for the wild-type sequence of regions of rpoB, gyrA and folP genes and probes for the prevalent mutations involved in acquired resistance to rifampin, fluoroquinolones and dapsone, respectively. The performances of the GenoType LepraeDR test were evaluated by comparing its results on 120 M. leprae strains, previously studied for resistance by the reference drug in vivo susceptibility method in the mouse footpad and for mutations in the gene regions described above by PCR-sequencing. The results of the test were 100% concordant with those of PCR sequencing and the mouse footpad test for the resistant strains: 16 strains resistant to rifampin, 22 to dapsone and 4 to ofloxacin with mutations (numbering system of the M. leprae genome) in rpoB (10 S456L, 1 S456F, 1 S456M + L458V, 1 H451Y, 1 G432S + H451D, 1 T433I + D441Y and 1 Q438V), in folP1 (8 P55L, 3 P55R, 7 T53I, 3 T53A, 1 T53V) and gyrA (4 A91V), respectively. Concordance was 98.3% for the susceptible strains, two strains showing a mutation at the codon 447 that in fact was not conferring resistance as shown by the in vivo method. Conclusions/Significance The GenoType LepraeDR test is a commercially available test that accurately detects for antibiotic resistance in leprosy cases. The test is easy to perform and could be implemented in endemic countries. PMID:22860144

  20. The use of genetic markers for detecting DNA polymorphism, genotype identification and phylogenetic relationships among banana cultivars.

    PubMed

    Venkatachalam, L; Sreedhar, R V; Bhagyalakshmi, N

    2008-06-01

    Genetic variations and relationships among 21 commercially important banana cultivars of South India were evaluated using 50 decamer RAPD primers and 12 ISSR primers. The primers were selected after a preliminary screening of several such primers for their ability to produce clear and reproducible patterns of multiple bands. The analyses resulted in the amplification of totally 641 bands of 200-3100bp, of which 382 bands were polymorphic, corresponding to nearly 60% genetic diversity. The RAPD and ISSR surveys between pairs of 21 cultivars revealed 60.15% and 56.73% of polymorphic bands, respectively. A strong linear relationship was observed between the Resolving power (Rp) of the primer and its ability to distinguish genotypes. Based on these data, a genetic similarity matrix was established and a dendrogram for each set of primers was developed by UPGMA. The genetic similarity coefficients in RAPD analysis ranged from 0.3177 to 0.7818 and in ISSR analysis from 0.1800 to 0.8462. A fingerprinting key was generated where the presence/absence of specific RAPD/ISSR bands were recorded for each cultivar. The presence of a specific RAPD (OPC-5(800)) band was observed for an endemic cultivar--Nanjanagudu Rasabale (NR). The study resulted in the identification and molecular classification of South Indian banana cultivars of which Robusta and Williams are global and others have either limited geographical distribution or purely endemic to South India. A group of eight cultivars was identified that are highly distinct from one another. The members of this group may be useful for generating 2X and 4X breeding populations for further use in breeding secondary triploid hybrids.

  1. Genotype-based association models of complex diseases to detect gene-gene and gene-environment interactions

    PubMed Central

    Fan, Ruzong; Manga, Prashiela

    2015-01-01

    A central problem in genetic epidemiology is to identify and rank genetic markers involved in a disease. Complex diseases, such as cancer, hypertension, diabetes, are thought to be caused by an interaction of a panel of genetic factors, that can be identified by markers, which modulate environmental factors. Moreover, the effect of each genetic marker may be small. Hence, the association signal may be missed unless a large sample is considered, or a priori biomedical data are used. Recent advances generated a vast variety of a priori information, including linkage maps and information about gene regulatory dependence assembled into curated pathway databases. We propose a genotype-based approach that takes into account linkage disequilibrium (LD) information between genetic markers that are in moderate LD while modeling gene-gene and gene-environment interactions. A major advantage of our method is that the observed genetic information enters a model directly thus eliminating the need to estimate haplotype-phase. Our approach results in an algorithm that is inexpensive computationally and does not suffer from bias induced by haplotype-phase ambiguity. We investigated our model in a series of simulation experiments and demonstrated that the proposed approach results in estimates that are nearly unbiased and have small variability. We applied our method to the analysis of data from a melanoma case-control study and investigated interaction between a set of pigmentation genes and environmental factors defined by age and gender. Furthermore, an application of our method is demonstrated using a study of Alcohol Dependence. PMID:26191336

  2. Simultaneous and sensitive detection of human immunodeficiency virus type 1 (HIV) drug resistant genotypes by multiplex oligonucleotide ligation assay.

    PubMed

    Ellis, Giovanina M; Vlaskin, Tatyana A; Koth, Andrew; Vaz, Louise E; Dross, Sandra E; Beck, Ingrid A; Frenkel, Lisa M

    2013-09-01

    Oligonucleotide ligation assay (OLA) is a highly specific and relatively simple method to detect point mutations encoding HIV-1 drug-resistance, which can detect mutants comprising ≥2-5% of the viral population. Nevirapine (NVP), tenofovir (TDF) and lamivudine (3TC) are antiretroviral (ARV) drugs used worldwide for treatment of HIV infection and prevention of mother-to-child-transmission. Adapting the OLA to detect multiple mutations associated with HIV resistance to these ARV simultaneously would provide an efficient tool to monitor drug resistance in resource-limited settings. Known proportions of mutant and wild-type plasmids were used to optimize a multiplex OLA for detection of K103N, Y181C, K65R, and M184V in HIV subtypes B and C, and V106M and G190A in subtype C. Simultaneous detection of two mutations was impaired if probes annealed to overlapping regions of the viral template, but was sensitive to ≥2-5% when testing codons using non-overlapping probes. PCR products from HIV-subtype B- and C-infected individuals were tested by multiplex-OLA and compared to results of single-codon OLA. Multiplex-OLA detected mutations at codon pairs 103/181, 106/190 and 65/184 reliably when compared to singleplex-OLA in clinical specimens. The multiplex-OLA is sensitive and specific and reduces the cost of screening for NVP, TDF and/or 3TC resistance. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Hybrid microarray based on double biomolecular markers of DNA and carbohydrate for simultaneous genotypic and phenotypic detection of cholera toxin-producing Vibrio cholerae.

    PubMed

    Shin, Hwa Hui; Seo, Jeong Hyun; Kim, Chang Sup; Hwang, Byeong Hee; Cha, Hyung Joon

    2016-05-15

    Life-threatening diarrheal cholera is usually caused by water or food contaminated with cholera toxin-producing Vibrio cholerae. For the prevention and surveillance of cholera, it is crucial to rapidly and precisely detect and identify the etiological causes, such as V. cholerae and/or its toxin. In the present work, we propose the use of a hybrid double biomolecular marker (DBM) microarray containing 16S rRNA-based DNA capture probe to genotypically identify V. cholerae and GM1 pentasaccharide capture probe to phenotypically detect cholera toxin. We employed a simple sample preparation method to directly obtain genomic DNA and secreted cholera toxin as target materials from bacterial cells. By utilizing the constructed DBM microarray and prepared samples, V. cholerae and cholera toxin were detected successfully, selectively, and simultaneously; the DBM microarray was able to analyze the pathogenicity of the identified V. cholerae regardless of whether the bacteria produces toxin. Therefore, our proposed DBM microarray is a new effective platform for identifying bacteria and analyzing bacterial pathogenicity simultaneously.

  4. Global Identification of Disease Associated Genes in Fragile X Cells

    DTIC Science & Technology

    2016-08-01

    AWARD NUMBER: W81XWH-15-1-0204 TITLE: Global Identification of Disease-Associated Genes in Fragile X Cells PRINCIPAL INVESTIGATOR: Wenyi Feng...Global Identification of Disease-Associated Genes in Fragile X Cells 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-15-1-0204 GRANT1171 2389...conflict. We have performed three biological replicate experiments to rigorously test if the Fragile X cell line produces more DSBs than the normal

  5. The Implicitome: A Resource for Rationalizing Gene-Disease Associations

    PubMed Central

    van der Horst, Eelke; Kaliyaperumal, Rajaram; Mina, Eleni; Tatum, Zuotian; Laros, Jeroen F. J.; van Mulligen, Erik M.; Schuemie, Martijn; Aten, Emmelien; Li, Tong Shu; Bruskiewich, Richard; Good, Benjamin M.; Su, Andrew I.; Kors, Jan A.; den Dunnen, Johan; van Ommen, Gert-Jan B.; Roos, Marco; ‘t Hoen, Peter A.C.; Mons, Barend; Schultes, Erik A.

    2016-01-01

    High-throughput experimental methods such as medical sequencing and genome-wide association studies (GWAS) identify increasingly large numbers of potential relations between genetic variants and diseases. Both biological complexity (millions of potential gene-disease associations) and the accelerating rate of data production necessitate computational approaches to prioritize and rationalize potential gene-disease relations. Here, we use concept profile technology to expose from the biomedical literature both explicitly stated gene-disease relations (the explicitome) and a much larger set of implied gene-disease associations (the implicitome). Implicit relations are largely unknown to, or are even unintended by the original authors, but they vastly extend the reach of existing biomedical knowledge for identification and interpretation of gene-disease associations. The implicitome can be used in conjunction with experimental data resources to rationalize both known and novel associations. We demonstrate the usefulness of the implicitome by rationalizing known and novel gene-disease associations, including those from GWAS. To facilitate the re-use of implicit gene-disease associations, we publish our data in compliance with FAIR Data Publishing recommendations [https://www.force11.org/group/fairgroup] using nanopublications. An online tool (http://knowledge.bio) is available to explore established and potential gene-disease associations in the context of other biomedical relations. PMID:26919047

  6. The Implicitome: A Resource for Rationalizing Gene-Disease Associations.

    PubMed

    Hettne, Kristina M; Thompson, Mark; van Haagen, Herman H H B M; van der Horst, Eelke; Kaliyaperumal, Rajaram; Mina, Eleni; Tatum, Zuotian; Laros, Jeroen F J; van Mulligen, Erik M; Schuemie, Martijn; Aten, Emmelien; Li, Tong Shu; Bruskiewich, Richard; Good, Benjamin M; Su, Andrew I; Kors, Jan A; den Dunnen, Johan; van Ommen, Gert-Jan B; Roos, Marco; 't Hoen, Peter A C; Mons, Barend; Schultes, Erik A

    2016-01-01

    High-throughput experimental methods such as medical sequencing and genome-wide association studies (GWAS) identify increasingly large numbers of potential relations between genetic variants and diseases. Both biological complexity (millions of potential gene-disease associations) and the accelerating rate of data production necessitate computational approaches to prioritize and rationalize potential gene-disease relations. Here, we use concept profile technology to expose from the biomedical literature both explicitly stated gene-disease relations (the explicitome) and a much larger set of implied gene-disease associations (the implicitome). Implicit relations are largely unknown to, or are even unintended by the original authors, but they vastly extend the reach of existing biomedical knowledge for identification and interpretation of gene-disease associations. The implicitome can be used in conjunction with experimental data resources to rationalize both known and novel associations. We demonstrate the usefulness of the implicitome by rationalizing known and novel gene-disease associations, including those from GWAS. To facilitate the re-use of implicit gene-disease associations, we publish our data in compliance with FAIR Data Publishing recommendations [https://www.force11.org/group/fairgroup] using nanopublications. An online tool (http://knowledge.bio) is available to explore established and potential gene-disease associations in the context of other biomedical relations.

  7. Evaluation of detection methods for genetically modified traits in genotypes resistant to European corn borer and herbicides.

    PubMed

    Ma, B L; Subedi, K; Evenson, L; Stewart, G

    2005-01-01

    Detection of genetically modified (GM) traits in corn (Zea mays L.) is urgently needed for preservation of genetic identity and marketing GM products. A laboratory study was conducted to evaluate the efficiency, accuracy, and reliability of different analytical methods to detect GM traits in corn. Samples with known fractions of GM concentrations (Bacillus thuringiensis [Bt], Liberty Link [LL] and stacked [Bt/LL] genes) from commercial seed companies and those derived from yellow kernels in a white corn ear, outcrossed by pollen of neighboring Bt hybrid, were tested by lateral flow quick test kits and by enzyme-linked immunosorbent assay (ELISA)-based test strips purchased from different companies. Liberty Link hybrids are resistant to glufosinate (Liberty or Basta) herbicides, whereas Bt corn is developed for the control of European corn borer (Ostrinia nubilalis). Selected samples of GM concentrations were also tested in a commercial laboratory using DNA-based polymerase chain reaction (PCR) test. The results demonstrated that qualitative lateral flow quick tests could detect samples containing 1% or higher Bt and 2% or higher LL concentrations within the recommended time limit of the test. Faint test lines for samples containing 0.5 to 1% Bt or 1 to 2% LL concentrations appeared if samples remained in the test cup overnight. ELISA test strips detected the Bt content semiquantitatively in the range of 0.5 to 2.0%. Grain samples derived from non-Bt corn outcrossed by neighboring Bt pollen had usually lower GM concentrations than commercial GM seed samples. Both ELISA- and DNA-based PCR tests distinguished samples with GM concentrations between 0.1 to 0.5%, but the precision of quantification at this range was very low and results were highly inconsistent.

  8. Design of allele-specific primers and detection of the human ABO genotyping to avoid the pseudopositive problem.

    PubMed

    Yaku, Hidenobu; Yukimasa, Tetsuo; Nakano, Shu-ichi; Sugimoto, Naoki; Oka, Hiroaki

    2008-11-01

    PCR experiments using DNA primers forming mismatch pairing with template lambda DNA at the 3' end were carried out in order to develop allele-specific primers capable of detecting SNP in genomes without generating pseudopositive amplification products, and thus avoiding the so-called pseudopositive problem. Detectable amounts of PCR products were obtained when primers forming a single or two mismatch pairings at the 3' end were used. In particular, 3' terminal A/C or T/C (primer/template) mismatches tended to allow PCR amplification to proceed, resulting in pseudopositive results in many cases. While less PCR product was observed for primers forming three terminal mismatch pairings, target DNA sequences were efficiently amplified by primers forming two mismatch pairings next to the terminal G/C base pairing. These results indicate that selecting a primer having a 3' terminal nucleotide that recognizes the SNP nucleotide and the next two nucleotides that form mismatch pairings with the template sequence can be used as an allele-specific primer that eliminates the pseudopositive problem. Trials with the human ABO genes demonstrated that this primer design is also useful for detecting a single base pair difference in gene sequences with a signal-to-noise ratio of at least 45.

  9. [Evaluation of the genotype MTBDR assay for rapid detection of rifampin and isoniazid resistance in clinical Mycobacterium tuberculosis complex clinical isolates].

    PubMed

    Aslan, Gönül; Tezcan, Seda; Emekdaş, Gürol

    2009-04-01

    Rapid identification of resistant Mycobacterium tuberculosis complex isolates is quite important for the establishment of early and appropriate therapy. The Genotype MTBDR (Hain Lifescience, Nehren, Germany) is a commercially available DNA strip assay designed for the rapid detection of rpoB and katG gene mutations in clinical isolates. This study was conducted to determine the mutation types of phenotypically drug resistant 26 M. tuberculosis complex clinical isolates [15 isoniazid (INH), 1 rifampin (RMP) and 10 INH and RMP resistant] by Genotype MTBDR (G-MTBDR) DNA strip assay and to compare the diagnostic performance of this test. Sixteen of 25 (64%) INH-resistant and 9 of 11 (81.8%) RMP-resistant clinical isolates were correctly identified with the presence of hybridization in mutation probe or lack of hybridization at least by one of the wild type probes, by G-MTBDR assay. Hybridization with mutation probes was detected in only 5 of the RMP resistant isolates. We observed rpoB MUT3 (S531L, Ser-->4Leu) mutation in 4 and rpoB MUT1 (D516V) in one of these isolates. In 56% (14/25) of the INH-resistant isolates, katG T1 (S315T1) hybridization pattern was observed at katG mutation probe. G-MTBDR assay couldn't identify two of the 11 (18.2%) RMP-resistant isolates and one of these iSolates was shown to have a mutation at codon 531 (TCG-GCG) and the other at codon 545 (CTG-->ATG), possibly not associated with resistance, by sequence analysis. In four of the eight (8/25; 32%) INH-resistant isolates not identified by G-MTBDR assay, DNA cycle sequencing revealed different nucleotide changes outside the most common mutation zone. One of these were at codon 293 (GCT-->ACT) in katG, one with dual mutation at 279 (GGC-->ACC) in katG and at 15th C-->T in inhA gene, one at 15th C-->T in inhA gene and one at 279 (GGC-->ACC) in katG gene region. DNA membrane strip assay can be a use ful tool for the rapid detection of resistant M. tuberculosis complex isolates and therefore

  10. Validation and comparison of different end point and real time RT-PCR assays for detection and genotyping of porcine reproductive and respiratory syndrome virus.

    PubMed

    Drigo, Michele; Franzo, Giovanni; Belfanti, Ilaria; Martini, Marco; Mondin, Alessandra; Ceglie, Letizia

    2014-06-01

    The accuracy and rapid diagnosis of PRRSV infection is a major prerequisite for every control and/or eradication strategy. In this study two real time RT-PCR based on different chemistry analysis (TaqMan Probes and SYBR Green) have been developed and validated before comparison to an end point two-step RT-PCR validated previously. All assays were aimed at discrimination between PRRSV genotypes. Furthermore, an exogenous internal control (IC) system had also been implemented in qRT-PCR. A rigorous analytical validation, executed on infected cell cultures and serum, demonstrated good sensitivity, specificity and repeatability. In particular RT-PCR was exceptionally sensitive and could detect a viral titre in the order of a magnitude of 1 copies/μL, 10-fold lower than other qRT-PCR described in this study. Optimal diagnostic performances have been demonstrated analyzing samples retrieved from an experimental infection, with RT-PCR again outperforming real time RT-PCR assays. All tests, showing substantial agreement between them, were able to detect early stages of viraemia (1 DPI) and some animals were classified as positive until the end of the study (76 DPI). Therefore, this supports the assays usefulness in animals with different clinical conditions and in a broad range of epidemiological scenarios. The benefits and disadvantages of different assays were also considered and discussed. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Genotypic Detection of rpoB and katG Gene Mutations Associated with Rifampicin and Isoniazid Resistance in Mycobacterium Tuberculosis Isolates: A Local Scenario (Kelantan).

    PubMed

    Ismail, Nurul-Ain; Ismail, Mohd Fazli; Noor, Siti Suraiya Md; Camalxaman, Siti Nazrina

    2016-01-01

    Drug resistant tuberculosis (DR-TB) remains a public health issue that is of major concern on a global scale. The characterisation of clinical isolates may provide key information regarding the underlying mechanisms of drug resistance, and helps to augment therapeutic options. This study aims to evaluate the frequency of gene mutations associated with Rifampicin (RIF) and Isoniazid (INH) resistance among nine clinical isolates. A total of nine drug resistant Mycobacterium tuberculosis clinical isolates were screened for genetic mutations in rpoB and katusing polymerase chain reaction (PCR) amplification and DNA sequencing. Genotypic analysis was performed to detect the mutations in the sequence of the target genes. Our findings reveal that 80% of the isolates possess mutations at codon 119 (His119Tyr) and 135 (Arg135Trp and Ser135Leu) within the rpoB gene; and 70% possess mutations in the katG gene at codon 238 with amino acid change (Leu238Arg). Findings from this study provide an overview of the current situation of RIF and INH resistance in a hospital Universiti Sains Malaysia (HUSM) located in Kelantan, Malaysia, which could facilitate molecular-based detection methods of drug-resistant strains. Further information regarding the molecular mechanisms involved in resistance in RR-/MDR-TB should be addressed in the near future.

  12. Genotypic Detection of rpoB and katG Gene Mutations Associated with Rifampicin and Isoniazid Resistance in Mycobacterium Tuberculosis Isolates: A Local Scenario (Kelantan)

    PubMed Central

    Ismail, Nurul-Ain; Ismail, Mohd Fazli; Noor, Siti Suraiya MD; Camalxaman, Siti Nazrina

    2016-01-01

    Background Drug resistant tuberculosis (DR-TB) remains a public health issue that is of major concern on a global scale. The characterisation of clinical isolates may provide key information regarding the underlying mechanisms of drug resistance, and helps to augment therapeutic options. This study aims to evaluate the frequency of gene mutations associated with Rifampicin (RIF) and Isoniazid (INH) resistance among nine clinical isolates. Methods A total of nine drug resistant Mycobacterium tuberculosis clinical isolates were screened for genetic mutations in rpoB and katusing polymerase chain reaction (PCR) amplification and DNA sequencing. Genotypic analysis was performed to detect the mutations in the sequence of the target genes. Results Our findings reveal that 80% of the isolates possess mutations at codon 119 (His119Tyr) and 135 (Arg135Trp and Ser135Leu) within the rpoB gene; and 70% possess mutations in the katG gene at codon 238 with amino acid change (Leu238Arg). Conclusion Findings from this study provide an overview of the current situation of RIF and INH resistance in a hospital Universiti Sains Malaysia (HUSM) located in Kelantan, Malaysia, which could facilitate molecular-based detection methods of drug-resistant strains. Further information regarding the molecular mechanisms involved in resistance in RR-/MDR-TB should be addressed in the near future. PMID:27540322

  13. PCR based detection of HPV 16 and 18 genotypes in normal oral mucosa of tobacco users and non-users.

    PubMed

    Pattanshetty, S; Kotrashetti, V S; Nayak, R; Bhat, K; Somannavar, P; Babji, D

    2014-08-01

    There is increasing evidence of a causal association between human papillomavirus (HPV) and oral squamous cell carcinoma (OSCC). Several studies have shown that HPV is associated with increased risk of oral cancer independent of exposure to tobacco and alcohol. The association is valid for HPVs 16 and 18, which generally are considered high risk types, because they have been detected in oral dysplastic lesions and cancers. We determined the baseline prevalence of HPVs 16 and 18 in normal oral mucosa of individuals with and without tobacco habit. PCR was used for DNA collected by oral smears to detect HPV 16/18 DNA in normal oral mucosa of 60 healthy individuals who were assigned to two groups of 30 subjects each. One group had a tobacco habit, the other did not. The tobacco user group comprised individuals who were tobacco chewers only. Sixty-five percent of individuals were positive for HPV 16/18 DNA, but HPV 16/18 positivity was less in individuals with tobacco habit than in those without tobacco habit. No significant association was found between the presence of HPVs and gender, age or duration of chewing habit, or between groups with and without a tobacco habit. We propose that HPVs16 and 18 commonly are present in normal oral mucosa and emphasize the importance of distinguishing clinical, subclinical and latent HPV infections when investigating HPVs and OSCC.

  14. HPV Genotypes in High Grade Cervical Lesions and Invasive Cervical Carcinoma as Detected by Two Commercial DNA Assays, North Carolina, 2001–2006

    PubMed Central

    Hariri, Susan; Steinau, Martin; Rinas, Allen; Gargano, Julia W.; Ludema, Christina; Unger, Elizabeth R.; Carter, Alicia L.; Grant, Kathy L.; Bamberg, Melanie; McDermott, James E.; Markowitz, Lauri E.; Brewer, Noel T.; Smith, Jennifer S.

    2012-01-01

    Background HPV typing using formalin fixed paraffin embedded (FFPE) cervical tissue is used to evaluate HPV vaccine impact, but DNA yield and quality in FFPE specimens can negatively affect test results. This study aimed to evaluate 2 commercial assays for HPV detection and typing using FFPE cervical specimens. Methods Four large North Carolina pathology laboratories provided FFPE specimens from 299 women ages18 and older diagnosed with cervical disease from 2001 to 2006. For each woman, one diagnostic block was selected and unstained serial sections were prepared for DNA typing. Extracts from samples with residual lesion were used to detect and type HPV using parallel and serial testing algorithms with the Linear Array and LiPA HPV genotyping assays. Findings LA and LiPA concordance was 0.61 for detecting any high-risk (HR) and 0.20 for detecting any low-risk (LR) types, with significant differences in marginal proportions for HPV16, 51, 52, and any HR types. Discordant results were most often LiPA-positive, LA-negative. The parallel algorithm yielded the highest prevalence of any HPV type (95.7%). HR type prevalence was similar using parallel (93.1%) and serial (92.1%) approaches. HPV16, 33, and 52 prevalence was slightly lower using the serial algorithm, but the median number of HR types per woman (1) did not differ by algorithm. Using the serial algorithm, HPV DNA was detected in >85% of invasive and >95% of pre-invasive lesions. The most common type was HPV16, followed by 52, 18, 31, 33, and 35; HPV16/18 was detected in 56.5% of specimens. Multiple HPV types were more common in lower grade lesions. Conclusions We developed an efficient algorithm for testing and reporting results of two commercial assays for HPV detection and typing in FFPE specimens, and describe HPV type distribution in pre-invasive and invasive cervical lesions in a state-based sample prior to HPV vaccine introduction. PMID:22479516

  15. Identification of highly related references about gene-disease association.

    PubMed

    Liu, Rey-Long; Shih, Chia-Chun

    2014-08-25

    Curation of gene-disease associations published in literature should be based on careful and frequent survey of the references that are highly related to specific gene-disease associations. Retrieval of the references is thus essential for timely and complete curation. We present a technique CRFref (Conclusive, Rich, and Focused References) that, given a gene-disease pair < g, d>, ranks high those biomedical references that are likely to provide conclusive, rich, and focused results about g and d. Such references are expected to be highly related to the association between g and d. CRFref ranks candidate references based on their scores. To estimate the score of a reference r, CRFref estimates and integrates three measures: degree of conclusiveness, degree of richness, and degree of focus of r with respect to < g, d>. To evaluate CRFref, experiments are conducted on over one hundred thousand references for over one thousand gene-disease pairs. Experimental results show that CRFref performs significantly better than several typical types of baselines in ranking high those references that expert curators select to develop the summaries for specific gene-disease associations. CRFref is a good technique to rank high those references that are highly related to specific gene-disease associations. It can be incorporated into existing search engines to prioritize biomedical references for curators and researchers, as well as those text mining systems that aim at the study of gene-disease associations.

  16. Identify Huntington's disease associated genes based on restricted Boltzmann machine with RNA-seq data.

    PubMed

    Jiang, Xue; Zhang, Han; Duan, Feng; Quan, Xiongwen

    2017-10-11

    Predicting disease-associated genes is helpful for understanding the molecular mechanisms during the disease progression. Since the pathological mechanisms of neurodegenerative diseases are very complex, traditional statistic-based methods are not suitable for identifying key genes related to the disease development. Recent studies have shown that the computational models with deep structure can learn automatically the features of biological data, which is useful for exploring the characteristics of gene expression during the disease progression. In this paper, we propose a deep learning approach based on the restricted Boltzmann machine to analyze the RNA-seq data of Huntington's disease, namely stacked restricted Boltzmann machine (SRBM). According to the SRBM, we also design a novel framework to screen the key genes during the Huntington's disease development. In this work, we assume that the effects of regulatory factors can be captured by the hierarchical structure and narrow hidden layers of the SRBM. First, we select disease-associated factors with different time period datasets according to the differentially activated neurons in hidden layers. Then, we select disease-associated genes according to the changes of the gene energy in SRBM at different time periods. The experimental results demonstrate that SRBM can detect the important information for differential analysis of time series gene expression datasets. The identification accuracy of the disease-associated genes is improved to some extent using the novel framework. Moreover, the prediction precision of disease-associated genes for top ranking genes using SRBM is effectively improved compared with that of the state of the art methods.

  17. Highly Sensitive Detection and Genotyping of HPV by PCR Multiplex and Luminex Technology in a Cohort of Colombian Women with Abnormal Cytology.

    PubMed

    García, Dabeiba A; Cid-Arregui, Angel; Schmitt, Markus; Castillo, Marcos; Briceño, Ignacio; Aristizábal, Fabio A

    2011-01-01

    Cancer of the uterine cervix (CC) is the second most common cancer in women worldwide. In Colombia, CC is the second most frequent cancer among the entire women population and the first among women aged between 15 and 44 years, with an estimated incidence of 24.9 cases/100,000 inhabitants. The main risk factor is infection with one or more high-risk human papillomavirus (HPV) types. The aim of this study was to estimate the genotype-specific prevalence of human papillomavirus (HPV) DNA in patients with cervical pathology using the multiplex PCR and Luminex xMAP technology. In addition, we compared genotyping with Luminex xMAP and with Reverse Line Blot (RLB). A cohort of 160 patients participated in the study, of which 25.6% had no cervical lesions, 35% presented cervical intraepithelial neoplasia of grade I (CIN I), 10% CIN II, 20.6% CIN III and 8.8% CC. The most frequent viral types in all lesion grades were HPV16 and HPV18. Infections by a unique virus were less frequent (19.4%) than multiple infections (80.6%). Single infections were found in 22% of women with no cervical lesions, and in 14.3% of CIN I, 18.7% CIN II, 21.2% CIN III and 28.6% of CC. Multiple infections were observed in 78.0% of cervical samples with negative histopathologic diagnosis, and in 85.7% of CIN I, 81.2% CIN II, 78.8% CIN III and 71.4% CC. All samples analyzed with Luminex xMAP were HPV-positive, while we could detect HPV in only 48.8% of cases with RLB. Of the samples positive by both methods, there was a 67.2% correlation in the viral type(s) detected. In conclusion, Luminex suspension array showed a remarkably higher sensitivity compared with RLB. Multiple infections were unexpectedly common, being HPV types 16 and 18 the most prevalent in all histopathologic grades.

  18. Highly Sensitive Detection and Genotyping of HPV by PCR Multiplex and Luminex Technology in a Cohort of Colombian Women with Abnormal Cytology

    PubMed Central

    García, Dabeiba A; Cid-Arregui, Angel; Schmitt, Markus; Castillo, Marcos; Briceño, Ignacio; Aristizábal, Fabio A

    2011-01-01

    Cancer of the uterine cervix (CC) is the second most common cancer in women worldwide. In Colombia, CC is the second most frequent cancer among the entire women population and the first among women aged between 15 and 44 years, with an estimated incidence of 24.9 cases/100,000 inhabitants. The main risk factor is infection with one or more high-risk human papillomavirus (HPV) types. The aim of this study was to estimate the genotype-specific prevalence of human papillomavirus (HPV) DNA in patients with cervical pathology using the multiplex PCR and Luminex xMAP technology. In addition, we compared genotyping with Luminex xMAP and with Reverse Line Blot (RLB). A cohort of 160 patients participated in the study, of which 25.6% had no cervical lesions, 35% presented cervical intraepithelial neoplasia of grade I (CIN I), 10% CIN II, 20.6% CIN III and 8.8% CC. The most frequent viral types in all lesion grades were HPV16 and HPV18. Infections by a unique virus were less frequent (19.4%) than multiple infections (80.6%). Single infections were found in 22% of women with no cervical lesions, and in 14.3% of CIN I, 18.7% CIN II, 21.2% CIN III and 28.6% of CC. Multiple infections were observed in 78.0% of cervical samples with negative histopathologic diagnosis, and in 85.7% of CIN I, 81.2% CIN II, 78.8% CIN III and 71.4% CC. All samples analyzed with Luminex xMAP were HPV-positive, while we could detect HPV in only 48.8% of cases with RLB. Of the samples positive by both methods, there was a 67.2% correlation in the viral type(s) detected. In conclusion, Luminex suspension array showed a remarkably higher sensitivity compared with RLB. Multiple infections were unexpectedly common, being HPV types 16 and 18 the most prevalent in all histopathologic grades. PMID:21769306

  19. Three-year risk of cervical precancer and cancer after the detection of low-risk human papillomavirus genotypes targeted by a commercial test.

    PubMed

    Castle, Philip E; Hunt, William C; Langsfeld, Erika; Wheeler, Cosette M

    2014-01-01

    To investigate the risk of cervical precancer and cancer associated with detection of human papillomavirus (HPV) 6, 11, and 42. We used data from the New Mexico Human Papillomavirus Pap Registry. A stratified sample of 59,644 residual cervical cytology specimens from a population of 379,000 underwent HPV genotyping. We measured the 3-year cumulative incidence of cervical intraepithelial neoplasia grade 2 or more severe (CIN 2+) and grade 3 or more severe (CIN 3+) after detection of single HPV 6, 11, or 42 infections or single or multiple infections of HPV 6, 11, or 42 ("HPV 6, 11, 42, or combinations"; n=581). The overall prevalence of a single infection of HPV 6, 11, or 42 was 0.8% (95% confidence interval [CI] 0.7-0.9%). The 3-year risks of CIN 2+ and CIN 3+ after HPV 6, 11, 42, or combinations infections (n=581) were 0.4% (CI 0.1-0.7%) for CIN 2+ and 0.0% for CIN 3+ (nota bene, no CI was calculable because no events occurred), respectively. By comparison, the 3-year risks of CIN 2+ and CIN 3+ after a negative HPV result (n=27,522) were 0.2% (95% CI 0.1-0.2%) and 0.1% (95% CI 0.0-0.1%), respectively. Detection of HPV 6, 11, 42, or combinations in the absence of high-risk HPV types does not identify women at increased 3-year risk for cervical precancer. Testing for HPV 6, 11, 42, or combinations of those types should be discontinued because it has no proven benefit to patients. II.

  20. Three-Year Risk of Cervical Precancer and Cancer After the Detection of Low-Risk Human Papillomavirus Genotypes Targeted by a Commercial Test

    PubMed Central

    Castle, Philip E.; Hunt, William C.; Langsfeld, Erika; Wheeler, Cosette M.

    2014-01-01

    Objective To investigate the risk of cervical precancer and cancer associated with detection of human papillomavirus (HPV) 6, 11, and 42. Methods We used data from the New Mexico HPV Pap Registry. A stratified sample of 59,644 residual cervical cytology specimens from a population of 379,000 underwent HPV genotyping. We measured the 3-year cumulative incidence of cervical intraepithelial neoplasia grade 2 or more severe (CIN2+) and grade 3 or more severe (CIN3+) after detection of single HPV6, 11, or 42 infections or single or multiple infections by of HPV6, 11, or 42 (“HPV6, 11, 42, or combinations”)(n=581). Results The overall prevalence of a single infection of HPV6, 11, or 42 was 0.8% (95% confidence interval [95%CI]: 0.7%, 0.9%). The 3-year risks of CIN2+ and CIN3+ after HPV6, 11, 42, or combinations infections (n = 581) were 0.4% (CI: 0.1%, 0.7%) for CIN2+ and 0.0% for CIN3+ (nota bene, no confidence interval was calculable because no events occurred), respectively. By comparison, the 3-year risks of CIN2+ and CIN3+ after a negative HPV result (n = 27,522) were 0.2% (95%CI: 0.1%, 0.2%) and 0.1% (95%CI: 0.0%, 0.1%), respectively. Conclusion Detection of HPV6, 11, 42, or combinations in the absence of high risk HPV types does not identify women at increased 3-year risk for cervical precancer. Testing for HPV6, 11, 42, or combinations of those types should be discontinued as it has no proven benefit to patients. PMID:24463663

  1. Genotypic and phenotypic detection of capsular polysaccharide and biofilm formation in Staphylococcus aureus isolated from bovine milk collected from Brazilian dairy farms.

    PubMed

    Salimena, Alessandra P S; Lange, Carla C; Camussone, Cecilia; Signorini, Marcelo; Calvinho, Luis F; Brito, Maria A V P; Borges, Cristiano A V; Guimarães, Alessandro S; Ribeiro, João B; Mendonça, Letícia C; Piccoli, Roberta H

    2016-12-01

    Staphylococcus aureus is a pathogen that frequently causes mastitis in bovine herds worldwide. This pathogen produces several virulence factors, including cell-associated adhesins, toxic and cytolytic exoproteins, and capsular polysaccharides. The aim of the present study was to test for the presence of genes involved in capsular polysaccharide production and biofilm formation in S. aureus isolated from bovine mastitis samples collected from 119 dairy herds located in three different Brazilian regions, as well as to assay the production of capsular polysaccharides and biofilm, in vitro. The detection of the cap, icaAD, and bap genes was performed using PCR. The detection and quantification of capsular polysaccharide production was performed using ELISA assays. The ability of the isolates to form a biofilm was examined using the polystyrene surface of microtiter plates. All 159 S. aureus isolates investigated harboured the cap gene: 80 % carried the cap5 gene and 20 % carried the cap8 gene. Sixty-nine percent of the isolates expressed capsular polysaccharide (CP) in vitro, 58 % expressed CP5 and 11 % expressed CP8. All of the isolates harboured the icaA and icaD genes, and 95.6 % of the isolates carried the bap gene. Of the 159 isolates analysed, 97.5 % were biofilm producers. A significant association between the capsular genotype and phenotype and the amount of biofilm formation was detected: cap5/CP5 isolates tended to form more biofilm and to produce a thinner CP layer than cap8/CP8 isolates. The results indicate a high potential for pathogenicity among S. aureus isolated from bovine milk collected from three different regions in Brazil.

  2. Clinical significance of residual viremia detected by two real-time PCR assays for response-guided therapy of HCV genotype 1 infection.

    PubMed

    Vermehren, Johannes; Aghemo, Alessio; Falconer, Karolin; Susser, Simone; Lunghi, Giovanna; Zeuzem, Stefan; Colombo, Massimo; Weiland, Ola; Sarrazin, Christoph

    2014-05-01

    The duration of current standard dual and protease inhibitor-based triple therapies for chronic hepatitis C is determined by assessment of early viral kinetics. Little is known about differences between HCV RNA assays for the use in response guided therapy. HCV RNA was assessed by two widely used real-time PCR-based assays, Cobas Ampliprep/Cobas TaqMan (CAP), and Real-Time HCV (ART) in 903 samples of hepatitis C genotype 1 patients treated with dual (n=169) or telaprevir-based triple therapy (n=164) in three European countries. Overall, CAP and ART were in excellent agreement for the determination of HCV-RNA concentrations (mean difference 0.21 log10 IU/ml). For treatment-naïve patients treated with peginterferon-alfa and ribavirin a lower rate of undetectable HCV-RNA at week 4 (RVR) was observed for ART (9%) vs. CAP (16%). Although 11/27 (41%) of patients with shortened treatment (24weeks) had detectable HCV-RNA <12IU/ml by ART at week 4 none of these patients experienced virologic relapse after treatment cessation. In patients who received triple therapy, 67% and 37% had undetectable HCV-RNA at week 4 by CAP and ART, respectively. However, 18/31 (58%) eligible patients for shortened treatment based on CAP had detectable HCV-RNA by ART at week 4. Again, relapse was not observed in these patients. Lower rates of undetectable HCV-RNA at week 4 were observed with ART compared to CAP in patients treated with dual and triple therapies. For ART, detectable <12IU/ml HCV-RNA levels at week 4 may be sufficient as part of the criteria used for selecting patients who receive a shortened treatment regimen. Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  3. Performance of a Cartridge-Based Assay for Detection of Clinically Significant Human Papillomavirus (HPV) Infection: Lessons from VALGENT (Validation of HPV Genotyping Tests)

    PubMed Central

    Geraets, Daan; Cuzick, Jack; Cadman, Louise; Moore, Catherine; Vanden Broeck, Davy; Padalko, Elisaveta; Quint, Wim; Arbyn, Marc

    2016-01-01

    The Validation of Human Papillomavirus (HPV) Genotyping Tests (VALGENT) studies offer an opportunity to clinically validate HPV assays for use in primary screening for cervical cancer and also provide a framework for the comparison of analytical and type-specific performance. Through VALGENT, we assessed the performance of the cartridge-based Xpert HPV assay (Xpert HPV), which detects 14 high-risk (HR) types and resolves HPV16 and HPV18/45. Samples from women attending the United Kingdom cervical screening program enriched with cytologically abnormal samples were collated. All had been previously tested by a clinically validated standard comparator test (SCT), the GP5+/6+ enzyme immunoassay (EIA). The clinical sensitivity and specificity of the Xpert HPV for the detection of cervical intraepithelial neoplasia grade 2 or higher (CIN2+) and CIN3+ relative to those of the SCT were assessed as were the inter- and intralaboratory reproducibilities according to international criteria for test validation. Type concordance for HPV16 and HPV18/45 between the Xpert HPV and the SCT was also analyzed. The Xpert HPV detected 94% of CIN2+ and 98% of CIN3+ lesions among all screened women and 90% of CIN2+ and 96% of CIN3+ lesions in women 30 years and older. The specificity for CIN1 or less (≤CIN1) was 83% (95% confidence interval [CI], 80 to 85%) in all women and 88% (95% CI, 86 to 91%) in women 30 years and older. Inter- and intralaboratory agreements for the Xpert HPV were 98% and 97%, respectively. The kappa agreements for HPV16 and HPV18/45 between the clinically validated reference test (GP5+/6+ LMNX) and the Xpert HPV were 0.92 and 0.91, respectively. The clinical performance and reproducibility of the Xpert HPV are comparable to those of well-established HPV assays and fulfill the criteria for use in primary cervical cancer screening. PMID:27385707

  4. A newly developed real-time PCR assay for detection and quantification of Fusarium oxysporum and its use in compatible and incompatible interactions with grafted melon genotypes.

    PubMed

    Haegi, Anita; Catalano, Valentina; Luongo, Laura; Vitale, Salvatore; Scotton, Michele; Ficcadenti, Nadia; Belisario, Alessandra

    2013-08-01

    A reliable and species-specific real-time quantitative polymerase chain reaction (qPCR) assay was developed for detection of the complex soilborne anamorphic fungus Fusarium oxysporum. The new primer pair, designed on the translation elongation factor 1-α gene with an amplicon of 142 bp, was highly specific to F. oxysporum without cross reactions with other Fusarium spp. The protocol was applied to grafted melon plants for the detection and quantification of F. oxysporum f. sp. melonis, a devastating pathogen of this cucurbit. Grafting technologies are widely used in melon to confer resistance against new virulent races of F. oxysporum f. sp. melonis, while maintaining the properties of valuable commercial varieties. However, the effects on the vascular pathogen colonization have not been fully investigated. Analyses were performed on 'Charentais-T' (susceptible) and 'Nad-1' (resistant) melon cultivars, both used either as rootstock and scion, and inoculated with F. oxysporum f. sp. melonis race 1 and race 1,2. Pathogen development was compared using qPCR and isolations from stem tissues. Early asymptomatic melon infections were detected with a quantification limit of 1 pg of fungal DNA. The qPCR protocol clearly showed that fungal development was highly affected by host-pathogen interaction (compatible or incompatible) and time (days postinoculation). The principal significant effect (P ≤ 0.01) on fungal development was due to the melon genotype used as rootstock, and this effect had a significant interaction with time and F. oxysporum f. sp. melonis race. In particular, the amount of race 1,2 DNA was significantly higher compared with that estimated for race 1 in the incompatible interaction at 18 days postinoculation. The two fungal races were always present in both the rootstock and scion of grafted plants in either the compatible or incompatible interaction.

  5. Detection and quantification of major toxigenic Microcystis genotypes in Moo-Tan reservoir and associated water treatment plant.

    PubMed

    Yen, Hung-Kai; Lin, Tsair-Fuh; Tseng, I-Cheng

    2012-02-01

    Two molecular methods, denaturing gradient gel electrophoresis (DGGE) and quantitative real-time polymerase chain reaction (qPCR) with the Universal ProbeLibrary (UPL) probe, were developed and used for the characterization and quantification of several microcystin producers in Moo-Tan Reservoir (MTR), Taiwan and its associated water treatment plant (Shih-Men Water Treatment Plant, SMWTP). Internal transcribed spacer (ITS) sequence, a highly diversified region between the 16S rRNA and 23S rRNA genes, was used to further identify the isolated strains from MTR and also used in DGGE for the detection of the specific DNA fragments and biomarkers for 11 strains observed in MTR. These ITS-DGGE biomarkers were successfully applied to monitor the community changes of potential toxigenic Microcystis sp. over a period of five years. Two highly specific primers were combined with UPL probes to measure microcystins synthesis gene (mcyB) and phycocyanin intergenic spacer region (cpcB) concentrations in water samples. The copy concentrations of UPL-mcyB and UPL-cpcB correlated well with MC-RR concentrations/water temperature and Microcystis sp. cell numbers in the water samples, respectively. For SMWTP, toxin concentrations were low, but the DGGE bands clearly demonstrated the presence of potential microcystin producers in both water treatment plants and finished water samples. It was demonstrated that toxigenic Microcystis sp. may penetrate through the treatment processes and pose a potential risk to human health in the drinking water systems.

  6. Detection and genotype analysis of Giardia duodenalis from asymptomatic Hungarian inhabitants and comparative findings in three distinct locations.

    PubMed

    Plutzer, Judit; Törökné, Andrea; Szénási, Zsuzsanna; Kucsera, István; Farkas, Kata; Karanis, Panagiotis

    2014-03-01

    The transmission route of giardiasis not yet understood and why some infected individuals remain asymptomatic while others become quite ill. The drinking water quality is supposedly responsible for the prevalence of asymptomatic Giardia duodenalis infections in different areas, therefore asymptomatic giardiasis has been investigated in three water supply areas of Hungary: three hundred stool samples from inhabitants of Budapest, Füzér and Mátrafüred were examined by immunological and molecular methods for the presence of G. duodenalis infections. Individuals were asked to fill out a validated questionnaire at the time of stool collection and the interview covered demographic data, family life, education and travel history.In Budapest and in Mátrafüred in one stool sample G. duodenalis Assemblage A, whereas in Füzér once G. duodenalis Assemblage A, once Assemblage B and twice mixed infection were detected. We found higher prevalence rate of 4% of G. duodenalis infections of asymptomatic people in the village Füzér, where the removal of the Giardia cysts of the drinking water treatment plant was not effective. This study throws a light the need to look into the possibility of other risks of Giardia infections such as water transmission routes. To our knowledge, this is the first study evaluating the prevalence of G. duodenalis infections in asymptomatic persons in Hungary.

  7. G8 rotaviruses with conserved genotype constellations detected in Malawi over 10 years (1997–2007) display frequent gene reassortment among strains co-circulating in humans

    PubMed Central

    Nakagomi, Toyoko; Doan, Yen Hai; Dove, Winifred; Ngwira, Bagrey; Iturriza-Gómara, Miren; Nakagomi, Osamu

    2013-01-01

    Rotavirus A, the most common cause of severe diarrhoea in children worldwide, occurs in five major VP7 (G) and VP4 (P) genotype combinations, comprising G1P[8], G2P[4], G3P[8], G4P[8] and G9P[8]. However, G8, a common bovine rotavirus genotype, has been reported frequently among children in African countries. Surveillance of rotavirus gastroenteritis conducted in a sentinel hospital in Blantyre, Malawi between 1997 and 2007 provided a rare opportunity to examine the whole genotype constellation of G8 strains and their evolution over time. A sample of 27 (9.0 %) of 299 G8 strains was selected to represent each surveillance year and a range of P genotypes, which shifted in predominance from P[6] to P[4] and P[8] during the study period. Following cell culture adaptation, whole genome sequencing demonstrated that the genetic background of 26 strains possessed the DS-1 genotype constellation. A single G8P[6] strain was a reassortant in which both NSP2 and NSP5 genes from strains with the Wa genotype constellation had been inserted into a strain with the DS-1 genotype background. Phylogenetic analysis suggested frequent reassortment among co-circulating strains with the DS-1 genotype constellation. Little evidence was identified to suggest the introduction of contemporary bovine rotavirus genes into any of the 27 G8 strains examined. In conclusion, Malawian G8 strains are closely related to other human strains with the DS-1 genotype constellation. They have evolved over the last decade through genetic reassortment with other human rotaviruses, changing their VP4 genotypes while maintaining a conserved genotype constellation for the remaining structural and non-structural proteins. PMID:23407423

  8. A Pragmatic Test for Detecting Association between a Dichotomous Trait and the Genotypes of Affected Families, Controls and Independent Cases

    PubMed Central

    Wang, Meng; Stewart, William C. L.

    2017-01-01

    The efficient analysis of hybrid designs [e.g., affected families, controls, and (optionally) independent cases] is attractive because it should have increased power to detect associations between genetic variants and disease. However, the computational complexity of such an analysis is not trivial, especially when the data contain pedigrees of arbitrary size and structure. To address this concern, we developed a pragmatic test of association that summarizes all of the available evidence in certain hybrid designs, irrespective of pedigree size or structure. Under the null hypothesis of no association, our proposed test statistic (POPFAM+) is the quadratic form of two correlated tests: a population-based test (e.g., wQLS), and a family-based test (e.g., PDT). We use the parametric bootstrap in conjunction with an estimate of the correlation to compute p-values, and we illustrate the potential for increased power when (1) the heritability of the trait is high; and, (2) the marker-specific association is driven by the over-representation of risk alleles in cases, and by the preferential transmission of risk alleles from heterozygous parents to their affected offspring. Based on simulation, we show that type I error is controlled, and that POPFAM+ is more powerful than wQLS or PDT alone. In a real data application, we used POPFAM+ to analyze 43 genes of a hybrid epilepsy study containing 85 affected families, 80 independent cases, 234 controls, and 118 reference samples from the International HapMap Project. The results of our analysis identified a promising epilepsy candidate gene for follow-up sequencing: malic enzyme 2 (ME2; min p < 0.0084). PMID:28536599

  9. Correlation of agar dilution and VITEK2 system for detection of resistance to macrolides, lincosamides and pristinamycin among Staphylococcus aureus and Staphylococcus epidermidis: association with genotypes.

    PubMed

    Bémer, P; Juvin, M-E; Corvec, S; Ros, A; Drugeon, H

    2005-08-01

    The performance of the VITEK2 system was evaluated against the agar dilution reference procedure for testing susceptibility of Staphylococcus aureus and Staphylococcus epidermidis to macrolides, lincosamides and streptogramins (MLS). Eighty clinical isolates were selected according to their resistance phenotype and genotype. Results for erythromycin and clindamycin showed 100% agreement; results for lincomycin showed agreement of 78%, with one very major error and 17 minor errors; and results for pristinamycin showed agreement of 46%, with one major error and 43 minor errors. Most isolates resistant to lincomycin and streptogramin A (L SgAr phenotype) were falsely susceptible to lincomycin, and intermediately-resistant or resistant to pristinamycin, with the VITEK2 system. No resistance gene was detected. Most (80%) isolates resistant constitutively to MLS (MLS(r)BC phenotype) were falsely intermediately-resistant to pristinamycin with the VITEK2 system. The erm(A) gene was more common than erm(C) in MLS(r)BC strains. Resistance to pristinamycin alone (SgA SgB PTr phenotype), or associated with either lincomycin resistance (L SgA SgB PTr phenotype) or constitutive MLS(B) resistance (MLS(BC) SgA PTr phenotype), was well-characterised without discordant results. Resistance to pristinamycin was always associated with resistance to streptogramin A, encoded by the vga(A), vga(B), vgb(A) and vat(A) genes in association with the erm(A) or erm(C) genes.

  10. Detection of porcine circovirus genotypes 2a and 2b in aborted foetuses from infected swine herds in the State of São Paulo, Brazil.

    PubMed

    de Castro, Alessandra M M G; Cruz, Taís F; Salgado, Vanessa R; Kanashiro, Tatiana M; Ferrari, Karen L; Araujo, João P; Brandão, Paulo E; Richtzenhain, Leonardo J

    2012-05-03

    Porcine circovirus type 2 (PCV2) has been associated with several disease complexes, including reproductive failure. The aim of this study was to identify the subtypes of PCV2 that are associated with reproductive failure in pigs from the State of São Paulo, Brazil and to investigate co-infections with other infectious organisms. Samples of 168 aborted foetuses or mummified foetuses from five farrow-to-finish swine farms known to be infected with PCV2 and located in the State of São Paulo were tested for PCV2 by polymerase chain reaction (PCR). Positive samples were additionally tested for porcine parvovirus (PPV), Leptospira spp. and Brucella spp. by PCR. PCV2 was detected in 18 of the samples (10.7%). PPV, Brucella spp. and Leptospira spp were found in 2, 10 and 0 cases, respectively. Eleven PCV2 strains were sequenced and determined to be either genotype 2a (n = 1) or 2b (n = 10). The findings indicate that the frequency of PCV2 infections in aborted porcine foetuses from the State of São Paulo is rather low (10.7%) and that co-infection with other pathogens is common and may be involved in PCV2 associated reproductive failure. No repeatable, characteristic amino acid motifs for regions of the PCV2 capsid protein seemed to be associated with abortion in sows.

  11. Detection of the Klebsiella pneumoniae carbapenemase (KPC) in K. pneumoniae Isolated from the Clinical Samples by the Phenotypic and Genotypic Methods

    PubMed Central

    Bina, Masoume; Pournajaf, Abazar; Mirkalantari, Shiva; Talebi, Malihe; Irajian, Gholamreza

    2015-01-01

    Background and Objective: The production of carbapenemases especially Klebsiella pneumoniae carbapenemase (KPC) is the most important mechanism of enzymatic resistance in isolated Enterobacteriaceae such as K. pneumoniae . The purpose of this study was detected of the carbapenemase producer K. pneumoniae strains with phenotypic and genotypic methods. Method: Out of 800 strains, 270 K. pneumoniae strains (33.7%), were obtained. Antibiotic susceptibility test was performed by disk diffusion method in accordance with CLSI guidelines. Carbapenem resistant strains were identified by the Modified Hodge Test based on CLSI instruction and PCR for surveying the presence of bla -KPC gene. Results: A total 270 K. pneumoniae strains were collected. Antibiotic susceptibility test results showed the highest and lowest resistance was related to piperacillin (60.6%) and carbapenems (14.6%) respectively. 80.5% (33 of 41) isolates were positive by MHT, but all of them (100%) were negative for amplification of the bla -KPC gene in the PCR method. Conclusion: The MHT was an appropriate method for approving carbapenemase production. Moreover, a laboratory could accept the carbapenemase production with PCR method for the bla-KPC gene, which has the additional profit of validating which KPC is present. PMID:26351485

  12. Identification of hepatitis C virus genotype 3 by a commercial assay challenged by natural polymorphisms detected in Spain from patients with diverse origins.

    PubMed

    Saludes, Verónica; Quer, Josep; Gregori, Josep; Bascuñana, Elisabet; García-Cehic, Damir; Esteban, Juan Ignacio; Ausina, Vicente; Martró, Elisa

    2016-05-01

    Hepatitis C virus (HCV) genotyping is crucial in clinical practise for determining the type and duration of antiviral therapy. Between 2009 and 2014, 24 (7.95%) of all HCV genotype 3 (HCV-3) cases obtained indeterminate results via the RealTime HCV Genotype II assay (Abbott) at a tertiary care center in Spain. HCV-3 is the second most common genotype worldwide. Moreover, it has been associated with a higher risk of liver disease progression and a lower response to the latest antivirals. Given the clinical significance of accurately identifying HCV-3, we aimed to characterize the genetic diversity of the HCV 5' untranslated region (5' UTR), the target of genotyping assays, by ultradeep pyrosequencing (UDPS). For the 24 indeterminate samples, the 5' UTR-core was amplified and subjected to UDPS with the 454/GS-Junior platform (Roche). The genotype/subtype of each identified haplotype was assigned by phylogenetic analysis. For comparison, three additional samples correctly identified as HCV-3 by the real-time assay were also analyzed. HCV genotyping based on 5' UTR-core UDPS was in agreement with NS5B Sanger sequencing in all cases, confirming the absence of mixed infections and recombination events. The generated 5' UTR sequences proved the presence of one to three polymorphisms at the probe-binding site of the Abbott assay, thereby differentiating indeterminate from correctly genotyped HCV-3 samples. The observed naturally occurring polymorphisms provide insight into regional differences observed with genotype 3, their impact on genotyping assay performance, and potential improvement and designing options. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Recombinant expression of the alternate reading frame protein (ARFP) of hepatitis C virus genotype 4a (HCV-4a) and detection of ARFP and anti-ARFP antibodies in HCV-infected patients.

    PubMed

    Shehat, Michael G; Bahey-El-Din, Mohammed; Kassem, Mervat A; Farghaly, Faten A; Abdul-Rahman, Medhat H; Fanaki, Nourhan H

    2015-08-01

    HCV is a single-stranded RNA virus with a single open reading frame (ORF) that is translated into a polyprotein that is then processed to form 10 viral proteins. An additional eleventh viral protein, the alternative reading frame protein (ARFP), was discovered relatively recently. This protein results from a translational frameshift in the core region during the expression of the viral proteins. Recombinant expression of different forms of ARFP was previously done for HCV genotypes 1 and 2, and more recently, genotype 3. However, none of the previous studies addressed the expression of ARFP of HCV genotype 4a, which is responsible for 80 % of HCV infections in the Middle East and Africa. Moreover, the direct detection of the ARFP antigen in HCV-infected patients was never studied before for any HCV genotype. In the present study, recombinant ARFP derived from HCV genotype 4a was successfully expressed in E. coli and purified using metal affinity chromatography. The recombinant ARFP protein and anti-ARFP antibodies were used for detection of ARFP antigen in patients' sera, employing competitive enzyme-linked immunosorbent assay (ELISA) procedures. Furthermore, the recombinant antigen was also used to detect and quantify anti-ARFP antibodies in HCV-infected Egyptian patients at different stages of pegylated interferon/ribavirin therapy, using an ELISA assay. The ARFP antigen was detectable in 69.4 % of RNA-positive sera, indicating that ARFP antigen is produced during the natural course of HCV infection. In addition, significant levels of anti-ARFP antibodies were present in 41 % of the serum samples tested. The important diagnostic value of the recombinant ARFP antigen was also demonstrated.

  14. The clinical significance of HCV core antigen detection during Telaprevir/Peg-Interferon/Ribavirin therapy in patients with HCV 1 genotype infection.

    PubMed

    Garbuglia, Anna Rosa; Lionetti, Raffaella; Lapa, Daniele; Taibi, Chiara; Visco-Comandini, Ubaldo; Montalbano, Marzia; D'Offizi, Gianpiero; Castiglione, Filippo; Capobianchi, Maria Rosaria; Paci, Paola

    2015-08-01

    Direct-acting antiviral drugs (DAA) regimen improve the SVR rate. However, adverse effects often lead to therapy interruption. This underlines the importance to find some predictive parameters of response in order to consider the possibility of a shorter time of antiviral treatment in the appearance of adverse effects without affecting the success of the therapy. We aimed to examine the HCVAg kinetics in the early phase of treatment and its predictive value of SVR in patients undergoing TPV/Peg-IFN/RBV treatment. Twenty-three patients infected by HCV genotype 1 (1a n=11; 1b n=12) were included in this prospective study. At baseline the median Log of HCVAg concentration in RVR and EVR patients were 3.15 fmol/L and 3.45 fmol/L, respectively with no significant differences. The baseline median HCV-RNA to HCVAg ratio was 233.77, this ratio was significantly lower when measured on day 1 (27.52) and on day 6 (24.84) (p<0.001). The two-tailed Fisher's exact test indicated that the SVR response is statistically significantly different in patients with detected HCVAg at week1 compared to patients with no detectable HCVAg (p=0.05). The sensitivity, specificity, and negative and positive predictive values (NPV, PPV) were 53.8, 87.5, 53.8 and 87.5%, respectively. The area under the ROC curve was 0.71 at day T6, the best cut-off of 3 fmol/L when evaluated with the HCVAg plasma concentration at day T6. Undetectable HCVAg in the early phase of TPV/Peg-IFN/RBV treatment could represent an important parameter for predicting SVR. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Detection of Serum Antibodies to Hepatitis E Virus Based on HEV Genotype 3 ORF2 Capsid Protein Expressed in Nicotiana benthamiana

    PubMed Central

    Mazalovska, Milena; Varadinov, Nikola; Koynarski, Tsvetoslav; Minkov, Ivan; Teoharov, Pavel; Lomonossoff, George P.

    2017-01-01

    Background Hepatitis E virus (HEV) causes epidemics in developing countries and is primarily transmitted through the fecal-oral route. There have been recent reports on the zoonotic spread of the virus, and several animal species, primarily pigs, have been recognized as reservoirs of HEV. Because of its possible spread, there is an urgent need of a method for the cost-effective production of HEV proteins that can be used as diagnostic antigens for the serological detection of anti-HEV antibodies. Methods The HEV open reading frame (ORF)2 protein was purified from plant tissue by using immobilized metal-anion chromatography (IMAC). The recombinant protein was used to develop an in-house ELISA for testing anti-HEV antibodies in both human and swine sera. Thirty-six serum samples collected from patients with serologically proven HEV infection with commercial kits were tested for anti-HEV IgG antibodies by using the plant-expressed protein. Forty-five serum samples collected from apparently healthy pigs in Bulgarian farms were also tested. Results We confirmed the transient expression and purification of a truncated version of the HEV genotype 3 capsid protein in Nicotiana benthamiana and its usefulness as a diagnostic antigen. ELISA showed the presence of anti-HEV IgG antibodies in 29 of the 36 human samples. The in-house ELISA showed anti-HEV IgG antibodies in 34 of the 45 pigs. Conclusions We describe a method for the production of HEV ORF2 protein in N. benthamiana and the usefulness of this protein for the serological detection of anti-HEV antibodies in both humans and swine. PMID:28445010

  16. Molecular detection and genotyping of enteroviruses from CSF samples of patients with suspected sepsis-like illness and/or aseptic meningitis from 2012 to 2015 in West Bank, Palestine

    PubMed Central

    Dumaidi, Kamal; Al-Jawabreh, Amer

    2017-01-01

    Background Human enteroviruses (HEVs) are the most frequently reported cause of aseptic meningitis with or without CSF pleocytosis in childhood. Rapid detection and genotype of HEVs is essential to determine the causative agent and variant causing sepsis-like illness and/or aseptic meningitis. Aim To investigate the molecular epidemiology of enteroviruses (EVs) among patients with sepsis-like illness and/or aseptic meningitis admitted to three major hospitals in West Bank, Palestine from 2012 to 2015. Methods During the study period, 356 CSF samples were collected from patients with sepsis-like illness and/or aseptic meningitis. Two RT-nested PCR assays targeting a partial part of 5'UTR for direct diagnosis and the VP1 region for genotyping by sequence analysis of the viral genome were used. Results HEV RNA was detected in 66 of 356 (18.5%) of CSF samples. Age distribution showed that 64% (42/66) were infants (<1 year), 18% were children between 1 and 5 years old, 12% were children between 5 and 10 years old, and 6% were more than 10 years old. Of the 66 EV cases, 12 were successfully genotyped. Five different EV genotypes were identified. All of them belonged to HEV-B species. The study showed that echovirus 6 genotype accounted for 42% of the sequenced cases. The HEV infections in the present study tended to show slight seasonal pattern with more cases occurring during spring and summer, yet still significant numbers were also reported in fall and winter seasons. Conclusion HEV was isolated from a significant number of children with sepsis-like illness and/or aseptic meningitis. In addition, the molecular method utilized for direct diagnosis and genotyping of HEV from CSF revealed that more than one HEV type circulated in the West Bank, Palestine during the study period. PMID:28225788

  17. Molecular detection and genotyping of enteroviruses from CSF samples of patients with suspected sepsis-like illness and/or aseptic meningitis from 2012 to 2015 in West Bank, Palestine.

    PubMed

    Dumaidi, Kamal; Al-Jawabreh, Amer

    2017-01-01

    Human enteroviruses (HEVs) are the most frequently reported cause of aseptic meningitis with or without CSF pleocytosis in childhood. Rapid detection and genotype of HEVs is essential to determine the causative agent and variant causing sepsis-like illness and/or aseptic meningitis. To investigate the molecular epidemiology of enteroviruses (EVs) among patients with sepsis-like illness and/or aseptic meningitis admitted to three major hospitals in West Bank, Palestine from 2012 to 2015. During the study period, 356 CSF samples were collected from patients with sepsis-like illness and/or aseptic meningitis. Two RT-nested PCR assays targeting a partial part of 5'UTR for direct diagnosis and the VP1 region for genotyping by sequence analysis of the viral genome were used. HEV RNA was detected in 66 of 356 (18.5%) of CSF samples. Age distribution showed that 64% (42/66) were infants (<1 year), 18% were children between 1 and 5 years old, 12% were children between 5 and 10 years old, and 6% were more than 10 years old. Of the 66 EV cases, 12 were successfully genotyped. Five different EV genotypes were identified. All of them belonged to HEV-B species. The study showed that echovirus 6 genotype accounted for 42% of the sequenced cases. The HEV infections in the present study tended to show slight seasonal pattern with more cases occurring during spring and summer, yet still significant numbers were also reported in fall and winter seasons. HEV was isolated from a significant number of children with sepsis-like illness and/or aseptic meningitis. In addition, the molecular method utilized for direct diagnosis and genotyping of HEV from CSF revealed that more than one HEV type circulated in the West Bank, Palestine during the study period.

  18. The National Tay Sachs and Allied Diseases Association.

    ERIC Educational Resources Information Center

    Zeitlin, Paula

    1986-01-01

    The National Tay-Sachs and Allied Diseases Association is involved in education, research, and prevention of Tay-Sachs, an inherited metabolic disorder which destroys the central nervous system, and over 30 related disorders. The group features a parent peer group network and a support group for carrier couples. (CL)

  19. National Tay-Sachs and Allied Diseases Association, Inc.

    ERIC Educational Resources Information Center

    Exceptional Parent, 1977

    1977-01-01

    Reviewed are the history and organization, purpose and programs, and public services of the National Tay-Sachs and Allied Diseases Association, an organization geared toward eradicating Tay-Sachs disease (a hereditary disorder affecting primarily Jewish infants which generally leads to deterioration and death by the child's fifth year). (SBH)

  20. National Tay-Sachs and Allied Diseases Association, Inc.

    ERIC Educational Resources Information Center

    Exceptional Parent, 1977

    1977-01-01

    Reviewed are the history and organization, purpose and programs, and public services of the National Tay-Sachs and Allied Diseases Association, an organization geared toward eradicating Tay-Sachs disease (a hereditary disorder affecting primarily Jewish infants which generally leads to deterioration and death by the child's fifth year). (SBH)

  1. The National Tay Sachs and Allied Diseases Association.

    ERIC Educational Resources Information Center

    Zeitlin, Paula

    1986-01-01

    The National Tay-Sachs and Allied Diseases Association is involved in education, research, and prevention of Tay-Sachs, an inherited metabolic disorder which destroys the central nervous system, and over 30 related disorders. The group features a parent peer group network and a support group for carrier couples. (CL)

  2. 59 FR- Disease Associated With Exposure to Certain Herbicide Agents

    Federal Register 2010, 2011, 2012, 2013, 2014

    1994-02-03

    ... Vietnam era and the subsequent development of Hodgkin's disease and porphyria cutanea tarda (PCT). The... consistent with chloracne Hodgkin's disease Non-Hodgkin's lymphoma Porphyria cutanea tarda Soft-tissue... AFFAIRS 38 CFR Part 3 RIN 2900-AG69 Disease Associated With Exposure to Certain Herbicide Agents...

  3. Lineage shift in Indian strains of Dengue virus serotype-3 (Genotype III), evidenced by detection of lineage IV strains in clinical cases from Kerala

    PubMed Central

    2013-01-01

    Background Local epidemiology of Dengue is defined by the genetic diversity of the circulating Dengue virus (DENV) strains. This important information is not available for the virus strains from most parts of the Indian subcontinent. The present study focused on the genetic diversity of the serotype 3 DENV strains (DENV-3) from India. Results A total of 22 DENV-3 strains identified by reverse-transcription PCR analysis of serum samples from 709 patients were studied. These samples were collected over a period of 4 years (2008–2011) from dengue fever suspected patients from Kerala, a dengue endemic state in South India. Comparison of a 1740bp nucleotide sequence of the viral Capsid-Pre-membrane-Envelope coding region of our strains and previously reported DENV-3 strains from India, South Asia and South America revealed non-synonymous substitutions that were genotype III-specific as well as sporadic. Evidence of positive selection was detected in the I81 amino acid residue of the envelope protein. Out of the 22 samples, three had I81A and 18 had I81V substitutions. In the phylogenetic analysis by maximum likelihood method the strains from Kerala clustered in two different lineages (lineage III and IV) within genotype III clade of DENV-3 strains. The ten strains that belonged to lineage IV had a signature amino acid substitution T219A in the envelope protein. Interestingly, all these strains were found to be closely related to a Singapore strain GU370053 isolated in 2007. Conclusions Our study identifies for the first time the presence of lineage IV strains in the Indian subcontinent. Results indicate the possibility of a recent exotic introduction and also a shift from the existing lineage III strains to lineage IV. Lineage shifts in DENV-3 strains have been attributed to dramatic increase in disease severity in many parts of the world. Hence the present observation could be significant in terms of the clinical severity of future dengue cases in the region. PMID

  4. PBHMDA: Path-Based Human Microbe-Disease Association Prediction

    PubMed Central

    Huang, Zhi-An; Chen, Xing; Zhu, Zexuan; Liu, Hongsheng; Yan, Gui-Ying; You, Zhu-Hong; Wen, Zhenkun

    2017-01-01

    With the advance of sequencing technology and microbiology, the microorganisms have been found to be closely related to various important human diseases. The increasing identification of human microbe-disease associations offers important insights into the underlying disease mechanism understanding from the perspective of human microbes, which are greatly helpful for investigating pathogenesis, promoting early diagnosis and improving precision medicine. However, the current knowledge in this domain is still limited and far from complete. Here, we present the computational model of Path-Based Human Microbe-Disease Association prediction (PBHMDA) based on the integration of known microbe-disease associations and the Gaussian interaction profile kernel similarity for microbes and diseases. A special depth-first search algorithm was implemented to traverse all possible paths between microbes and diseases for inferring the most possible disease-related microbes. As a result, PBHMDA obtained a reliable prediction performance with AUCs (The area under ROC curve) of 0.9169 and 0.8767 in the frameworks of both global and local leave-one-out cross validations, respectively. Based on 5-fold cross validation, average AUCs of 0.9082 ± 0.0061 further demonstrated the efficiency of the proposed model. For the case studies of liver cirrhosis, type 1 diabetes, and asthma, 9, 7, and 9 out of predicted microbes in the top 10 have been confirmed by previously published experimental literatures, respectively. We have publicly released the prioritized microbe-disease associations, which may help to select the most potential pairs for further guiding the experimental confirmation. In conclusion, PBHMDA may have potential to boost the discovery of novel microbe-disease associations and aid future research efforts toward microbe involvement in human disease mechanism. The code and data of PBHMDA is freely available at http://www.escience.cn/system/file?fileId=85214. PMID:28275370

  5. PBHMDA: Path-Based Human Microbe-Disease Association Prediction.

    PubMed

    Huang, Zhi-An; Chen, Xing; Zhu, Zexuan; Liu, Hongsheng; Yan, Gui-Ying; You, Zhu-Hong; Wen, Zhenkun

    2017-01-01

    With the advance of sequencing technology and microbiology, the microorganisms have been found to be closely related to various important human diseases. The increasing identification of human microbe-disease associations offers important insights into the underlying disease mechanism understanding from the perspective of human microbes, which are greatly helpful for investigating pathogenesis, promoting early diagnosis and improving precision medicine. However, the current knowledge in this domain is still limited and far from complete. Here, we present the computational model of Path-Based Human Microbe-Disease Association prediction (PBHMDA) based on the integration of known microbe-disease associations and the Gaussian interaction profile kernel similarity for microbes and diseases. A special depth-first search algorithm was implemented to traverse all possible paths between microbes and diseases for inferring the most possible disease-related microbes. As a result, PBHMDA obtained a reliable prediction performance with AUCs (The area under ROC curve) of 0.9169 and 0.8767 in the frameworks of both global and local leave-one-out cross validations, respectively. Based on 5-fold cross validation, average AUCs of 0.9082 ± 0.0061 further demonstrated the efficiency of the proposed model. For the case studies of liver cirrhosis, type 1 diabetes, and asthma, 9, 7, and 9 out of predicted microbes in the top 10 have been confirmed by previously published experimental literatures, respectively. We have publicly released the prioritized microbe-disease associations, which may help to select the most potential pairs for further guiding the experimental confirmation. In conclusion, PBHMDA may have potential to boost the discovery of novel microbe-disease associations and aid future research efforts toward microbe involvement in human disease mechanism. The code and data of PBHMDA is freely available at http://www.escience.cn/system/file?fileId=85214.

  6. Inflammatory bowel disease associations with HLA Class II genes

    SciTech Connect

    Castro, R.; Yang, H.; Targan, S.

    1994-09-01

    A PCR-SSOP assay has been used to analyze HLA-Class II DRB1 and DQB1 alleles in 378 Caucasians from a population in Southern California. The data has been analyzed separately for the Ashkenasi Jews and non-Jewish patients (n=286) and controls (n=92). Two common clinical forms of inflammatory bowel disease (IBD) have been studied: ulcerative colitis (UC) and Crohn`s disease (CD). In CD, we observed a susceptible effect with the rare DR1 allele - DRB*0103 [O.R.=4.56; 95% CI (0.96, 42.97); p=0.03]; a trend for an increase in DRB1*0103 was also observed in UC patients. A susceptible effect with DRB1*1502 [O.R.=5.20; 95% CI (1.10, 48.99); p=0.02] was observed in non-Jewish UC patients. This susceptible effect was restricted to UC ANCA-positive (antineutrophil cytoplasmic antibodies) patients. In addition, a significant association with DRB1*1101-DQB1*0301 [O.R.=9.46; 95% CI (1.30, 413.87); p=0.01] was seen with UC among non-Jewish patients: this haplotype was increased with CD among non-Jewish patients. Two protective haplotypes were detected among CD non-Jewish patients: DRB1*1301-DQB1*0603 [O.R.=0.34; 95% CI (0.09, 1.09); p=0.04], and DRB*0404-DQB1*0302 [O.R.=<0.08; 95% CI (0.0, 0.84); p=0.01]. When the same data were analyzed at the serology level, we observed a positive association in UC with DR2 [O.R.6.77; 95% CI (2.47, 22.95); p=2 x 10{sup -4}], and a positive association in CD with DR1 [O.R.=2.63; 95% CI (1.14, 6.62); p=0.01] consistent with previous reports. Thus, some IBD disease associations appear to be common to both UC and CD, while some are unique to one disease.

  7. Evaluation of the GenoType Mycobacteria Direct assay for direct detection of the Mycobacterium tuberculosis complex obtained from sputum samples.

    PubMed

    Kiraz, Nuri; Saglik, Imran; Kiremitci, Abdurrahman; Kasifoglu, Nilgun; Akgun, Yurdanur

    2010-08-01

    An increase in the prevalence of tuberculosis (TB) in recent years has accelerated the search for novel tools for the rapid diagnosis of TB infection. This study evaluated the GenoType Mycobacteria Direct (GTMD) assay (Hain Lifescience) for direct detection of the Mycobacterium tuberculosis complex (MTBC) from sputum samples and compared it with conventional methods. The GTMD test is a commercial assay produced using strip techniques and works based on a nucleic acid sequence-based amplification technique. This test allows 23S rRNA amplification-based detection of MTBC, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii and Mycobacterium malmoense directly from decontaminated clinical samples within 6 h. In the present study, 115 sputum samples were processed to detect acid-fast bacilli (AFB) using two microscopy methods (carbol fuchsin and fluorescent staining), two culture methods [Löwenstein-Jensen (LJ) and BACTEC 12B media] and the GTMD test. The results showed that 86 of the samples were positive by direct microscopy, 84 were positive by BACTEC 12B culture, 73 were positive by LJ culture and 95 were positive by the GTMD test. All of the isolates turned out to be MTBC. Moreover, the sensitivity and specificity of the GTMD test for MTBC in patients were 97 and 58 %, respectively, taking the culture combination as the gold standard. When the test was compared with culture of samples from anti-TB-treated patients, the sensitivity and specificity for the test were 100 and 15 %, respectively. Low specificity in treated people might arise from depressed proliferation of AFB. As the two methods target the same living bacilli, the difference is obviously notable. When the culture results and clinical findings of the patients were evaluated together (true-positive specimens), the sensitivity and specificity values of the GTMD test for all patients were 97 and 90 %, respectively. However, both of these values increased to 100 % for the patients

  8. Mapping of disease-associated variants in admixed populations

    PubMed Central

    2011-01-01

    Recent developments in high-throughput genotyping and whole-genome sequencing will enhance the identification of disease loci in admixed populations. We discuss how a more refined estimation of ancestry benefits both admixture mapping and association mapping, making disease loci identification in admixed populations more powerful. High-throughput genotyping and sequencing will enable refined estimation of ancestry, thus enhancing disease loci identification in admixed populations PMID:21635713

  9. Detection of the G3 genotype of Echinococcus granulosus from hydatid cysts of Chilean cattle using COX1 and ND1 mitochondrial markers.

    PubMed

    Espinoza, Sandra; Salas, Ana María; Vargas, Alex; Freire, Victoria; Diaz, Eric; Sánchez, Gittith; Venegas, Juan

    2014-01-01

    For a deeper understanding of the phylogenetic relationships of Echinococcus genotypes and species in different intermediate hosts, we analyzed samples from human and bovine hydatid cysts. For this, segments of the cytochrome oxidase (COX1) and NADH dehydrogenase (ND1) mitochondrial genes were used. To obtain sufficient amounts of the ND1 marker to be sequenced properly, a new variant of the PCR assay was implemented. Phylogenetic analysis with both markers showed that most of the analyzed samples correspond to genotype G1. However, a sample from cysts of a bovine lung (Q21), with the COX1 marker, was grouped in a node together with a sample belonging to genotype G3. In the phylogenetic tree obtained with the ND1 marker, this sample was grouped with sequences of genotypes G3, G2, and G4. Analyzing the single nucleotide polymorphic (SNP) sites of both markers, it was observed that the Q21 sequence is almost identical to the G3 sequence and differ in only one SNP from the G2 sequence, and is completely different from G4. These results are noteworthy, since neither G2 nor G3 genotypes have been described previously in Chile, raising the possibility that the G3 genotype is present in these latitudes. This information is highly relevant; it can be employed to uncover additional unknown details of transmission cycles of this important parasite.

  10. Text mining facilitates database curation - extraction of mutation-disease associations from Bio-medical literature.

    PubMed

    Ravikumar, Komandur Elayavilli; Wagholikar, Kavishwar B; Li, Dingcheng; Kocher, Jean-Pierre; Liu, Hongfang

    2015-06-06

    Advances in the next generation sequencing technology has accelerated the pace of individualized medicine (IM), which aims to incorporate genetic/genomic information into medicine. One immediate need in interpreting sequencing data is the assembly of information about genetic variants and their corresponding associations with other entities (e.g., diseases or medications). Even with dedicated effort to capture such information in biological databases, much of this information remains 'locked' in the unstructured text of biomedical publications. There is a substantial lag between the publication and the subsequent abstraction of such information into databases. Multiple text mining systems have been developed, but most of them focus on the sentence level association extraction with performance evaluation based on gold standard text annotations specifically prepared for text mining systems. We developed and evaluated a text mining system, MutD, which extracts protein mutation-disease associations from MEDLINE abstracts by incorporating discourse level analysis, using a benchmark data set extracted from curated database records. MutD achieves an F-measure of 64.3% for reconstructing protein mutation disease associations in curated database records. Discourse level analysis component of MutD contributed to a gain of more than 10% in F-measure when compared against the sentence level association extraction. Our error analysis indicates that 23 of the 64 precision errors are true associations that were not captured by database curators and 68 of the 113 recall errors are caused by the absence of associated disease entities in the abstract. After adjusting for the defects in the curated database, the revised F-measure of MutD in association detection reaches 81.5%. Our quantitative analysis reveals that MutD can effectively extract protein mutation disease associations when benchmarking based on curated database records. The analysis also demonstrates that incorporating

  11. Protein-driven inference of miRNA–disease associations

    PubMed Central

    Mørk, Søren; Pletscher-Frankild, Sune; Palleja Caro, Albert; Gorodkin, Jan; Jensen, Lars Juhl

    2014-01-01

    Motivation: MicroRNAs (miRNAs) are a highly abundant class of non-coding RNA genes involved in cellular regulation and thus also diseases. Despite miRNAs being important disease factors, miRNA–disease associations remain low in number and of variable reliability. Furthermore, existing databases and prediction methods do not explicitly facilitate forming hypotheses about the possible molecular causes of the association, thereby making the path to experimental follow-up longer. Results: Here we present miRPD in which miRNA–Protein–Disease associations are explicitly inferred. Besides linking miRNAs to diseases, it directly suggests the underlying proteins involved, which can be used to form hypotheses that can be experimentally tested. The inference of miRNAs and diseases is made by coupling known and predicted miRNA–protein associations with protein–disease associations text mined from the literature. We present scoring schemes that allow us to rank miRNA–disease associations inferred from both curated and predicted miRNA targets by reliability and thereby to create high- and medium-confidence sets of associations. Analyzing these, we find statistically significant enrichment for proteins involved in pathways related to cancer and type I diabetes mellitus, suggesting either a literature bias or a genuine biological trend. We show by example how the associations can be used to extract proteins for disease hypothesis. Availability and implementation: All datasets, software and a searchable Web site are available at http://mirpd.jensenlab.org. Contact: lars.juhl.jensen@cpr.ku.dk or gorodkin@rth.dk PMID:24273243

  12. Inferring drug-disease associations based on known protein complexes.

    PubMed

    Yu, Liang; Huang, Jianbin; Ma, Zhixin; Zhang, Jing; Zou, Yapeng; Gao, Lin

    2015-01-01

    Inferring drug-disease associations is critical in unveiling disease mechanisms, as well as discovering novel functions of available drugs, or drug repositioning. Previous work is primarily based on drug-gene-disease relationship, which throws away many important information since genes execute their functions through interacting others. To overcome this issue, we propose a novel methodology that discover the drug-disease association based on protein complexes. Firstly, the integrated heterogeneous network consisting of drugs, protein complexes, and disease are constructed, where we assign weights to the drug-disease association by using probability. Then, from the tripartite network, we get the indirect weighted relationships between drugs and diseases. The larger the weight, the higher the reliability of the correlation. We apply our method to mental disorders and hypertension, and validate the result by using comparative toxicogenomics database. Our ranked results can be directly reinforced by existing biomedical literature, suggesting that our proposed method obtains higher specificity and sensitivity. The proposed method offers new insight into drug-disease discovery. Our method is publicly available at http://1.complexdrug.sinaapp.com/Drug_Complex_Disease/Data_Download.html.

  13. Characterization of Disease-Associated Mutations in Human Transmembrane Proteins

    PubMed Central

    Molnár, János; Szakács, Gergely; Tusnády, Gábor E.

    2016-01-01

    Transmembrane protein coding genes are commonly associated with human diseases. We characterized disease causing mutations and natural polymorphisms in transmembrane proteins by mapping missense genetic variations from the UniProt database on the transmembrane protein topology listed in the Human Transmembrane Proteome database. We found characteristic differences in the spectrum of amino acid changes within transmembrane regions: in the case of disease associated mutations the non-polar to non-polar and non-polar to charged amino acid changes are equally frequent. In contrast, in the case of natural polymorphisms non-polar to charged amino acid changes are rare while non-polar to non-polar changes are common. The majority of disease associated mutations result in glycine to arginine and leucine to proline substitutions. Mutations to positively charged amino acids are more common in the center of the lipid bilayer, where they cause more severe structural and functional anomalies. Our analysis contributes to the better understanding of the effect of disease associated mutations in transmembrane proteins, which can help prioritize genetic variations in personal genomic investigations. PMID:26986070

  14. Detection of Hepatitis B Virus (HBV) Genotype E Carried—Even in the Presence of High Titers of Anti-HBs Antibodies—by an Argentinean Patient of African Descent Who Had Received Vaccination against HBV

    PubMed Central

    Mathet, Verónica L.; Cuestas, María L.; Ruiz, Vanesa; Minassian, María L.; Rivero, Cintia; Trinks, Julieta; Daleoso, Graciela; León, Liliana M.; Sala, Andrea; Libellara, Beatriz; Corach, Daniel; Oubiña, José R.

    2006-01-01

    Genotype E hepatitis B virus (HBV) was detected in two Argentine sisters exhibiting an African mitochondrial lineage. One of them (who had been vaccinated against HBV) exhibited anti-HBs cocirculating antibodies without HBsAg escape mutants, while her unvaccinated sister showed a D144A HBsAg escape mutant without anti-HBs antibodies. Both sisters carried an unusual L209V substitution within HBsAg. PMID:16954295

  15. Detection of hepatitis B virus (HBV) genotype E carried--even in the presence of high titers of anti-HBs antibodies--by an Argentinean patient of African descent who had received vaccination against HBV.

    PubMed

    Mathet, Verónica L; Cuestas, María L; Ruiz, Vanesa; Minassian, María L; Rivero, Cintia; Trinks, Julieta; Daleoso, Graciela; León, Liliana M; Sala, Andrea; Libellara, Beatriz; Corach, Daniel; Oubiña, José R

    2006-09-01

    Genotype E hepatitis B virus (HBV) was detected in two Argentine sisters exhibiting an African mitochondrial lineage. One of them (who had been vaccinated against HBV) exhibited anti-HBs cocirculating antibodies without HBsAg escape mutants, while her unvaccinated sister showed a D144A HBsAg escape mutant without anti-HBs antibodies. Both sisters carried an unusual L209V substitution within HBsAg.

  16. Development of an endpoint genotyping assay to detect the Mycoplasma pneumoniae 23S rRNA gene and distinguish the existence of macrolide resistance-associated mutations at position 2063.

    PubMed

    Suzuki, Yu; Seto, Junji; Shimotai, Yoshitaka; Ikeda, Tatsuya; Yahagi, Kazue; Mizuta, Katsumi; Matsuzaki, Yoko; Hongo, Seiji

    2016-12-01

    The prevalence of macrolide-resistant Mycoplasma pneumoniae harboring a mutation in the 23S rRNA gene is increasing, and rapid detection assays are needed for clinical management. We developed an endpoint genotyping assay to detect the M. pneumoniae 23S rRNA gene and determine the existence of macrolide resistance-associated mutations at position 2063 (A2063G, A2063T and A2063C mutations). This A2063B genotyping assay detected more than 50 copies/reaction of the M. pneumoniae gene in every nucleotide mutation at position 2063. Of 42 clinical specimens, 3 were positive without mutation, 6 were positive with the A2063G mutation, and 33 were negative. The results were confirmed using nested PCR with the sequencing of the M. pneumoniae 23S rRNA gene, and a high sensitivity (90%), specificity (100%), and coincidence ratio (kappa coefficient=0.93) were obtained. Therefore, the A2063B genotyping assay is useful for the rapid discrimination of macrolide resistance mutations at position 2063.

  17. Rotavirus genotypes in Belarus, 2008-2012.

    PubMed

    Semeiko, Galina V; Yermalovich, Marina A; Poliakova, Nadezhda; Mijatovic-Rustempasic, Slavica; Kerin, Tara K; Wasley, Annemarie; Videbaek, Dovile; Gentsch, Jon R; Bowen, Michael D; Samoilovich, Elena O

    2014-12-01

    This study describes group A rotavirus (RVA) genotype prevalence in Belarus from 2008 to 2012. In 2008, data from 3 sites in Belarus (Brest, Mogilev, Minsk) indicated that G4P[8] was the predominant genotype. Data from Minsk (2008-2012) showed that G4P[8] was the predominant RVA genotype in all years except in 2011 when G3P[8] was most frequently detected. Other RVA genotypes common in Europe (G1P[8], G2P[4]) were detected each year of the study. This study reveals the dominance of genotype G4P[8] in Belarus and helps to establish the baseline genotype prevalence prior to RVA vaccine introduction in the country.

  18. Detection and genetic characterization of porcine group A rotaviruses in asymptomatic pigs in smallholder farms in East Africa: predominance of P[8] genotype resembling human strains.

    PubMed

    Amimo, J O; Junga, J O; Ogara, W O; Vlasova, A N; Njahira, M N; Maina, S; Okoth, E A; Bishop, R P; Saif, L J; Djikeng, A

    2015-02-25

    ] strains (Ug-049 and Ug-453) had G5-P[13]-I5-R1-C1-M1-A8-N1-T7-E1-H1 while the untypeable strain (Ug-218) had G5-P[?]-I5-R1-C1-M1-A8-N1-T1-E1-H? In conclusion, P[6] and P[8] genotypes detected were genetically closely related to human strains suggesting the possibility of interspecies transmission. Further studies are required to determine the role of RVA in swine enteric disease burden and to determine the genetic/antigenic heterogeneity of the circulating strains for development of accurate diagnostic tools and to implement appropriate prophylaxis programs.

  19. Signal to cut-off (S/CO) ratio and detection of HCV genotype 1 by real-time PCR one-step method: is there any direct relationship?

    PubMed

    Albertoni, Guilherme; Arnoni, C P; Araújo, P R B; Carvalho, F O; Barreto, J A

    2010-01-01

    Polymerase chain reaction (PCR) methods play an essential role in providing data related to diagnosis, monitoring and treatment of hepatitis C virus (HCV) infection. EIA results are reported as ''reactive'' or ''non reactive'' and EIA S/CO ratio may also be reported as ''high'' or ''low.'' This study aimed to evaluate the performance of a real-time RT-PCR and assess whether there is relationship between S/CO and PCR results. Sera from blood donors were analyzed by Enzyme-Linked Immunosorbent Assay (ELISA) and RT-PCR assay to detect HCV infection. The RT-PCR assay to genotypes 1a/b showed an acceptable linear response in serial dilutions. The samples were divided into two groups based on their serological results: group A--S/CO ratio < 3 (60 samples) and group B--S/CO ratio > 3 (41 samples). Viral loads were confirmed positive in group B samples in 90%, and in group A samples were confirmed positive in only 13% by RT-PCR. The methodology used was able to detect the presence of RNA-HCV genotype I in 90% of the samples serologically positive in group B. All negative samples were sent to search for other genotypes of HCV (genotypes 2-6) and were confirmed as negative. These data suggests that these negative samples may have HCV RNA viral load below the detection limit of our test (310 IU/ mL), or a false positive result in serological test, or spontaneous viral clearance occurred.

  20. Detection Rate of High-Grade Cervical Neoplasia and Cost-Effectiveness of High-Risk Human Papillomavirus Genotyping with Reflex Liquid-based Cytology in Cervical Cancer Screening.

    PubMed

    Tay, Sun Kuie; Lin, Lynette Eo; Goh, Ronald Ch

    2017-07-01

    This study aimed to determine the prevalence of cervical intraepithelial neoplasia grade 3 or worse (≥CIN3) and cost-effectiveness of human papillomavirus (HPV) genotyping with reflex liquid-based cytology (LBC) for cervical cancer screening in Singapore. Women who were ≥25 years old and undertook co-testing with LBC and HPV-genotyping (Cobas-4800, Roche, USA) for HPV-16, HPV-18 and 12 high-risk HPV types in a single institution were studied retrospectively. A single cervical smear in ThinPrep® PreservCyt® solution (Hologic, USA) was separated for tests in independent cytology and molecular pathology laboratories. The results were reviewed by a designated gynaecologist according to institutional clinical management protocols. Those who tested positive for HPV-16 and/or HPV-18 (regardless of cytology results), cytology showing low-grade squamous intraepithelial lesions (LSIL) or high-grade SIL (HSIL), or atypical squamous cells of undetermined significance (ASCUS) with positive 12 high-risk HPV types were referred for colposcopy. Colposcopy was performed by experienced colposcopists. Cervical biopsy, either directed punch biopsies or excisional biopsy, was determined by a colposcopist. The diagnosis of ≥CIN3 was reviewed by a gynaecologic pathologist. Cost-effectiveness of HPV-based screening in terms of disease and financial burden was analysed using epidemiological, clinical and financial input data from Singapore. Of 1866 women studied, 167 (8.9%) had abnormal cytology (≥ASCUS) and 171 (9.2%) tested positive for high-risk HPV. Twenty-three CIN were detected. Three of the 10 ≥CIN3 cases had negative cytology but positive HPV-16. Compared to cytology, HPV genotyping detected more ≥CIN3 (OR: 1.43). HPV+16/18 genotyping with reflex LBC was superior in terms of cost-effectiveness to LBC with reflex HPV, both for disease detection rate and cost per case of ≥CIN2 detected. Compared to cytology, HPV+16/18 genotyping with reflex LBC detected more ≥CIN3

  1. Rapid detection of HCV genotyping 1a, 1b, 2a, 3a, 3b and 6a in a single reaction using two-melting temperature codes by a real-time PCR-based assay.

    PubMed

    Athar, Muhammad Ammar; Xu, Ye; Xie, Xiaoting; Xu, Zhenxing; Ahmad, Vakil; Hayder, Zulfiqar; Hussain, Syed Sajid; Liao, Yiqun; Li, Qingge

    2015-09-15

    The genotype of the hepatitis C virus (HCV) is an important indicator for antiviral therapeutic response. We hereby described development of a rapid HCV genotyping approach that enabled the identification of the six most common HCV subtypes of Asia, i.e., 1a, 1b, 2a, 3a, 3b, and 6a, in a single reaction. Using two dual-labeled, self-quenched probes that target the core region of the HCV genome, the exact subtype could be accurately identified by two-melting temperature codes determined from the two respective probes in a real-time PCR assay. Analytical sensitivity studies using armored RNA samples representing each of the six HCV subtypes showed that 5 copies/reaction of HCV RNA could be detected. The assay was evaluated using 244 HCV-positive serum samples and the results were compared with sequencing analysis. Of the 224 samples, subtype 3a (127, 52.3%) was the dominant, followed by 1b (51, 20.9%), 3b (47, 19.3%), 2a (8, 3.3%), 6a (4, 1.6%) and the least was subtype 1a (1, 0.4%). Moreover, 6 (2.5%) mixed infection samples were also detected. These results were fully concordant with sequencing analysis. We concluded that this real-time PCR-based assay could provide a rapid and reliable tool for routine HCV genotyping in most Asian countries.

  2. Graves' disease associated with alopecia areata developing after Hashimoto's thyroiditis.

    PubMed

    Aşık, Mehmet; Binnetoğlu, Emine; Şen, Hacer; Tekeli, Zeliha; Uysal, Fatma; Ukinç, Kubilay

    2013-01-01

    Graves' disease and Hashimoto's thyroiditis are the most common autoimmune thyroid diseases. Hypothyroidism can develop in patients with Graves' disease, either spontaneously or as a result of radioactive iodine therapy or surgery. However, it is rare for patients with Hashimoto's thyroiditis to subsequently develop Graves' disease. We report a case of alopecia areata associated with Graves' disease in a 41-year-old woman who had previously been diagnosed with Hashimoto's disease. Alopecia areata is an autoimmune disease associated with other autoimmune diseases such as thyroid disorders, anemia, and other skin disorders.

  3. [Crohn's disease associated with Berger's disease. A rare complication].

    PubMed

    López Barbarín, J M; Lafuente Martínez, P; García Campos, F; Ibarra Peña, B; Díaz de Otazu, R

    1990-10-01

    We report the case of a male patient with Crohn's disease associated with IgA nephropathy. He was treated surgically for the intestinal disorder and then with corticoids and sulfasalazine. Six years after treatment the patient was asymptomatic. As the intestinal situation improved there was concomitant normalization of urinary sediment, maintaining renal function. The fact that the digestive mucosa is one of the body's major sources of secretory IgA may account for the existence of a common for Crohn's disease and certain forms of IgA nephropathy.

  4. Detection of neonatal unit clusters of Candida parapsilosis fungaemia by microsatellite genotyping: Results from laboratory-based sentinel surveillance, South Africa, 2009-2010.

    PubMed

    Magobo, Rindidzani E; Naicker, Serisha D; Wadula, Jeannette; Nchabeleng, Maphoshane; Coovadia, Yacoob; Hoosen, Anwar; Lockhart, Shawn R; Govender, Nelesh P

    2017-05-01

    Neonatal candidaemia is a common, deadly and costly hospital-associated disease. To determine the genetic diversity of Candida parapsilosis causing fungaemia in South African neonatal intensive care units (NICUs). From February 2009 through to August 2010, cases of candidaemia were reported through laboratory-based surveillance. C. parapsilosis isolates from neonatal cases were submitted for identification by internal transcribed spacer (ITS) region sequencing, antifungal susceptibility testing and microsatellite genotyping. Cluster analysis was performed using Unweighted Pair Group Method with Arithmetic Mean (UPGMA). Of 1671 cases with a viable Candida isolate, 393 (24%) occurred among neonates. Isolates from 143 neonatal cases were confirmed as C. parapsilosis sensu stricto. Many isolates were resistant to fluconazole (77/143; 54%) and voriconazole (20/143; 14%). Of 79 closely-related genotypes, 18 were represented by ≥2 isolates; 61 genotypes had a single isolate each. Seven clusters, comprised of 82 isolates, were identified at five hospitals in three provinces. Isolates belonging to certain clusters were significantly more likely to be fluconazole resistant: all cluster 7 isolates and the majority of cluster 4 (78%), 5 (89%) and 6 (67%) isolates (P<.001). Candida parapsilosis-associated candidaemia in public-sector NICUs was caused by closely related genotypes and there was molecular evidence of undetected outbreaks as well as intra-hospital transmission. © 2017 Blackwell Verlag GmbH.

  5. [Prevalence of non-vaccinable high risk genotypes of human papillomavirus in the Early Detection of Cervical Cancer Program in Cantabria].

    PubMed

    Paz-Zulueta, María; Fernández-Feito, Ana; Amparán Ruiz, Marina; Azofra Olave, Asunción; Martín Seco, Yolanda; Ojugas Zabala, Sonia; Otero García, Aurora; Royano Rasines, Carmen; Sarabia-Lavín, Raquel; Torres Manrique, Blanca; Santibáñez Margüello, Miguel

    2016-01-01

    To estimate the prevalence of infection with non-vaccinable high risk genotypes of human papillomavirus (HPV). Cross-sectional study. Seven randomly selected health centres in Cantabria (Northern Spain). All women with an evaluable vaginal smear (n=3,359) between 2010 and 2011. The variables collected were cytological diagnosis, PCR results, and family planning method. The vaginal smear results were classified with the Bethesda system. The classification by Muñoz et al. was used for typing as oncogenic risk HPV. Proportions and odds ratio (OR) were estimated with corresponding confidence intervals at 95% (95% CI). The prevalence of HPV infection was 2.71% (95% CI: 2.15 to 3.27). The prevalence of high oncogenic risk HPV genotypes was 2.26%; (95% CI: 1.75 to 2.78). The most frequent genotype was 16 (28.89%). More than half of the women were positive for one of the non-vaccinable high risk genotypes: 51 (18.89%) and 58 (13.33%) and 68 (12.22%) or 31 (11.11%). At least two non-vaccinable high-risk genotypes co-existed in 23.33% of women. Younger women (≤30 years) had twice the risk of any HPV infection: OR 2.01 (95% CI: 1.02 to 3.96); and were twice as likely to use condoms compared to hormonal contraceptives, OR 2.09 (95% CI: 1.64 to 2.67). According to the high percentage of non-vaccinable high oncogenic risk HPV types, there should be a re-think of the prevention strategy in the population, who may have a false sense of protection. Copyright © 2015 Elsevier España, S.L.U. All rights reserved.

  6. Phenotypic and genotypic detection of ESBL mediated cephalosporin resistance in Klebsiella pneumoniae: emergence of high resistance against cefepime, the fourth generation cephalosporin.

    PubMed

    Grover, S S; Sharma, Meenakshi; Chattopadhya, D; Kapoor, Hema; Pasha, S T; Singh, Gajendra

    2006-10-01

    common resistance phenotypes probably belonged to a single strain as evident by MIC patterns, genotypic characterization and resistance profile to non-cephalosporin group of antimicrobials thereby pointing out the possibility of an outbreak. PCR may be regarded as a reliable method for detection of ESBL since in addition to the strains that could be identified as ESBL producers by DDST and E-test ESBL; PCR could demonstrate ESBL production among additional 32 strains (15 in phase I and 17 in phase II). Continued uses of cephalosporin group appear to be a potential risk factor for emergence of ESBL producing K. pneumoniae strains. In addition, as noted in the present study, the rise of resistance to cefepime that has been introduced recently in this country for therapeutic use could be of concern.

  7. Comparison of Modified Impedance Whole Blood Platelet Aggregation Method Detecting Platelet Function in ACS Patients with Different CYP2C19 Genotypes.

    PubMed

    Cui, Chanjuan; Qiao, Rui; Zhang, Jie

    2016-01-01

    A reliable laboratory test to monitor onclopidogrel platelet reactivity (PR) is very necessary. In addition, genetic factors also play an important part in onclopidogrel PR. This study aimed to modify the original impedance whole blood platelet aggregation assay associated with the release assay to monitor onclopidogrel PR and assess their relationship with genotype. We adjusted the concentration of calcium in the in vitro reaction system of platelet aggregation to modify the original impedance whole blood platelet aggregation assay. Meanwhile, chronolume, which quantified the adenosine triphosphate (ATP) released from platelet dense granules, is added to this reaction system to reflect the platelet release function. In the modified assay, platelet magnified activation time (MAT) and the maximal platelet ATP release value (RV) were used to reflect platelet function parameters. In the original assay, the electrical resistance (omega) and RV were used to reflect platelet function parameters. Onclopidogrel PR was detected by the original impedance whole blood platelet aggregation assay, modified assay, and flow cytometric vasodilator stimulated phosphoprotein (VASP) assay in 168 patients with acute coronary syndromes (ACS). CYP2C19*2 and CYP2C19*3 polymorphisms were also detected in all of these patients. This modified method showed that when 12.5 microL CaCl2 (0.2 mmol/L) was added to the reaction system, MAT was appropriate (93 +/- 23 seconds). The CVs for the modified impedance assay and release assay were 9.31% and 6.13%, respectively. The mean VASP-PRI in the patient group treated with clopidogrel was significantly lower than that in the control group without antiplatelet therapy (54.88 +/- 16.81% vs. 79.86 +/- 10.24%, p < 0.001). MAT of the modified method in VASP PRI > 50% group were shorter than that in the PRI < 50% group [185 (154-241) vs. 214 (184-250), p < 0.051. Meanwhile, the RV of the modified method in VASP PRI > 50% group were higher than that in the

  8. Genotypic diversity of european Phytophthora ramorum isolates based on SSR analysis

    Treesearch

    Kris Van Poucke; Annelies Vercauteren; Martine Maes; Sabine Werres; Kurt Heungens

    2013-01-01

    in Scotland were genotyped using seven microsatellite markers as described by Vercauteren et al. (2010). Thirty multilocus genotypes were identified within the Scottish population, with 51 percent of the isolates belonging to the main European genotype EU1MG1 and 13 unique detected genotypes. Ten of those genotypes were site specific, often represented by...

  9. PheWAS: demonstrating the feasibility of a phenome-wide scan to discover gene–disease associations

    PubMed Central

    Denny, Joshua C.; Ritchie, Marylyn D.; Basford, Melissa A.; Pulley, Jill M.; Bastarache, Lisa; Brown-Gentry, Kristin; Wang, Deede; Masys, Dan R.; Roden, Dan M.; Crawford, Dana C.

    2010-01-01

    Motivation: Emergence of genetic data coupled to longitudinal electronic medical records (EMRs) offers the possibility of phenome-wide association scans (PheWAS) for disease–gene associations. We propose a novel method to scan phenomic data for genetic associations using International Classification of Disease (ICD9) billing codes, which are available in most EMR systems. We have developed a code translation table to automatically define 776 different disease populations and their controls using prevalent ICD9 codes derived from EMR data. As a proof of concept of this algorithm, we genotyped the first 6005 European–Americans accrued into BioVU, Vanderbilt's DNA biobank, at five single nucleotide polymorphisms (SNPs) with previously reported disease associations: atrial fibrillation, Crohn's disease, carotid artery stenosis, coronary artery disease, multiple sclerosis, systemic lupus erythematosus and rheumatoid arthritis. The PheWAS software generated cases and control populations across all ICD9 code groups for each of these five SNPs, and disease-SNP associations were analyzed. The primary outcome of this study was replication of seven previously known SNP–disease associations for these SNPs. Results: Four of seven known SNP–disease associations using the PheWAS algorithm were replicated with P-values between 2.8 × 10−6 and 0.011. The PheWAS algorithm also identified 19 previously unknown statistical associations between these SNPs and diseases at P < 0.01. This study indicates that PheWAS analysis is a feasible method to investigate SNP–disease associations. Further evaluation is needed to determine the validity of these associations and the appropriate statistical thresholds for clinical significance. Availability:The PheWAS software and code translation table are freely available at http://knowledgemap.mc.vanderbilt.edu/research. Contact: josh.denny@vanderbilt.edu PMID:20335276

  10. Comparison of SPF10 real-time PCR and conventional PCR in combination with the INNO-LiPA HPV Genotyping Extra assay for the detection and typing of human papillomavirus in cervical samples.

    PubMed

    Micalessi, M I; Boulet, G A; Pillet, S; Jacquet, J; Pozzetto, B; Bogers, J J; Bourlet, T

    2013-12-01

    The novel SPF10 real-time PCR assay allows the simultaneous amplification and detection of the HPV target. That way, LiPA analysis of the HPV-negative samples can be avoided, reducing workload and cost. This study aims to evaluate the performance of the SPF10 real-time PCR in combination with the LiPA assay for HPV detection and typing in cervical samples. Thirty-nine cervical samples were subjected to the SPF10 conventional PCR in combination with the LiPA assay. Subsequently, the SPF10 real-time PCR was performed to enable the comparison between the SPF10 conventional and the real-time PCR results. In case of discrepancy, the samples were subjected to the CLART HPV2 assay. As a result, 27 out of 39 samples were identified as HPV-positive by the SPF10 real-time PCR and were genotyped further by the LiPA assay. Twenty samples (74.1%) showed an absolute agreement between the conventional and real-time SPF10 PCR (concordant), three (11.1%) displayed additional or fewer types (compatible), two (7.4%) did not show any similarity between both assays (discordant) and the remaining two (7.4%) were LiPA-negative. The two assays showed an excellent strength of agreement for individual (κ=0.932) and multiple genotype detection (κ=0.834). In conclusion, the two SPF10 PCR methods are comparable. Therefore, the SPF10 real-time PCR with subsequent LiPA could be used for the detection and genotyping of HPV in cervical samples.

  11. Recovery of DNA for the detection and genotyping of human papillomavirus from clinical cervical specimens stored for up to 2 years in a universal collection medium with denaturing reagent.

    PubMed

    Campos, Elisabete A; Simões, José Antonio; Rabelo-Santos, Silvia H; Sarian, Luis Otávio; Pitta, Denise Rocha; Levi, José Eduardo; Derchain, Sophie

    2008-02-01

    The recovery and stability of DNA for the detection and genotyping of HPV in UCM-containing specimens, after exposure to denaturing reagents and stored for up to 2 years were evaluated. Samples were collected from 60 women who had cervical cytology specimens harboring cervical intraepithelial neoplasia (CIN) 2 or 3. All samples were stored in UCM and had been frozen at -20 degrees C following the addition of the denaturing reagent (sodium hydroxide) and the removal of the aliquot required for Hybrid Capture 2 testing for the identification of HPV DNA. The samples had been stored for 6, 12 and 24 months (20 samples for each storage time). HPV DNA extraction was performed according to a protocol designed specifically and the presence and quality of DNA was confirmed by human beta-globin detection using the consensus primers G73 and G74. HPV DNA was amplified using the consensus primers PGMY09 and PGMY11, and reverse line-blot hybridization was used to detect type-specific amplicons for 37 HPV types. The DNA extracted from the denatured specimen was recovered in 57/60 (95%) of the samples. HPV DNA was detected in 56/57 (98%) of the recovered samples. Twenty-six of the 56 samples recovered (48%) were genotyped successfully.

  12. Genotyping of Portuguese Chlamydia trachomatis urogenital isolates.

    PubMed Central

    Borrego, M J; Gomes, J P; Lefebvre, J F; Eb, F; Orfila, J; Catry, M A

    1997-01-01

    OBJECTIVE: To determine the prevalence of the different Chlamydia trachomatis genotypes in Portuguese patients. METHODS: Urogenital isolates (n = 240) derived from attenders of various clinics in the Lisbon area were differentiated into genovars by genotyping with restriction fragment length polymorphism (RFLP) analysis of the PCR amplified omp1 gene. RESULTS: Genotype E was the most common for both men (47.9%) and women (43.8%). Genotypes D and F were the second most prevalent for men (11.3%) and genotype H was the second most prevalent for women (19.5%). Genotypes F, G, D, in women and H, G, I, in men, were found in a lower percentage of cases. Genotypes B, Ba, J, K, L1 and L2 were very rarely detected. CONCLUSIONS: With one exception, the overall distribution of Chlamydia trachomatis genotypes in our study is similar to what has been observed in other western countries. The only exception is the unusual prevalence of genotype H among women. The clinical manifestations associated with this and other genotypes were similar. PMID:9582485

  13. Use of whole blood for analysis of disease-associated biomarkers.

    PubMed

    May, Jennifer E; Pemberton, Roy M; Hart, John P; McLeod, Julie; Wilcock, Gordon; Doran, Olena

    2013-06-01

    Analyses for diagnosis and monitoring of pathological conditions often rely on blood samples, partly due to relative ease of collection. However, many interfering substances largely preclude the use of whole blood itself, necessitating separation of plasma or serum. We present a feasibility study demonstrating potential use of fresh or frozen whole blood to detect soluble biomarkers using an enzyme-linked immunosorbent assay (ELISA)-based method. Good correlation between levels of soluble CD25 in plasma and whole blood of healthy individuals or Alzheimer's patients was established. These results provide a basis for development of a novel biosensor approach for disease-associated biomarker detection in whole blood. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Diastolic Dysfunction of Hypertrophic Cardiomyopathy Genotype-Positive Subjects Without Hypertrophy Is Detected by Tissue Doppler Imaging: A Systematic Review and Meta-analysis.

    PubMed

    Liu, Wen; Sun, Dandan; Yang, Jun

    2017-10-01

    To evaluate whether diastolic dysfunction derived by tissue Doppler imaging (TDI) would be an earlier manifestation in genotype-positive hypertrophic cardiomyopathy (HCM) subjects without left ventricular hypertrophy (LVH). We systematically searched Pubmed, Medline, and Web of Science with an upper date limit of June 2016 for studies evaluating the diastolic function of HCM genotype-positive subjects without hypertrophy (G+/LVH-). Based on the inclusion criteria, eligible studies were selected. The quality of selected studies was assessed by the Newcastle-Ottawa Scale before being included in the meta-analysis. The statistic data such as weighted mean difference (WMD) and 95% confidence interval (CI) were calculated by Stata 12.0 software. Seventeen studies were included in the systematic review, and 12 were finally involved in the meta-analysis. The G+/LVH- subjects showed decreased Ea derived by TDI on both the interventricular septum (WMD [95% CI] = -1.822 [-3.104, -0.541]) and lateral wall (WMD [95% CI] = -2.269 [-3.820, -0.719]), and increased E/Ea on both interventricular septum (WMD [95% CI] = 1.363 [0.552, 2.174]) and lateral (WMD [95% CI] = 1.339 [0.386, 2.293]) wall. Tissue Doppler imaging-derived diastolic dysfunction can be found in HCM genotype-positive subjects without hypertrophy. © 2017 by the American Institute of Ultrasound in Medicine.

  15. The pathogenesis of Hirschsprung's disease-associated enterocolitis.

    PubMed

    Austin, Kelly Miller

    2012-11-01

    Hirschsprung's disease-associated enterocolitis (HAEC) remains the most life-threatening complication in Hirschsprung disease (HD) patients. The pathogenesis of HAEC has not been determined and many hypotheses regarding the etiology of HAEC have been proposed. These include a possible causal relationship between the abnormal enteric nervous system development in HD and the development of enterocolitis. Based on the complex genetic causes of HD that have been discovered and the resultant heterogeneous group of patients that exists, the causes of HAEC are likely multiple. New insights regarding the relationship of the role of the enteric nervous system and its interaction between intestinal barrier function, innate host immunity, and commensal microflora have been discovered, which may shed light on this perplexing problem. This review presents current known risk factors of HAEC and the proposed theories and supporting evidence for the potential etiologies of HAEC.

  16. Natural antisense transcripts of Alzheimer's disease associated genes.

    PubMed

    Guo, Jin-Hu; Cheng, Hai-Peng; Yu, Long; Zhao, Shouyuan

    2006-04-01

    Natural antisense transcripts (NATs), also named endogenous antisense transcripts, are a class of genes whose role in controlling gene expression is becoming more and more relevant. NATs might play important roles in gene expression and translation regulation. Present work investigated the presence of NATs of Alzheimer's disease associated genes including PRESENILIN1, PRESENILIN2, BACE1, BACE2, APP, APOE, TAU (MAPT), PRION, alpha-SYNUCLEIN (SNCA), NICASTRIN, PEN2, APH1A, APH1B as well as CD147 (BASIGIN), and the results revealed that APP, BACE2, APH1A, TAU, CD147 and alpha-SYNUCLEIN contain natural antisense transcripts. These NATs were characterized according to the sense-antisense overlapping information and potential functional mechanisms were proposed. Present findings provide preliminary but important information about transcription regulation of AD associated genes, which would further our understanding of the gene expression regulation of AD, and also suggest a novel potential strategy for the therapy of AD.

  17. First detection and genotyping of Giardia intestinalis in stool samples collected from children in Ghazni Province, eastern Afghanistan and evaluation of the PCR assay in formalin-fixed specimens.

    PubMed

    Lass, Anna; Karanis, Panagiotis; Korzeniewski, Krzysztof

    2017-08-01

    It is estimated that faecal-orally transmitted diseases are common in Afghanistan, as a consequence of poor hygienic standards of life and widespread contamination of water and food with both human and animal faeces. However, there is little information in the literature concerning infections caused by intestinal parasites in the Afghan population. In this study, we report the occurrence of Giardia intestinalis assemblages (A and B) in formalin-fixed stool samples collected from 245 Afghan schoolchildren living in Ghazni Province in eastern Afghanistan. Detection of the parasite's DNA and genotyping was performed using real-time PCR, specific to the β-giardin gene of G. intestinalis. Positive results were recorded in 52 (21.2%) samples. Genotyping was successful in 39 faecal samples and showed the predominance of assemblage B of G. intestinalis in this population (15 assemblage A and 24 assemblage B). Co-infection with both genotypes A and B was detected in four samples. Additionally, we evaluated the effect of 10% buffered formalin fixative on the detection of G. intestinalis DNA using real-time PCR and nested PCR characterised by different lengths of PCR products (74 and 479 bp, respectively). The human faeces containing the Giardia cysts were tested for 16 weeks. Amplification of G. intestinalis DNA with real-time PCR was possible up to 6 weeks of preservation of stool sample in formalin, compared to only 2 weeks with nested PCR. This suggests that real-time PCR is a more suitable tool in cases where stool samples have to be kept in formalin for longer periods of time.

  18. Comparison of Hybrid Capture II, Linear Array, and a Bead-Based Multiplex Genotyping Assay for Detection of Human Papillomavirus in Women with Negative Pap Test Results and Atypical Squamous Cells of Undetermined Significance

    PubMed Central

    Comar, Manola; Iannacone, Michelle R.; Casalicchio, Giorgia; McKay-Chopin, Sandrine; Tommasino, Massimo

    2012-01-01

    Many methods with different levels of analytical sensitivity and clinical specificity have been developed to detect the presence of high-risk (HR) types of the human papillomavirus (HPV) in cervical samples. The Hybrid Capture II (HC-II) assay is broadly used for primary screening. In addition, several HPV genotyping assays, based on PCR methods, display higher sensitivity than the HC-II and are also used in screening programs. We evaluated the performance of three HPV DNA tests, namely, the HC-II, the Linear Array (LA) HPV genotyping assay, and an HPV type-specific E7 PCR bead-based multiplex genotyping assay (TS-MPG) that is a laboratory-developed method for the detection of HPV, in 94 women with atypical squamous cells of undetermined significance (ASC-US) and in cytological samples from 86 women with a negative Pap test. The HPV prevalence with the TS-MPG assay was increased compared to the prevalence with the LA and HC-II assays. The HPV DNA prevalence in women with ASC-US was greater with the TS-MPG assay (46.2%) than with the LA (36.3%) and HC-II (29.7%) assays. The HPV DNA prevalence in the control group was greater with the TS-MPG assay (32.1%) than with the LA assay (10.7%). Two women with ASC-US who were HPV DNA negative by the HC-II and positive by the TS-MPG or/and LA assays had lesions that progressed to low-grade squamous intraepithelial and high-grade squamous intraepithelial lesions. This study shows that the TS-MPG assay exhibited higher analytical sensitivity than the LA and HC-II assays for the detection of HPV DNA, which reduces the potential to incorrectly identify a woman's HPV infection status. PMID:23035194

  19. Application of a competitive real time PCR for detection of Marteilia refringens genotype "O" and "M" in two geographical locations: The Ebro Delta, Spain and the Rhine-Meuse Delta, the Netherlands.

    PubMed

    Carrasco, Noèlia; Voorbergen-Laarman, Michal; Lacuesta, Beatriz; Furones, Dolors; Engelsma, Marc Y

    2017-07-19

    Species belonging to the genus Marteilia are protozoan parasites of bivalves. The species Marteilia refringens, jeopardizing the health of European bivalves, is included on the list of OIE notifiable pathogens. Two genotypes of Marteilia refringens are distinguished: type "O" affecting mainly oysters, and type "M" affecting mainly mussels. Historically, detection of Marteilia species is primarily carried out by histology. In recent years molecular assays are more frequently used for the detection of mollusc pathogens, also in routine monitoring. In the present work, a competitive real-time PCR assay was developed for rapid and sensitive detection of M. refringens and discrimination between "M" and "O" genotypes of M. refringens. The real-time PCR assay was shown to be analytically sensitive and specific and has a high repeatability and efficiency. Subsequent application of the assay on collected bivalves from two geographical locations, the Ebro Delta in Mediterranean Spain and the Rhine-Meuse Delta in the Netherlands resulted in detection of M. refringens type M in Mytilus galloprovincialis and M. refringens type O in Ostrea edulis from Spain. In two O. edulis specimen both M. refringens type O and type M were detected. In the Netherlands M. refringens was not observed in any of the tested Mytilus edulis and O. edulis. The results obtained by real time PCR were in correspondence with the results obtained by histopathology and a substantial agreement with the results obtained by conventional PCR. In conclusion, the developed real time PCR assay facilitates rapid detection and subtyping of M. refringens and could be applied for further studies on epidemiology of the parasite, geographical distribution and host specificity. Copyright © 2017. Published by Elsevier Inc.

  20. Transformation of QTL genotypic effects to allelic effects

    PubMed Central

    Nagamine, Yoshitaka

    2005-01-01

    The genotypic and allelic effect models are equivalent in terms of QTL detection in a simple additive model, but the QTL allelic model has the advantage of providing direct information for marker-assisted selection. However, the allelic matrix is four times as large as the genotypic IBD matrix, causing computational problems, especially in genome scans examining multiple positions. Transformation from genotypic to allelic effects, after estimating the genotypic effects with a smaller IBD matrix, can solve this problem. Although the validity of transformation from genotypic to allelic effects has been disputed, this work proves that transformation can successfully yield unique allelic effects when genotypic and allelic IBD matrixes exist. PMID:16093016

  1. Prediction of miRNA-disease Associations using an Evolutionary Tuned Latent Semantic Analysis.

    PubMed

    Pallez, Denis; Gardès, Julien; Pasquier, Claude

    2017-09-05

    MicroRNAs, small non-coding elements implied in gene regulation, are very interesting biomarkers for various diseases such as cancers. They represent potential prodigious biotechnologies for early diagnosis and gene therapies. However, experimental verification of microRNA-disease associations are time-consuming and costly, so that computational modeling is a proper solution. Previously, we designed MiRAI, a predictive method based on distributional semantics, to identify new associations between microRNA molecules and human diseases. Our preliminary results showed very good prediction scores compared to other available methods. However, MiRAI performances depend on numerous parameters that cannot be tuned manually. In this study, a parallel evolutionary algorithm is proposed for finding an optimal configuration of our predictive method. The automatically parametrized version of MiRAI achieved excellent performance. It highlighted new miRNA-disease associations, especially the potential implication of mir-188 and mir-795 in various diseases. In addition, our method allowed to detect several putative false associations contained in the reference database.

  2. Revealing disease-associated pathways by network integration of untargeted metabolomics.

    PubMed

    Pirhaji, Leila; Milani, Pamela; Leidl, Mathias; Curran, Timothy; Avila-Pacheco, Julian; Clish, Clary B; White, Forest M; Saghatelian, Alan; Fraenkel, Ernest

    2016-09-01

    Uncovering the molecular context of dysregulated metabolites is crucial to understand pathogenic pathways. However, their system-level analysis has been limited owing to challenges in global metabolite identification. Most metabolite features detected by untargeted metabolomics carried out by liquid-chromatography-mass spectrometry cannot be uniquely identified without additional, time-consuming experiments. We report a network-based approach, prize-collecting Steiner forest algorithm for integrative analysis of untargeted metabolomics (PIUMet), that infers molecular pathways and components via integrative analysis of metabolite features, without requiring their identification. We demonstrated PIUMet by analyzing changes in metabolism of sphingolipids, fatty acids and steroids in a Huntington's disease model. Additionally, PIUMet enabled us to elucidate putative identities of altered metabolite features in diseased cells, and infer experimentally undetected, disease-associated metabolites and dysregulated proteins. Finally, we established PIUMet's ability for integrative analysis of untargeted metabolomics data with proteomics data, demonstrating that this approach elicits disease-associated metabolites and proteins that cannot be inferred by individual analysis of these data.

  3. One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples

    PubMed Central

    Mijatovic-Rustempasic, Slavica; Esona, Mathew D.; Tam, Ka Ian; Quaye, Osbourne; Bowen, Michael D.

    2016-01-01

    Background. Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8–100% sensitivity, 99.7–100% specificity, 85–95% efficiency and a limit of detection of 4–60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81–92% efficiency and limit of detection of 150–600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8

  4. [Hepatitis C virus genotypes circulating in Colombia].

    PubMed

    Santos, Óscar; Gómez, Alberto; Vizcaíno, Viviana; Casas, María Consuelo; Ramírez, María Del Pilar; Olaya, Patricia

    2017-01-24

    Chronic hepatitis C virus infection is a worldwide public health problem; it has been estimated that over 180 million people are infected with this virus worldwide. Its precise incidence and prevalence (i.e., epidemiology) and the most frequent circulating genotypes in Colombia are unknown. To describe the hepatitis C virus (HCV) genotypes and subtypes in infected Colombian patients. We recovered the data on 1,538 HCV isolates from 1,527 patients in two Colombian reference laboratories typed by PAGE or qPCR. Patients' mean age was 53 years; 70% of them were 40 to 70 years old, and 52%, females; 57% of all tests were ordered in Bogotá and 80% of cases were from Cundinamarca, Valle and Atlántico departments. Genotype 1 was detected in 88.6% of cases, distributed as follows: 70% subtype 1b, 13.5% subtype 1a and 5.1%, undetermined subtypes. Genotype 2 was found in 5.4% of the patients, genotype 3 in 2% and genotype 4 in 4%. Mixed genotypes were found in 0.8% of the samples. Genotype 1 is the most common HCV genotype circulating in Colombia, and subtype 1b the most frequent.

  5. Mutation detection in the ABCC6 gene and genotype-phenotype analysis in a large international case series affected by pseudoxanthoma elasticum.

    PubMed

    Pfendner, Ellen G; Vanakker, Olivier M; Terry, Sharon F; Vourthis, Sophia; McAndrew, Patricia E; McClain, Monica R; Fratta, Sarah; Marais, Anna-Susan; Hariri, Susan; Coucke, Paul J; Ramsay, Michele; Viljoen, Denis; Terry, Patrick F; De Paepe, Anne; Uitto, Jouni; Bercovitch, Lionel G

    2007-10-01

    Pseudoxanthoma elasticum (PXE), an autosomal recessive disorder with considerable phenotypic variability, mainly affects the eyes, skin and cardiovascular system, characterised by dystrophic mineralization of connective tissues. It is caused by mutations in the ABCC6 (ATP binding cassette family C member 6) gene, which encodes MRP6 (multidrug resistance-associated protein 6). To investigate the mutation spectrum of ABCC6 and possible genotype-phenotype correlations. Mutation data were collected on an international case series of 270 patients with PXE (239 probands, 31 affected family members). A denaturing high-performance liquid chromatography-based assay was developed to screen for mutations in all 31 exons, eliminating pseudogene coamplification. In 134 patients with a known phenotype and both mutations identified, genotype-phenotype correlations were assessed. In total, 316 mutant alleles in ABCC6, including 39 novel mutations, were identified in 239 probands. Mutations were found to cluster in exons 24 and 28, corresponding to the second nucleotide-binding fold and the last intracellular domain of the protein. Together with the recurrent R1141X and del23-29 mutations, these mutations accounted for 71.5% of the total individual mutations identified. Genotype-phenotype analysis failed to reveal a significant correlation between the types of mutations identified or their predicted effect on the expression of the protein and the age of onset and severity of the disease. This study emphasises the principal role of ABCC6 mutations in the pathogenesis of PXE, but the reasons for phenotypic variability remain to be explored.

  6. Inductive matrix completion for predicting gene-disease associations.

    PubMed

    Natarajan, Nagarajan; Dhillon, Inderjit S

    2014-06-15

    Most existing methods for predicting causal disease genes rely on specific type of evidence, and are therefore limited in terms of applicability. More often than not, the type of evidence available for diseases varies-for example, we may know linked genes, keywords associated with the disease obtained by mining text, or co-occurrence of disease symptoms in patients. Similarly, the type of evidence available for genes varies-for example, specific microarray probes convey information only for certain sets of genes. In this article, we apply a novel matrix-completion method called Inductive Matrix Completion to the problem of predicting gene-disease associations; it combines multiple types of evidence (features) for diseases and genes to learn latent factors that explain the observed gene-disease associations. We construct features from different biological sources such as microarray expression data and disease-related textual data. A crucial advantage of the method is that it is inductive; it can be applied to diseases not seen at training time, unlike traditional matrix-completion approaches and network-based inference methods that are transductive. Comparison with state-of-the-art methods on diseases from the Online Mendelian Inheritance in Man (OMIM) database shows that the proposed approach is substantially better-it has close to one-in-four chance of recovering a true association in the top 100 predictions, compared to the recently proposed Catapult method (second best) that has <15% chance. We demonstrate that the inductive method is particularly effective for a query disease with no previously known gene associations, and for predicting novel genes, i.e. genes that are previously not linked to diseases. Thus the method is capable of predicting novel genes even for well-characterized diseases. We also validate the novelty of predictions by evaluating the method on recently reported OMIM associations and on associations recently reported in the literature

  7. Inductive matrix completion for predicting gene–disease associations

    PubMed Central

    Natarajan, Nagarajan; Dhillon, Inderjit S.

    2014-01-01

    Motivation: Most existing methods for predicting causal disease genes rely on specific type of evidence, and are therefore limited in terms of applicability. More often than not, the type of evidence available for diseases varies—for example, we may know linked genes, keywords associated with the disease obtained by mining text, or co-occurrence of disease symptoms in patients. Similarly, the type of evidence available for genes varies—for example, specific microarray probes convey information only for certain sets of genes. In this article, we apply a novel matrix-completion method called Inductive Matrix Completion to the problem of predicting gene-disease associations; it combines multiple types of evidence (features) for diseases and genes to learn latent factors that explain the observed gene–disease associations. We construct features from different biological sources such as microarray expression data and disease-related textual data. A crucial advantage of the method is that it is inductive; it can be applied to diseases not seen at training time, unlike traditional matrix-completion approaches and network-based inference methods that are transductive. Results: Comparison with state-of-the-art methods on diseases from the Online Mendelian Inheritance in Man (OMIM) database shows that the proposed approach is substantially better—it has close to one-in-four chance of recovering a true association in the top 100 predictions, compared to the recently proposed Catapult method (second best) that has <15% chance. We demonstrate that the inductive method is particularly effective for a query disease with no previously known gene associations, and for predicting novel genes, i.e. genes that are previously not linked to diseases. Thus the method is capable of predicting novel genes even for well-characterized diseases. We also validate the novelty of predictions by evaluating the method on recently reported OMIM associations and on associations recently

  8. A previously undescribed ostreid herpes virus 1 (OsHV-1) genotype detected in the pacific oyster, Crassostrea gigas, in Ireland.

    PubMed

    Lynch, S A; Carlsson, J; Reilly, A O; Cotter, E; Culloty, S C

    2012-10-01

    Significant mortalities of the Pacific oyster, Crassostrea gigas, have been reported worldwide since the 1950s. The impact these re-occurring mortality events have had on the C. gigas industry has highlighted the necessity to determine the factors that may be causing these mortalities. This study investigated the possible role of ostreid herpes virus (OsHV-1) in C. gigas mortalities over 2 successive summers at 2 study areas in Ireland. A single sample of adult C. gigas, which had been experiencing mortalities at one of the sites was screened. Successive cohorts of C. gigas spat obtained from a hatchery outside Ireland was relayed to both sites in 2003 and in 2004. Spat were screened each year prior to relaying. Samples were collected every 2 weeks and mortality counts were recorded and observed at both sites. Polymerase chain reaction (PCR) analysis and subsequent sequencing indicated that a previously undocumented variant genotype of OsHV-1 was present in the single cohort of adult C. gigas and in seed and juveniles at both sites, in both years. Analysis suggests that the Irish OsHV-1 μvar variant genotype is closely related to OsHV-1 μvar, first described in France in 2008.

  9. An analysis of human microbe-disease associations.

    PubMed

    Ma, Wei; Zhang, Lu; Zeng, Pan; Huang, Chuanbo; Li, Jianwei; Geng, Bin; Yang, Jichun; Kong, Wei; Zhou, Xuezhong; Cui, Qinghua

    2017-01-01

    The microbiota living in the human body has critical impacts on our health and disease, but a systems understanding of its relationships with disease remains limited. Here, we use a large-scale text mining-based manually curated microbe-disease association data set to construct a microbe-based human disease network and investigate the relationships between microbes and disease genes, symptoms, chemical fragments and drugs. We reveal that microbe-based disease loops are significantly coherent. Microbe-based disease connections have strong overlaps with those constructed by disease genes, symptoms, chemical fragments and drugs. Moreover, we confirm that the microbe-based disease analysis is able to predict novel connections and mechanisms for disease, microbes, genes and drugs. The presented network, methods and findings can be a resource helpful for addressing some issues in medicine, for example, the discovery of bench knowledge and bedside clinical solutions for disease mechanism understanding, diagnosis and therapy. © The Author 2016. Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

  10. Moyamoya disease associated with pulmonary sarcoidosis--case report.

    PubMed

    Takenaka, K; Ito, M; Kumagai, M; Yamakawa, H; Sugimoto, Y; Yamakawa, H; Nishimura, Y; Sakai, N

    1998-09-01

    A 61-year-old female presented with a unique case of moyamoya disease associated with pulmonary sarcoidosis. She was admitted for sudden onset of left temporalgia with episode of numbness on face, tongue, and upper extremity on the right side. The next morning, she had symptoms of Gerstmann syndrome and her ability to speak was disturbed. Her medical history included radical resection of lung cancer on the right side. She had no symptoms of pulmonary sarcoidosis. Neuroimaging showed an infarction in the left occipital lobe. Angiography showed occlusions of the bilateral internal carotid arteries at the supraclinoid portions. Subsequently, a left superficial temporal artery-middle cerebral artery anastomosis with encephalo-myo-synangiosis was performed. Ninety-three days after admission, she suddenly developed dyspnea which resulted in death 3 hours later. Autopsy findings showed typical epithelioid granulomas of sarcoid type in the lymph nodes of the peribronchus, lung, and liver. Thrombotic emboli were found in the bilateral pulmonary arteries, and marked fibrous intimal thickening in the bilateral internal carotid arteries. Immunological reaction with inflammatory events may cause pathological changes in patients with moyamoya disease or sarcoidosis. The co-incidence in this case suggests that some common inflammatory events may be involved in the pathogenesis of these diseases.

  11. Cancer-disease associations: A visualization and animation through medical big data.

    PubMed

    Iqbal, Usman; Hsu, Chun-Kung; Nguyen, Phung Anh Alex; Clinciu, Daniel Livius; Lu, Richard; Syed-Abdul, Shabbir; Yang, Hsuan-Chia; Wang, Yao-Chin; Huang, Chu-Ya; Huang, Chih-Wei; Chang, Yo-Cheng; Hsu, Min-Huei; Jian, Wen-Shan; Li, Yu-Chuan Jack

    2016-04-01

    Cancer is the primary disease responsible for death and disability worldwide. Currently, prevention and early detection represents the best hope for cure. Knowing the expected diseases that occur with a particular cancer in advance could lead to physicians being able to better tailor their treatment for cancer. The aim of this study was to build an animated visualization tool called as Cancer Associations Map Animation (CAMA), to chart the association of cancers with other disease over time. The study population was collected from the Taiwan National Health Insurance Database during the period January 2000 to December 2002, 782 million outpatient visits were used to compute the associations of nine major cancers with other diseases. A motion chart was used to quantify and visualize the associations between diseases and cancers. The CAMA motion chart that was built successfully facilitated the observation of cancer-disease associations across ages and genders. The CAMA system can be accessed online at http://203.71.86.98/web/runq16.html. The CAMA animation system is an animated medical data visualization tool which provides a dynamic, time-lapse, animated view of cancer-disease associations across different age groups and gender. Derived from a large, nationwide healthcare dataset, this exploratory data analysis tool can detect cancer comorbidities earlier than is possible by manual inspection. Taking into account the trajectory of cancer-specific comorbidity development may facilitate clinicians and healthcare researchers to more efficiently explore early stage hypotheses, develop new cancer treatment approaches, and identify potential effect modifiers or new risk factors associated with specific cancers. Copyright © 2016. Published by Elsevier Ireland Ltd.

  12. Genotype 3b of human parvovirus B19 detected from hospitalized children with solid malignancies in a North Indian tertiary care hospital.

    PubMed

    Jain, Amita; Jain, Parul; Prakash, Shantanu; Kumar, Archana; Khan, Danish N; Seth, Akansha; Gupta, Shikha; Kant, Ravi

    2016-11-01

    Human parvovirus B19 (B19V) infection is known to cause serious consequences in immuno-compromized individuals. The present cross sectional study was designed to estimate the prevalence and genotype distribution of B19V in children receiving chemotherapy for solid malignancies at a tertiary care hospital in North India during October 2013 to May 2015. Serum samples from all the patients were tested for anti-B19V IgM and IgG antibodies and for B19V-DNA as soon as received. Samples testing positive for B19V-DNA were subjected to viral load estimation and to genotype determination by sequencing. Total 96 children were enrolled of which 9 (9.3%), 32 (33.3%), and 25 (26%) tested positive for anti-B19V IgM, anti-B19V IgG, and B19V-DNA, respectively. The viral load of B19V-DNA positive children ranged from 5.5 × 10(2) to 3.5 × 10(12) copies/ml. Accordingly children were divided into three groups: group I, with acute infection (n = 25); group II, previously exposed (n = 27), and group III, negative for B19V infection or with inappropriate antibody response (n = 44). B19V positivity was significantly associated (P-value < 0.0001) with a history of blood transfusion in the past 6 months, severe anemia (hemoglobin levels <6 gm%) and thrombocytopenia (platelets <150,000/cu.mm.). Sequence analysis of 21 of 25 DNA positive samples showed that all of them were Genotype 3b that clustered into three groups. All the sequences within each cluster were identical. The nucleotide identity of the sequences suggests a nosocomial outbreak of B19V during the study period. Children on chemotherapy for solid tumors should be routinely screened for B19V infection by both serology and PCR. J. Med. Virol. 88:1922-1929, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  13. Detection of 15 novel mutations in 52 children from 40 families with the Marfan or Loeys-Dietz syndrome and phenotype-genotype correlations.

    PubMed

    Pees, C; Michel-Behnke, I; Hagl, M; Laccone, F

    2014-12-01

    We report about 52 pediatric patients of 40 different families with confirmed Marfan syndrome (MFS) in 49 patients and Loeys-Dietz syndrome (LDS) in 3 patients. We found 39 different mutations, 15 of them being novel. Phenotype-genotype correlation in the 49 MFS patients showed that the majority of patients carrying mutations in exons 1-21 had ectopic lens (80%). Patients having mutations in exons 23-32 had a higher probability of aortic root dilation, in 50% even above a z score of 3. We found three children with neonatal MFS form, two of them with novel mutations. Of the three LDS patients, only one presented with the typical phenotype of LDS type 1. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. Diseases Associated with Defective Responses to DNA Damage

    PubMed Central

    O’Driscoll, Mark

    2012-01-01

    Within the last decade, multiple novel congenital human disorders have been described with genetic defects in known and/or novel components of several well-known DNA repair and damage response pathways. Examples include disorders of impaired nucleotide excision repair, DNA double-strand and single-strand break repair, as well as compromised DNA damage-induced signal transduction including phosphorylation and ubiquitination. These conditions further reinforce the importance of multiple genome stability pathways for health and development in humans. Furthermore, these conditions inform our knowledge of the biology of the mechanics of genome stability and in some cases provide potential routes to help exploit these pathways therapeutically. Here, I will review a selection of these exciting findings from the perspective of the disorders themselves, describing how they were identified, how genotype informs phenotype, and how these defects contribute to our growing understanding of genome stability pathways. PMID:23209155

  15. Prevalence of hepatitis viruses in patients with acute hepatitis and characterization of the detected genotype 4 hepatitis E virus sequences in Mongolia.

    PubMed

    Tsatsralt-Od, Bira; Baasanjav, Nachin; Nyamkhuu, Dulmaa; Ohnishi, Hiroshi; Takahashi, Masaharu; Okamoto, Hiroaki

    2016-02-01

    Hepatitis E is considered to be a worldwide public health problem. Although the prevalence of hepatitis E virus (HEV) antibodies in healthy individuals is noted to be 11%, no patients with acute hepatitis E have previously been identified in Mongolia. Three hundred two consecutive patients (183 males and 119 females; median age of 22.0 [Interquartile range: 18.3-25.0] years) who were clinically diagnosed with sporadic acute hepatitis during 2012-2013 in Ulaanbaatar, Mongolia, were studied. By serological and/or molecular approaches, 77 (25.5%), 93 (30.8%), 19 (6.3%), 48 (15.9%), and 12 (4.0%) of the patients were diagnosed with acute hepatitis of types A, B, C, D (superinfection of hepatitis delta virus on a background of chronic hepatitis B virus infection) and E, respectively, while the cause of hepatitis was unknown in the remaining 53 patients (17.5%). The 12 hepatitis E patients had no history of travel abroad in the 3 months before the onset of disease, and lived separately in fixed or movable houses with water supplied via pipe, tank or well, denying transmission from a common water supply. The 12 HEV isolates obtained from the patients showed high nucleotide identities of 99.7-100%, and a representative HEV isolate, MNE13-227, was closest to the Chinese isolates of genotype 4, with the highest identity of 97.3% in the 304-nt ORF2 sequence and 92.1% over the entire genome. The present study revealed the occurrence of autochthonous acute hepatitis E in Mongolia, caused by a monophyletic genotype 4 HEV strain.

  16. Construction of High Density Sweet Cherry (Prunus avium L.) Linkage Maps Using Microsatellite Markers and SNPs Detected by Genotyping-by-Sequencing (GBS).

    PubMed

    Guajardo, Verónica; Solís, Simón; Sagredo, Boris; Gainza, Felipe; Muñoz, Carlos; Gasic, Ksenija; Hinrichsen, Patricio

    2015-01-01

    Linkage maps are valuable tools in genetic and genomic studies. For sweet cherry, linkage maps have been constructed using mainly microsatellite markers (SSRs) and, recently, using single nucleotide polymorphism markers (SNPs) from a cherry 6K SNP array. Genotyping-by-sequencing (GBS), a new methodology based on high-throughput sequencing, holds great promise for identification of high number of SNPs and const