Molecular beacon probes-base multiplex NASBA Real-time for detection of HIV-1 and HCV.

PubMed

Mohammadi-Yeganeh, S; Paryan, M; Mirab Samiee, S; Kia, V; Rezvan, H

2012-06-01

Developed in 1991, nucleic acid sequence-based amplification (NASBA) has been introduced as a rapid molecular diagnostic technique, where it has been shown to give quicker results than PCR, and it can also be more sensitive. This paper describes the development of a molecular beacon-based multiplex NASBA assay for simultaneous detection of HIV-1 and HCV in plasma samples. A well-conserved region in the HIV-1 pol gene and 5'-NCR of HCV genome were used for primers and molecular beacon design. The performance features of HCV/HIV-1 multiplex NASBA assay including analytical sensitivity and specificity, clinical sensitivity and clinical specificity were evaluated. The analysis of scalar concentrations of the samples indicated that the limit of quantification of the assay was <1000 copies/ml for HIV-1 and <500 copies/ml for HCV with 95% confidence interval. Multiplex NASBA assay showed a 98% sensitivity and 100% specificity. The analytical specificity study with BLAST software demonstrated that the primers do not attach to any other sequences except for that of HIV-1 or HCV. The primers and molecular beacon probes detected all HCV genotypes and all major variants of HIV-1. This method may represent a relatively inexpensive isothermal method for detection of HIV-1/HCV co-infection in monitoring of patients.

Molecular assays reveal the presence and diversity of genes encoding pea footrot pathogenicity determinants in Nectria haematococca and in agricultural soils.

PubMed

Etebu, E; Osborn, A M

2009-05-01

The aim of this study was to develop molecular assays for investigating the presence and diversity of pathogenicity genes from the pea footrot pathogen Nectria haematococca (anamorph Fusarium solani f.sp. pisi) in soils. Polymerase chain reaction (PCR) assays were developed to amplify four N. haematococca pathogenicity genes (PDA, PEP1, PEP3 and PEP5) from isolates and soil-DNA from five agricultural fields with a prior footrot history. A collection of 15 fungi isolated on medium selective for Fusarium spp. exhibited variation in their virulence to peas as assessed via a disease index (DI: 0-5; no virulence to the highest virulence). PCR analyses showed that three isolates in which all four pathogenicity genes were detected resulted in the highest DI (>3.88). All four pathogenicity genes were detected in soil-DNA obtained from all five fields with a footrot disease history, but were not amplified from soils, which had no footrot history. Denaturing gradient gel electrophoresis and/or sequence analysis revealed diversity amongst the pathogenicity genes. The PCR assays developed herein enable the specific detection of pathogenic N. haematococca in soils without recourse to culture. Molecular assays that specifically target pathogenicity genes have the capacity to assess the presence of the footrot-causing pathogen in agricultural soils.

A molecular approach for the rapid, selective and sensitive detection of Exophiala jeanselmei in environmental samples: development and performance assessment of a real-time PCR assay.

PubMed

Libert, X; Chasseur, C; Packeu, A; Bureau, F; Roosens, N H; De Keersmaecker, S J C

2016-02-01

Exophiala jeanselmei is an opportunistic pathogenic black yeast growing in humid environments such as water reservoirs of air-conditioning systems. Because this fungal contaminant could be vaporized into the air and subsequently cause health problems, its monitoring is recommended. Currently, this monitoring is based on culture and microscopic identification which are complex, sometimes ambiguous and time-demanding, i.e., up to 21 days. Therefore, molecular, culture-independent methods could be more advantageous for the monitoring of E. jeanselmei. In this study, we developed a SYBR®green real-time PCR assay based on the internal transcribed spacer 2 from the 18S ribosomal DNA complex for the specific detection of E. jeanselmei. The selectivity (100 %), PCR efficiency (95.5 %), dynamic range and repeatability of this qPCR assay were subsequently evaluated. The limit of detection for this qPCR assay was determined to be 1 copy of genomic DNA of E. jeanselmei. Finally, water samples collected from cooling reservoirs were analyzed using this qPCR assay to deliver a proof of concept for the molecular detection of E. jeanselmei in environmental samples. The results obtained by molecular analysis were compared with those of classical methods (i.e., culture and microscopic identification) used in routine analysis and were 100 % matching. This comparison demonstrated that this SYBR®green qPCR assay can be used as a molecular alternative for monitoring and routine investigation of samples contaminated by E. jeanselmei, while eliminating the need for culturing and thereby considerably decreasing the required analysis time to 2 days.

The yeast p53 functional assay: a new tool for molecular epidemiology. Hopes and facts.

PubMed

Fronza, G; Inga, A; Monti, P; Scott, G; Campomenosi, P; Menichini, P; Ottaggio, L; Viaggi, S; Burns, P A; Gold, B; Abbondandolo, A

2000-04-01

The assumption of molecular epidemiology that carcinogens leave fingerprints has suggested that analysis of the frequency, type, and site of mutations in genes frequently altered in carcinogenesis may provide clues to the identification of the factors contributing to carcinogenesis. In this mini-review, we revise the development, and validation of the yeast-based p53 functional assay as a new tool for molecular epidemiology. We show that this assay has some very interesting virtues but also has some drawbacks. The yeast functional assay can be used to determine highly specific mutation fingerprints in the human p53 cDNA sequence. Discrimination is possible when comparing mutation spectra induced by sufficiently different mutagens. However, we also reported that the same carcinogen may induce distinguishable mutation spectra due to known influencing factors.

Current use and acceptability of novel diagnostic tests for active tuberculosis: a worldwide survey

PubMed Central

Amicosante, Massimo; D’Ambrosio, Lia; Munoz, Marcela; Mello, Fernanda Carvalho de Queiroz; Tebruegge, Marc; Chegou, Novel Njweipi; Seghrouchni, Fouad; Centis, Rosella; Goletti, Delia; Bothamley, Graham; Migliori, Giovanni Battista

2017-01-01

ABSTRACT Objective: To determine the current use and potential acceptance (by tuberculosis experts worldwide) of novel rapid tests for the diagnosis of tuberculosis that are in line with World Health Organization target product profiles. Methods: A multilingual survey was disseminated online between July and November of 2016. Results: A total of 723 individuals from 114 countries responded to the survey. Smear microscopy was the most commonly used rapid tuberculosis test (available to 90.9% of the respondents), followed by molecular assays (available to 70.7%). Only a small proportion of the respondents in middle- and low-income countries had access to interferon-gamma-release assays. Serological and lateral flow immunoassays were used by more than a quarter (25.4%) of the respondents. Among the respondents who had access to molecular tests, 46.7% were using the Xpert assay overall, that proportion being higher in lower middle-income countries (55.6%) and low-income countries (76.6%). The data also suggest that there was some alignment of pricing for molecular assays. Respondents stated they would accept novel rapid tuberculosis tests if available, including molecular assays (acceptable to 86.0%) or biomarker-based serological assays (acceptable to 81.7%). Simple biomarker-based assays were more commonly deemed acceptable in middle- and low-income countries. Conclusions: Second-generation molecular assays have become more widely available in high- and low-resource settings. However, the development of novel rapid tuberculosis tests continues to be considered important by tuberculosis experts. Our data also underscore the need for additional training and education of end users. PMID:29160384

Current use and acceptability of novel diagnostic tests for active tuberculosis: a worldwide survey.

PubMed

Amicosante, Massimo; D'Ambrosio, Lia; Munoz, Marcela; Mello, Fernanda Carvalho de Queiroz; Tebruegge, Marc; Chegou, Novel Njweipi; Seghrouchni, Fouad; Centis, Rosella; Goletti, Delia; Bothamley, Graham; Migliori, Giovanni Battista

2017-01-01

To determine the current use and potential acceptance (by tuberculosis experts worldwide) of novel rapid tests for the diagnosis of tuberculosis that are in line with World Health Organization target product profiles. A multilingual survey was disseminated online between July and November of 2016. A total of 723 individuals from 114 countries responded to the survey. Smear microscopy was the most commonly used rapid tuberculosis test (available to 90.9% of the respondents), followed by molecular assays (available to 70.7%). Only a small proportion of the respondents in middle- and low-income countries had access to interferon-gamma-release assays. Serological and lateral flow immunoassays were used by more than a quarter (25.4%) of the respondents. Among the respondents who had access to molecular tests, 46.7% were using the Xpert assay overall, that proportion being higher in lower middle-income countries (55.6%) and low-income countries (76.6%). The data also suggest that there was some alignment of pricing for molecular assays. Respondents stated they would accept novel rapid tuberculosis tests if available, including molecular assays (acceptable to 86.0%) or biomarker-based serological assays (acceptable to 81.7%). Simple biomarker-based assays were more commonly deemed acceptable in middle- and low-income countries. Second-generation molecular assays have become more widely available in high- and low-resource settings. However, the development of novel rapid tuberculosis tests continues to be considered important by tuberculosis experts. Our data also underscore the need for additional training and education of end users.

A molecular-beacon-based asymmetric PCR assay for easy visualization of amplicons in the diagnosis of trichomoniasis.

PubMed

Sonkar, Subash C; Sachdev, Divya; Mishra, Prashant K; Kumar, Anita; Mittal, Pratima; Saluja, Daman

2016-12-15

The currently available nucleic acid amplification tests (NAATs) for trichomoniasis are accurate, quick and confirmative with superior sensitivity than traditional culture-based microbiology assays. However, these assays are associated with problems of carry over contamination, false positive results, requirement of technical expertise for performance and detection of end product. Hence, a diagnostic assay with easy visualization of the amplified product will be profitable. An in-house, rapid, sensitive, specific molecular-beacon-based PCR assay, using primers against pfoB gene of Trichomonas vaginalis, was developed and evaluated using dry ectocervical swabs (n=392) from symptomatic females with vaginal discharge. Total DNA was isolated and used as template for the PCR assays. The performance and reproducibility of PCR assay was evaluated by composite reference standard (CRS). For easy visualization of the amplified product, molecular-beacon was designed and amplicons were visualized directly using fluorescent handheld dark reader or by Micro-Plate Reader. Molecular-beacons are single-stranded hairpin shaped nucleic acid probes composed of a stem, with fluorophore/quencher pair and a loop region complementary to the desired DNA. The beacon-based PCR assay designed in the present study is highly specific as confirmed by competition experiments and extremely sensitive with detection limit of 20fg of genomic DNA (3-4 pathogens). The minimum infrastructure requirement and ease to perform the assay makes this method highly useful for resource poor countries for better disease management. Copyright © 2016 Elsevier B.V. All rights reserved.

Planar Supported Membranes with Mobile SNARE Proteins and Quantitative Fluorescence Microscopy Assays to Study Synaptic Vesicle Fusion

PubMed Central

Kiessling, Volker; Liang, Binyong; Kreutzberger, Alex J. B.; Tamm, Lukas K.

2017-01-01

Synaptic vesicle membrane fusion, the process by which neurotransmitter gets released at the presynaptic membrane is mediated by a complex interplay between proteins and lipids. The realization that the lipid bilayer is not just a passive environment where other molecular players like SNARE proteins act, but is itself actively involved in the process, makes the development of biochemical and biophysical assays particularly challenging. We summarize in vitro assays that use planar supported membranes and fluorescence microscopy to address some of the open questions regarding the molecular mechanisms of SNARE-mediated membrane fusion. Most of the assays discussed in this mini-review were developed in our lab over the last 15 years. We emphasize the sample requirements that we found are important for the successful application of these methods. PMID:28360838

EVALUATING THE ABILITY OF IN VITRO ASSAYS BASED ON KEY EVENTS IN NEURODEVELOPMENT TO PREDICT DEVELOPMENTAL NEUROTOXICITY (DNT)

EPA Science Inventory

USABecause of the multitude of potential molecular targets for chemical disruption in the developing nervous system, our laboratory has developed in vitro assays that measure chemical disruption of key neurodevelopmental processes. These include proliferation, differentiation, ap...

STRESS PATHWAY-BASED REPORTER ASSAYS TO ASSESS TOXICITY OF ENVIRONMENTAL CHEMICALS.

EPA Science Inventory

There is an increasing need for assays for the rapid and efficient assessment of toxicities of large numbers of environmental chemicals. To meet this need, we are developing cell-based reporter assays that measure the activation of key molecular stress pathways. We are using pro...

Molecular Assays for Determining Mycobacterium leprae Viability in Tissues of Experimentally Infected Mice

PubMed Central

Davis, Grace L.; Ray, Nashone A.; Lahiri, Ramanuj; Gillis, Thomas P.; Krahenbuhl, James L.; Williams, Diana L.; Adams, Linda B.

2013-01-01

Background The inability of Mycobacterium leprae to grow on axenic media has necessitated specialized techniques in order to determine viability of this organism. The purpose of this study was to develop a simple and sensitive molecular assay for determining M. leprae viability directly from infected tissues. Methodology/Principle Findings Two M. leprae-specific quantitative reverse transcription PCR (qRT-PCR) assays based on the expression levels of esxA, encoding the ESAT-6 protein, and hsp18, encoding the heat shock 18 kDa protein, were developed and tested using infected footpad (FP) tissues of both immunocompetent and immunocompromised (athymic nu/nu) mice. In addition, the ability of these assays to detect the effects of anti-leprosy drug treatment on M. leprae viability was determined using rifampin and rifapentine, each at 10 mg/kg for 1, 5, or 20 daily doses, in the athymic nu/nu FP model. Molecular enumeration (RLEP PCR) and viability determinations (qRT-PCR) were performed via Taqman methodology on DNA and RNA, respectively, purified from ethanol-fixed FP tissue and compared with conventional enumeration (microscopic counting of acid fast bacilli) and viability assays (radiorespirometry, viability staining) which utilized bacilli freshly harvested from the contralateral FP. Both molecular and conventional assays demonstrated growth and high viability of M. leprae in nu/nu FPs over a 4 month infection period. In contrast, viability was markedly decreased by 8 weeks in immunocompetent mice. Rifapentine significantly reduced bacterial viability after 5 treatments, whereas rifampin required up to 20 treatments for the same efficacy. Neither drug was effective after a single treatment. In addition, host gene expression was monitored with the same RNA preparations. Conclusions hsp18 and esxA qRT-PCR are sensitive molecular indicators, reliably detecting viability of M. leprae in tissues without the need for bacterial isolation or immediate processing, making these assays applicable for in vivo drug screening and promising for clinical and field applications. PMID:24179562

Molecular assays for determining Mycobacterium leprae viability in tissues of experimentally infected mice.

PubMed

Davis, Grace L; Ray, Nashone A; Lahiri, Ramanuj; Gillis, Thomas P; Krahenbuhl, James L; Williams, Diana L; Adams, Linda B

2013-01-01

The inability of Mycobacterium leprae to grow on axenic media has necessitated specialized techniques in order to determine viability of this organism. The purpose of this study was to develop a simple and sensitive molecular assay for determining M. leprae viability directly from infected tissues. Two M. leprae-specific quantitative reverse transcription PCR (qRT-PCR) assays based on the expression levels of esxA, encoding the ESAT-6 protein, and hsp18, encoding the heat shock 18 kDa protein, were developed and tested using infected footpad (FP) tissues of both immunocompetent and immunocompromised (athymic nu/nu) mice. In addition, the ability of these assays to detect the effects of anti-leprosy drug treatment on M. leprae viability was determined using rifampin and rifapentine, each at 10 mg/kg for 1, 5, or 20 daily doses, in the athymic nu/nu FP model. Molecular enumeration (RLEP PCR) and viability determinations (qRT-PCR) were performed via Taqman methodology on DNA and RNA, respectively, purified from ethanol-fixed FP tissue and compared with conventional enumeration (microscopic counting of acid fast bacilli) and viability assays (radiorespirometry, viability staining) which utilized bacilli freshly harvested from the contralateral FP. Both molecular and conventional assays demonstrated growth and high viability of M. leprae in nu/nu FPs over a 4 month infection period. In contrast, viability was markedly decreased by 8 weeks in immunocompetent mice. Rifapentine significantly reduced bacterial viability after 5 treatments, whereas rifampin required up to 20 treatments for the same efficacy. Neither drug was effective after a single treatment. In addition, host gene expression was monitored with the same RNA preparations. hsp18 and esxA qRT-PCR are sensitive molecular indicators, reliably detecting viability of M. leprae in tissues without the need for bacterial isolation or immediate processing, making these assays applicable for in vivo drug screening and promising for clinical and field applications.

Molecular markers in disease detection and follow-up of patients with non-muscle invasive bladder cancer.

PubMed

Maas, Moritz; Walz, Simon; Stühler, Viktoria; Aufderklamm, Stefan; Rausch, Steffen; Bedke, Jens; Stenzl, Arnulf; Todenhöfer, Tilman

2018-05-01

Diagnosis and surveillance of non-muscle invasive bladder cancer (NMIBC) is mainly based on endoscopic bladder evaluation and urine cytology. Several assays for determining additional molecular markers (urine-, tissue- or blood-based) have been developed in recent years but have not been included in clinical guidelines so far. Areas covered: This review gives an update on different molecular markers in the urine and evaluates their role in patients with NMIBC in disease detection and surveillance. Moreover, the potential of recent approaches such as DNA methylation assays, multi-panel RNA gene expression assays and cell-free DNA analysis is assessed. Expert commentary: Most studies on various molecular urine markers have mainly focused on a potential replacement of cystoscopy. New developments in high throughput technologies and urine markers may offer further advantages as they may represent a non-invasive approach for molecular characterization of the disease. This opens new options for individualized surveillance strategies and may help to choose the best therapeutic option. The implementation of these technologies in well-designed clinical trials is essential to further promote the use of urine diagnostics in the management of patients with NMIBC.

Phenotypic screening for developmental neurotoxicity ...

EPA Pesticide Factsheets

There are large numbers of environmental chemicals with little or no available information on their toxicity, including developmental neurotoxicity. Because of the resource-intensive nature of traditional animal tests, high-throughput (HTP) methods that can rapidly evaluate chemicals for the potential to affect the developing brain are being explored. Typically, HTP screening uses biochemical and molecular assays to detect the interaction of a chemical with a known target or molecular initiating event (e.g., the mechanism of action). For developmental neurotoxicity, however, the mechanism(s) is often unknown. Thus, we have developed assays for detecting chemical effects on the key events of neurodevelopment at the cellular level (e.g., proliferation, differentiation, neurite growth, synaptogenesis, network formation). Cell-based assays provide a test system at a level of biological complexity that encompasses many potential neurotoxic mechanisms. For example, phenotypic assessment of neurite outgrowth at the cellular level can detect chemicals that target kinases, ion channels, or esterases at the molecular level. The results from cell-based assays can be placed in a conceptual framework using an Adverse Outcome Pathway (AOP) which links molecular, cellular, and organ level effects with apical measures of developmental neurotoxicity. Testing a wide range of concentrations allows for the distinction between selective effects on neurodevelopmental and non-specific

Real-time monitoring of disintegration activity of catalytic core domain of HIV-1 integrase using molecular beacon.

PubMed

Zhang, Da-wei; Zhao, Ming-ming; He, Hong-qiu; Guo, Shun-xing

2013-09-15

HIV-1 integrase, an essential enzyme for retroviral replication, is a validated target for anti-HIV therapy development. The catalytic core domain of integrase (IN-CCD) is capable of catalyzing disintegration reaction. In this work, a hairpin-shaped disintegration substrate was designed and validated by enzyme-linked immunosorbent assay; a molecular beacon-based assay was developed for disintegration reaction of IN-CCD. Results showed that the disintegration substrate could be recognized and catalyzed by IN-CCD, and the disintegration reaction can be monitored according to the increase of fluorescent signal. The assay can be applied to real-time detection of disintegration with advantages of simplicity, high sensitivity, and excellent specificity. Copyright © 2013 Elsevier Inc. All rights reserved.

A review of current and future molecular diagnostic tests for use in the microbiology laboratory.

PubMed

Jannes, Geert; De Vos, Daniel

2006-01-01

Nucleic acid-based diagnostics gradually are replacing or complementing culture-based, biochemical, and immunological assays in routine microbiology laboratories. Similar to conventional tests, the first-generation deoxyribonucleic acid assays determined only a single analyte. Recent improvements in detection technologies have paved the way for the development of multiparameter assays using macroarrays or micro-arrays, while the introduction of closed-tube real-time polymerase chain reaction systems has resulted in the development of rapid microbial diagnostics with a reduced contamination risk. The use of these new molecular technologies is not restricted to detection and identification of microbial pathogens but also can be used for genotyping, allowing one to determine antibiotic resistance or to perform microbial fingerprinting.

Use of Bispecific Antibodies in Molecular Velcro Assays Whose Specificity Approaches the Theoretical Limit of Immunodetection for Bordetella pertussis

PubMed Central

Tang, X. L.; Peppler, M. S.; Irvin, R. T.; Suresh, M. R.

2004-01-01

A bispecific monoclonal antibody (bsMAb) that detects Bordetella pertussis, the causative agent of whooping cough, and horseradish peroxidase (HRPO) has been developed by use of the quadroma technology. A quadroma, P123, was produced by fusing two well-characterized hybridomas against the bacterium and the enzyme and was subcloned to obtain a stable bsMAb-secreting cell line. The quadroma was theoretically expected to produce up to 10 different molecular species of immunoglobulins, so secreted bispecific antibody was complexed with excess HRPO and the HRPO-bsMAb complex was purified in one step by benzhydroxamic acid-agarose affinity cochromatography. An ultrasensitive homosandwich molecular “velcro” enzyme-linked immunosorbent assay for the detection of B. pertussis whole bacteria with HRPO-bsMAb was established in both microplate and nasopharyngeal swab formats. This assay demonstrates a high sensitivity that approaches the theoretical limit of detection of one bacterium. This new nanoprobe can be used to develop a new generation of assays that are simple, inexpensive alternatives to quantitative PCR and that can be used by clinical laboratories. This strategy of homosandwich assays with solid-phase monospecific antibodies and solution-phase bsMAb with specificity for the same repeating surface determinants can be applied to generate ultrasensitive immunodiagnostic assays for viruses and bacteria. PMID:15242951

DNA Methyltransferase Activity Assays: Advances and Challenges

PubMed Central

Poh, Wan Jun; Wee, Cayden Pang Pee; Gao, Zhiqiang

2016-01-01

DNA methyltransferases (MTases), a family of enzymes that catalyse the methylation of DNA, have a profound effect on gene regulation. A large body of evidence has indicated that DNA MTase is potentially a predictive biomarker closely associated with genetic disorders and genetic diseases like cancer. Given the attention bestowed onto DNA MTases in molecular biology and medicine, highly sensitive detection of DNA MTase activity is essential in determining gene regulation, epigenetic modification, clinical diagnosis and therapeutics. Conventional techniques such as isotope labelling are effective, but they often require laborious sample preparation, isotope labelling, sophisticated equipment and large amounts of DNA, rendering them unsuitable for uses at point-of-care. Simple, portable, highly sensitive and low-cost assays are urgently needed for DNA MTase activity screening. In most recent technological advances, many alternative DNA MTase activity assays such as fluorescent, electrochemical, colorimetric and chemiluminescent assays have been proposed. In addition, many of them are coupled with nanomaterials and/or enzymes to significantly enhance their sensitivity. Herein we review the progress in the development of DNA MTase activity assays with an emphasis on assay mechanism and performance with some discussion on challenges and perspectives. It is hoped that this article will provide a broad coverage of DNA MTase activity assays and their latest developments and open new perspectives toward the development of DNA MTase activity assays with much improved performance for uses in molecular biology and clinical practice. PMID:26909112

Assay for Arf GTP-binding Proteins | NCI Technology Transfer Center | TTC

Cancer.gov

The National Cancer Institute's Laboratory of Cellular and Molecular Biology is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize an antibody-based proteomics assay.

A novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods.

PubMed

Nixon, Gavin J; Svenstrup, Helle F; Donald, Carol E; Carder, Caroline; Stephenson, Judith M; Morris-Jones, Stephen; Huggett, Jim F; Foy, Carole A

2014-12-01

Molecular diagnostic measurements are currently underpinned by the polymerase chain reaction (PCR). There are also a number of alternative nucleic acid amplification technologies, which unlike PCR, work at a single temperature. These 'isothermal' methods, reportedly offer potential advantages over PCR such as simplicity, speed and resistance to inhibitors and could also be used for quantitative molecular analysis. However there are currently limited mechanisms to evaluate their quantitative performance, which would assist assay development and study comparisons. This study uses a sexually transmitted infection diagnostic model in combination with an adapted metric termed isothermal doubling time (IDT), akin to PCR efficiency, to compare quantitative PCR and quantitative loop-mediated isothermal amplification (qLAMP) assays, and to quantify the impact of matrix interference. The performance metric described here facilitates the comparison of qLAMP assays that could assist assay development and validation activities.

Verification of performance specifications for a US Food and Drug Administration-approved molecular microbiology test: Clostridium difficile cytotoxin B using the Becton, Dickinson and Company GeneOhm Cdiff assay.

PubMed

Schlaberg, Robert; Mitchell, Michael J; Taggart, Edward W; She, Rosemary C

2012-01-01

US Food and Drug Administration (FDA)-approved diagnostic tests based on molecular genetic technologies are becoming available for an increasing number of microbial pathogens. Advances in technology and lower costs have moved molecular diagnostic tests formerly performed for research purposes only into much wider use in clinical microbiology laboratories. To provide an example of laboratory studies performed to verify the performance of an FDA-approved assay for the detection of Clostridium difficile cytotoxin B compared with the manufacturer's performance standards. We describe the process and protocols used by a laboratory for verification of an FDA-approved assay, assess data from the verification studies, and implement the assay after verification. Performance data from the verification studies conducted by the laboratory were consistent with the manufacturer's performance standards and the assay was implemented into the laboratory's test menu. Verification studies are required for FDA-approved diagnostic assays prior to use in patient care. Laboratories should develop a standardized approach to verification studies that can be adapted and applied to different types of assays. We describe the verification of an FDA-approved real-time polymerase chain reaction assay for the detection of a toxin gene in a bacterial pathogen.

Detection and Characterization of Viral Species/Subspecies Using Isothermal Recombinase Polymerase Amplification (RPA) Assays.

PubMed

Glais, Laurent; Jacquot, Emmanuel

2015-01-01

Numerous molecular-based detection protocols include an amplification step of the targeted nucleic acids. This step is important to reach the expected sensitive detection of pathogens in diagnostic procedures. Amplifications of nucleic acid sequences are generally performed, in the presence of appropriate primers, using thermocyclers. However, the time requested to amplify molecular targets and the cost of the thermocycler machines could impair the use of these methods in routine diagnostics. Recombinase polymerase amplification (RPA) technique allows rapid (short-term incubation of sample and primers in an enzymatic mixture) and simple (isothermal) amplification of molecular targets. RPA protocol requires only basic molecular steps such as extraction procedures and agarose gel electrophoresis. Thus, RPA can be considered as an interesting alternative to standard molecular-based diagnostic tools. In this paper, the complete procedures to set up an RPA assay, applied to detection of RNA (Potato virus Y, Potyvirus) and DNA (Wheat dwarf virus, Mastrevirus) viruses, are described. The proposed procedure allows developing species- or subspecies-specific detection assay.

Development of a multiplex assay for genus- and species-specific detection of Phytophthora based on differences in mitochondrial gene order

Treesearch

G. J. Bilodeau; F. N. Martin; M. D. Coffey; C. L. Blomquist

2014-01-01

A molecular diagnostic assay for Phytophthora spp. that is specific, sensitive, has both genus- and species-specific detection capabilities multiplexed, and can be used to systematically develop markers for detection of a wide range of species would facilitate research and regulatory efforts. To address this need, a marker system was developed...

Serodiagnosis of parasitic diseases.

PubMed Central

Maddison, S E

1991-01-01

In this review on serodiagnosis of parasitic diseases, antibody detection, antigen detection, use of monoclonal antibodies in parasitic serodiagnosis, molecular biological technology, and skin tests are discussed. The focus at the Centers for Disease Control on developing improved antigens, a truly quantitative FAST-enzyme-linked immunosorbent assay, and the very specific immunoblot assays for antibody detection is highlighted. The last two assays are suitable for field studies. Identification of patient response in terms of immunoglobulin class or immunoglobulin G subclass isotypes or both is discussed. Immunoglobulin isotypes may asist in defining the stage of some diseases. In other instances, use of a particular anti-isotype conjugate may increase the specificity of the assay. Monoclonal antibodies have played important roles in antigen purification and identification, in competitive antibody assays with increased sensitivity and specificity, and in assays for antigen detection in serum, body fluids, or excreta. Molecular biological technology has allowed significant advances in the production of defined parasitic serodiagnostic antigens. PMID:1747862

Evidence of clinical utility: an unmet need in molecular diagnostics for patients with cancer.

PubMed

Parkinson, David R; McCormack, Robert T; Keating, Susan M; Gutman, Steven I; Hamilton, Stanley R; Mansfield, Elizabeth A; Piper, Margaret A; Deverka, Patricia; Frueh, Felix W; Jessup, J Milburn; McShane, Lisa M; Tunis, Sean R; Sigman, Caroline C; Kelloff, Gary J

2014-03-15

This article defines and describes best practices for the academic and business community to generate evidence of clinical utility for cancer molecular diagnostic assays. Beyond analytical and clinical validation, successful demonstration of clinical utility involves developing sufficient evidence to demonstrate that a diagnostic test results in an improvement in patient outcomes. This discussion is complementary to theoretical frameworks described in previously published guidance and literature reports by the U.S. Food and Drug Administration, Centers for Disease Control and Prevention, Institute of Medicine, and Center for Medical Technology Policy, among others. These reports are comprehensive and specifically clarify appropriate clinical use, adoption, and payer reimbursement for assay manufacturers, as well as Clinical Laboratory Improvement Amendments-certified laboratories, including those that develop assays (laboratory developed tests). Practical criteria and steps for establishing clinical utility are crucial to subsequent decisions for reimbursement without which high-performing molecular diagnostics will have limited availability to patients with cancer and fail to translate scientific advances into high-quality and cost-effective cancer care. See all articles in this CCR Focus section, "The Precision Medicine Conundrum: Approaches to Companion Diagnostic Co-development." ©2014 AACR.

Using next-generation sequencing to develop molecular diagnostics for Pseudoperonospora cubensis, the cucurbit downy mildew pathogen

USDA-ARS?s Scientific Manuscript database

Advances in Next Generation Sequencing (NGS) allow for rapid development of genomics resources needed to generate molecular diagnostics assays for infectious agents. NGS approaches are particularly helpful for organisms that cannot be cultured, such as the downy mildew pathogens, a group of biotrop...

A technological update of molecular diagnostics for infectious diseases

PubMed Central

Liu, Yu-Tsueng

2008-01-01

Identification of a causative pathogen is essential for the choice of treatment for most infectious diseases. Many FDA approved molecular assays; usually more sensitive and specific compared to traditional tests, have been developed in the last decade. A new trend of high throughput and multiplexing assays are emerging thanks to technological developments for the human genome sequencing project. The applications of microarray and ultra high throughput sequencing technologies for diagnostic microbiology are reviewed. The race for the $1000 genome technology by 2014 will have a profound impact in diagnosis and treatment of infectious diseases in the near future. PMID:18782035

Molecular Subgroup of Primary Prostate Cancer Presenting with Metastatic Biology.

PubMed

Walker, Steven M; Knight, Laura A; McCavigan, Andrena M; Logan, Gemma E; Berge, Viktor; Sherif, Amir; Pandha, Hardev; Warren, Anne Y; Davidson, Catherine; Uprichard, Adam; Blayney, Jaine K; Price, Bethanie; Jellema, Gera L; Steele, Christopher J; Svindland, Aud; McDade, Simon S; Eden, Christopher G; Foster, Chris; Mills, Ian G; Neal, David E; Mason, Malcolm D; Kay, Elaine W; Waugh, David J; Harkin, D Paul; Watson, R William; Clarke, Noel W; Kennedy, Richard D

2017-10-01

Approximately 4-25% of patients with early prostate cancer develop disease recurrence following radical prostatectomy. To identify a molecular subgroup of prostate cancers with metastatic potential at presentation resulting in a high risk of recurrence following radical prostatectomy. Unsupervised hierarchical clustering was performed using gene expression data from 70 primary resections, 31 metastatic lymph nodes, and 25 normal prostate samples. Independent assay validation was performed using 322 radical prostatectomy samples from four sites with a mean follow-up of 50.3 months. Molecular subgroups were identified using unsupervised hierarchical clustering. A partial least squares approach was used to generate a gene expression assay. Relationships with outcome (time to biochemical and metastatic recurrence) were analysed using multivariable Cox regression and log-rank analysis. A molecular subgroup of primary prostate cancer with biology similar to metastatic disease was identified. A 70-transcript signature (metastatic assay) was developed and independently validated in the radical prostatectomy samples. Metastatic assay positive patients had increased risk of biochemical recurrence (multivariable hazard ratio [HR] 1.62 [1.13-2.33]; p=0.0092) and metastatic recurrence (multivariable HR=3.20 [1.76-5.80]; p=0.0001). A combined model with Cancer of the Prostate Risk Assessment post surgical (CAPRA-S) identified patients at an increased risk of biochemical and metastatic recurrence superior to either model alone (HR=2.67 [1.90-3.75]; p<0.0001 and HR=7.53 [4.13-13.73]; p<0.0001, respectively). The retrospective nature of the study is acknowledged as a potential limitation. The metastatic assay may identify a molecular subgroup of primary prostate cancers with metastatic potential. The metastatic assay may improve the ability to detect patients at risk of metastatic recurrence following radical prostatectomy. The impact of adjuvant therapies should be assessed in this higher-risk population. Copyright © 2017 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Validation of Immunohistochemical Assays for Integral Biomarkers in the NCI-MATCH EAY131 Clinical Trial.

PubMed

Khoury, Joseph D; Wang, Wei-Lien; Prieto, Victor G; Medeiros, L Jeffrey; Kalhor, Neda; Hameed, Meera; Broaddus, Russell; Hamilton, Stanley R

2018-02-01

Biomarkers that guide therapy selection are gaining unprecedented importance as targeted therapy options increase in scope and complexity. In conjunction with high-throughput molecular techniques, therapy-guiding biomarker assays based upon immunohistochemistry (IHC) have a critical role in cancer care in that they inform about the expression status of a protein target. Here, we describe the validation procedures for four clinical IHC biomarker assays-PTEN, RB, MLH1, and MSH2-for use as integral biomarkers in the nationwide NCI-Molecular Analysis for Therapy Choice (NCI-MATCH) EAY131 clinical trial. Validation procedures were developed through an iterative process based on collective experience and adaptation of broad guidelines from the FDA. The steps included primary antibody selection; assay optimization; development of assay interpretation criteria incorporating biological considerations; and expected staining patterns, including indeterminate results, orthogonal validation, and tissue validation. Following assay lockdown, patient samples and cell lines were used for analytic and clinical validation. The assays were then approved as laboratory-developed tests and used for clinical trial decisions for treatment selection. Calculations of sensitivity and specificity were undertaken using various definitions of gold-standard references, and external validation was required for the PTEN IHC assay. In conclusion, validation of IHC biomarker assays critical for guiding therapy in clinical trials is feasible using comprehensive preanalytic, analytic, and postanalytic steps. Implementation of standardized guidelines provides a useful framework for validating IHC biomarker assays that allow for reproducibility across institutions for routine clinical use. Clin Cancer Res; 24(3); 521-31. ©2017 AACR . ©2017 American Association for Cancer Research.

The use of chemical and molecular microbial indicators for faecal source identification.

PubMed

Gilpin, B; James, T; Nourozi, F; Saunders, D; Scholes, P; Savill, M

2003-01-01

Identifying the source of faecal pollution is important to enable appropriate management of faecal pollution of water. We are developing and evaluating a combination of these microbial and chemical indicators better able to identify the source of faecal pollution. These assays make use of a combination of direct PCR, culturing, and colony hybridisation to identify source specific species of Bifidobacterium, Rhodococcus and Bacteroides. In conjunction with assays for (a) fluorescent whitening agents and (b) faecal sterols and stanols, these indicators were able to identify human derived faecal pollution in river water containing inputs from septic tanks, municipal oxidation ponds, farmed animals and feral animals. Differentiating amongst the animal sources was more difficult and will require development of molecular assays for organisms specific to each animal group.

Using "Pseudomonas Putida xylE" Gene to Teach Molecular Cloning Techniques for Undergraduates

ERIC Educational Resources Information Center

Dong, Xu; Xin, Yi; Ye, Li; Ma, Yufang

2009-01-01

We have developed and implemented a serial experiment in molecular cloning laboratory course for undergraduate students majored in biotechnology. "Pseudomonas putida xylE" gene, encoding catechol 2, 3-dioxygenase, was manipulated to learn molecular biology techniques. The integration of cloning, expression, and enzyme assay gave students…

Diagnosis of Neisseria gonorrhoeae Using Molecular Beacon

PubMed Central

Patel, Achchhe Lal; Sonkar, Subash Chandra; Kumari, Indu; Saluja, Daman

2015-01-01

Neisseria gonorrhoeae is an important sexually transmitted diseases (STD) causing pathogen worldwide. Due to absence of an affordable diagnostic assay, routine screening of gonococcal infection becomes impossible in developing countries where infection rates are maximum. Treatment is given on the basis of symptoms alone which leads to spread of infection. Thus, development of a rapid, sensitive, specific, and PCR based visual diagnostic assay suitable for developing countries, required for better disease management, is aimed at in present study. Endocervical swabs were collected from patients visiting gynecology department of various hospitals in Delhi. In-house PCR based assay was developed and modified to visual assay using molecular beacon for end-point detection. It was evaluated against Roche AMPLICOR NG kit and rmp gene. Specificity of beacon was confirmed by competition experiments. Diagnostic test was 98.21% specific and 99.59% sensitive whereas negative and positive predicted value were 99.40% and 98.78%, respectively. We also observed that twice the concentration (2X) of premix was stable at 4°C for 4 months and dry swab samples gave concordant results with that of wet swabs. These features make the test best suitable for routine diagnosis of genital infections in developing countries. PMID:25802857

Molecular classification of breast cancer: what the pathologist needs to know.

PubMed

Rakha, Emad A; Green, Andrew R

2017-02-01

Breast cancer is a heterogeneous disease featuring distinct histological, molecular and clinical phenotypes. Although traditional classification systems utilising clinicopathological and few molecular markers are well established and validated, they remain insufficient to reflect the diverse biological and clinical heterogeneity of breast cancer. Advancements in high-throughput molecular techniques and bioinformatics have contributed to the improved understanding of breast cancer biology, refinement of molecular taxonomies and the development of novel prognostic and predictive molecular assays. Application of such technologies is already underway, and is expected to change the way we manage breast cancer. Despite the enormous amount of work that has been carried out to develop and refine breast cancer molecular prognostic and predictive assays, molecular testing is still in evolution. Pathologists should be aware of the new technology and be ready for the challenge. In this review, we provide an update on the application of molecular techniques with regard to breast cancer diagnosis, prognosis and outcome prediction. The current contribution of emerging technology to our understanding of breast cancer is also highlighted. Copyright © 2016 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.

Targeted DNA sequencing and in situ mutation analysis using mobile phone microscopy

NASA Astrophysics Data System (ADS)

Kühnemund, Malte; Wei, Qingshan; Darai, Evangelia; Wang, Yingjie; Hernández-Neuta, Iván; Yang, Zhao; Tseng, Derek; Ahlford, Annika; Mathot, Lucy; Sjöblom, Tobias; Ozcan, Aydogan; Nilsson, Mats

2017-01-01

Molecular diagnostics is typically outsourced to well-equipped centralized laboratories, often far from the patient. We developed molecular assays and portable optical imaging designs that permit on-site diagnostics with a cost-effective mobile-phone-based multimodal microscope. We demonstrate that targeted next-generation DNA sequencing reactions and in situ point mutation detection assays in preserved tumour samples can be imaged and analysed using mobile phone microscopy, achieving a new milestone for tele-medicine technologies.

Development of a multiplex assay for genus and species-specific detection of Phytophthora based on differences in mitochondrial gene order

USDA-ARS?s Scientific Manuscript database

The availability of a molecular diagnostic assay for Phytophthora that is specific, sensitive, has both genus and species specific detection capabilities multiplexed and can be used to systematically develop markers for detection of a wide range of species would facilitate research and regulatory ef...

Development of a rapid diagnostic assay for the detection of tomato chlorotic dwarf viroid based on isothermal reverse-transcription-recombinase polymerase amplification

USDA-ARS?s Scientific Manuscript database

A molecular diagnostic assay utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 39 °C and target-specific primers and probe were developed for the rapid, sensitive, and specific detection of tomato chlorotic dwarf viroid (TCDVd) in ...

Development of a Molecular-Beacon Assay To Detect the G1896A Precore Mutation in Hepatitis B Virus-Infected Individuals

PubMed Central

Waltz, Therese L.; Marras, Salvatore; Rochford, Gemma; Nolan, John; Lee, Eugenia; Melegari, Margherita; Pollack, Henry

2005-01-01

The 1896 precore (PC) mutation is the most frequent cause of hepatitis B virus e-antigen (HBeAg)-negative chronic hepatitis B virus (HBV) infection. Detection of the 1896 PC mutation has application in studies monitoring antiviral therapy and the natural history of the disease. Identification of this mutation is usually performed by direct sequencing, which is both costly and laborious. The aim of this study was to develop a rapid, high-throughput assay to detect the 1896 PC mutation using real-time PCR and molecular-beacon technology. The assay was initially standardized on oligonucleotide targets and plasmids containing the wild-type (WT) and PC mutation and then tested on plasma samples from children with HBV DNA of >106 copies/ml. Nine individuals were HBeAg negative and suspected to harbor HBeAg mutations, while 12 children were HBeAg positive and selected as controls. Ninety percent (19 of 21) of plasma samples tested with molecular beacons were in complete agreement with sequencing results. The remaining 10% (2 of 21) of samples were identified as heterogeneous mixtures of WT and mutant virus by molecular beacons, though sequencing found only a homogeneous mutant in both cases. Overall, the 1896 PC mutation was detected by this assay in 55.5% of the children with HBeAg-negative infection. In summary, this assay is a rapid, sensitive, and specific technique that effectively discriminates WT from 1896 PC mutant HBV and may be useful in clinical and epidemiological studies. PMID:15634980

Development of real-time PCR assays for the detection of Moraxella macacae associated with bloody nose syndrome in rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques

PubMed Central

Whitehouse, Chris A.; Chase, Kitty; Embers, Monica E.; Kulesh, David A.; Ladner, Jason T.; Palacios, Gustavo F.; Minogue, Timothy D.

2016-01-01

Background Moraxella macacae is a recently described bacterial pathogen that causes epistaxis or so-called bloody nose syndrome in captive macaques. The aim of this study was to develop specific molecular diagnostic assays for M. macacae and to determine their performance characteristics. Methods We developed six real-time PCR assays on the Roche LightCycler. The accuracy, precision, selectivity, and limit of detection (LOD) were determined for each assay, in addition to further validation by testing nasal swabs from macaques presenting with epistaxis at the Tulane National Primate Research Center. Results All assays exhibited 100% specificity and were highly sensitive with an LOD of 10 fg for chromosomal assays and 1 fg for the plasmid assay. Testing of nasal swabs from 10 symptomatic macaques confirmed the presence of M. macacae in these animals. Conclusions We developed several accurate, sensitive, and species-specific real-time PCR assays for the detection of M. macacae in captive macaques. PMID:26365904

Fundamental approaches in molecular biology for communication sciences and disorders.

PubMed

Bartlett, Rebecca S; Jetté, Marie E; King, Suzanne N; Schaser, Allison; Thibeault, Susan L

2012-08-01

This contemporary tutorial will introduce general principles of molecular biology, common deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein assays and their relevance in the field of communication sciences and disorders. Over the past 2 decades, knowledge of the molecular pathophysiology of human disease has increased at a remarkable pace. Most of this progress can be attributed to concomitant advances in basic molecular biology and, specifically, the development of an ever-expanding armamentarium of technologies for analysis of DNA, RNA, and protein structure and function. Details of these methodologies, their limitations, and examples from the communication sciences and disorders literature are presented. Results/Conclusions The use of molecular biology techniques in the fields of speech, language, and hearing sciences is increasing, facilitating the need for an understanding of molecular biology fundamentals and common experimental assays.

Detection of enterotoxigenic Clostridium perfringens in meat samples by using molecular methods.

PubMed

Kaneko, Ikuko; Miyamoto, Kazuaki; Mimura, Kanako; Yumine, Natsuko; Utsunomiya, Hirotoshi; Akimoto, Shigeru; McClane, Bruce A

2011-11-01

To prevent food-borne bacterial diseases and to trace bacterial contamination events to foods, microbial source tracking (MST) methods provide important epidemiological information. To apply molecular methods to MST, it is necessary not only to amplify bacterial cells to detection limit levels but also to prepare DNA with reduced inhibitory compounds and contamination. Isolates carrying the Clostridium perfringens enterotoxin gene (cpe) on the chromosome or a plasmid rank among the most important food-borne pathogens. Previous surveys indicated that cpe-positive C. perfringens isolates are present in only ∼5% of nonoutbreak food samples and then only at low numbers, usually less than 3 cells/g. In this study, four molecular assays for the detection of cpe-positive C. perfringens isolates, i.e., ordinary PCR, nested PCR, real-time PCR, and loop-mediated isothermal amplification (LAMP), were developed and evaluated for their reliability using purified DNA. For use in the artificial contamination of meat samples, DNA templates were prepared by three different commercial DNA preparation kits. The four molecular assays always detected cpe when >10³ cells/g of cpe-positive C. perfringens were present, using any kit. Of three tested commercial DNA preparation kits, the InstaGene matrix kit appeared to be most suitable for the testing of a large number of samples. By using the InstaGene matrix kit, the four molecular assays efficiently detected cpe using DNA prepared from enrichment culture specimens of meat samples contaminated with low numbers of cpe-positive C. perfringens vegetative cells or spores. Overall, the current study developed molecular assay protocols for MST to detect the contamination of foods with low numbers of cells, and at a low frequency, of cpe-positive C. perfringens isolates.

Rapid and accurate identification by real-time PCR of biotoxin-producing dinoflagellates from the family gymnodiniaceae.

PubMed

Smith, Kirsty F; de Salas, Miguel; Adamson, Janet; Rhodes, Lesley L

2014-03-07

The identification of toxin-producing dinoflagellates for monitoring programmes and bio-compound discovery requires considerable taxonomic expertise. It can also be difficult to morphologically differentiate toxic and non-toxic species or strains. Various molecular methods have been used for dinoflagellate identification and detection, and this study describes the development of eight real-time polymerase chain reaction (PCR) assays targeting the large subunit ribosomal RNA (LSU rRNA) gene of species from the genera Gymnodinium, Karenia, Karlodinium, and Takayama. Assays proved to be highly specific and sensitive, and the assay for G. catenatum was further developed for quantification in response to a bloom in Manukau Harbour, New Zealand. The assay estimated cell densities from environmental samples as low as 0.07 cells per PCR reaction, which equated to three cells per litre. This assay not only enabled conclusive species identification but also detected the presence of cells below the limit of detection for light microscopy. This study demonstrates the usefulness of real-time PCR as a sensitive and rapid molecular technique for the detection and quantification of micro-algae from environmental samples.

Application of statistical process control to qualitative molecular diagnostic assays.

PubMed

O'Brien, Cathal P; Finn, Stephen P

2014-01-01

Modern pathology laboratories and in particular high throughput laboratories such as clinical chemistry have developed a reliable system for statistical process control (SPC). Such a system is absent from the majority of molecular laboratories and where present is confined to quantitative assays. As the inability to apply SPC to an assay is an obvious disadvantage this study aimed to solve this problem by using a frequency estimate coupled with a confidence interval calculation to detect deviations from an expected mutation frequency. The results of this study demonstrate the strengths and weaknesses of this approach and highlight minimum sample number requirements. Notably, assays with low mutation frequencies and detection of small deviations from an expected value require greater sample numbers to mitigate a protracted time to detection. Modeled laboratory data was also used to highlight how this approach might be applied in a routine molecular laboratory. This article is the first to describe the application of SPC to qualitative laboratory data.

Molecular and Clinical Aspects of Protein Aggregation Assays in Neurodegenerative Diseases.

PubMed

Villar-Piqué, Anna; Schmitz, Matthias; Candelise, Niccolò; Ventura, Salvador; Llorens, Franc; Zerr, Inga

2018-02-10

The presence of protein deposits is a common pathological hallmark in patients suffering from neurodegenerative conditions and other proteinopathies. Deciphering the molecular basis of protein misfolding and aggregation is a crucial step towards the full comprehension of the factors that trigger the onset of these diseases and for the development of efficient therapeutical strategies. In this regard, in vitro aggregation assays for misfolded proteins offer an excellent tool to study pathological processes of protein deposition under controlled conditions, where confounders can be easily discriminated. These methods are generally cost-effective and have been proved useful in many fields, including drug discovery and clinical diagnostics. Here, we review the bases of in vitro aggregation and seeding assays, recapitulate their main applications and offer a critical evaluation of their limitations. Comprehending the molecular mechanisms behind these assays and combining them with in vivo or cell-based experiments will maximize their potential and allow the necessary improvement to overcome some of the current drawbacks.

The past and the future of Alzheimer's disease CSF biomarkers-a journey toward validated biochemical tests covering the whole spectrum of molecular events.

PubMed

Blennow, Kaj; Zetterberg, Henrik

2015-01-01

This paper gives a short review on cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease (AD), from early developments to high-precision validated assays on fully automated lab analyzers. We also discuss developments on novel biomarkers, such as synaptic proteins and Aβ oligomers. Our vision for the future is that assaying a set of biomarkers in a single CSF tube can monitor the whole spectrum of AD molecular pathogenic events. CSF biomarkers will have a central position not only for clinical diagnosis, but also for the understanding of the sequence of molecular events in the pathogenic process underlying AD and as tools to monitor the effects of novel drug candidates targeting these different mechanisms.

Rapid and inexpensive body fluid identification by RNA profiling-based multiplex High Resolution Melt (HRM) analysis

PubMed Central

Hanson, Erin K.; Ballantyne, Jack

2014-01-01

Positive identification of the nature of biological material present on evidentiary items can be crucial for understanding the circumstances surrounding a crime. However, traditional protein-based methods do not permit the identification of all body fluids and tissues, and thus molecular based strategies for the conclusive identification of all forensically relevant biological fluids and tissues need to be developed. Messenger RNA (mRNA) profiling is an example of such a molecular-based approach. Current mRNA body fluid identification assays involve capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the analysis time. For qRT-PCR assays, only 3-4 markers can be included in a single reaction since each requires a different fluorescent dye. To simplify mRNA profiling assays, and reduce the time and cost of analysis, we have developed single- and multiplex body fluid High Resolution Melt (HRM) assays for the identification of common forensically relevant biological fluids and tissues. The incorporated biomarkers include IL19 (vaginal secretions), IL1F7 (skin), ALAS2 (blood), MMP10 (menstrual blood), HTN3 (saliva) and TGM4 (semen).  The HRM assays require only unlabeled PCR primers and a single saturating intercalating fluorescent dye (Eva Green). Each body-fluid-specific marker can easily be identified by the presence of a distinct melt peak. Usually, HRM assays are used to detect variants or isoforms for a single gene target. However, we have uniquely developed duplex and triplex HRM assays to permit the simultaneous detection of multiple targets per reaction. Here we describe the development and initial performance evaluation of the developed HRM assays. The results demonstrate the potential use of HRM assays for rapid, and relatively inexpensive, screening of biological evidence. PMID:24715968

Rapid and inexpensive body fluid identification by RNA profiling-based multiplex High Resolution Melt (HRM) analysis.

PubMed

Hanson, Erin K; Ballantyne, Jack

2013-01-01

Positive identification of the nature of biological material present on evidentiary items can be crucial for understanding the circumstances surrounding a crime. However, traditional protein-based methods do not permit the identification of all body fluids and tissues, and thus molecular based strategies for the conclusive identification of all forensically relevant biological fluids and tissues need to be developed. Messenger RNA (mRNA) profiling is an example of such a molecular-based approach. Current mRNA body fluid identification assays involve capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the analysis time. For qRT-PCR assays, only 3-4 markers can be included in a single reaction since each requires a different fluorescent dye. To simplify mRNA profiling assays, and reduce the time and cost of analysis, we have developed single- and multiplex body fluid High Resolution Melt (HRM) assays for the identification of common forensically relevant biological fluids and tissues. The incorporated biomarkers include IL19 (vaginal secretions), IL1F7 (skin), ALAS2 (blood), MMP10 (menstrual blood), HTN3 (saliva) and TGM4 (semen).  The HRM assays require only unlabeled PCR primers and a single saturating intercalating fluorescent dye (Eva Green). Each body-fluid-specific marker can easily be identified by the presence of a distinct melt peak. Usually, HRM assays are used to detect variants or isoforms for a single gene target. However, we have uniquely developed duplex and triplex HRM assays to permit the simultaneous detection of multiple targets per reaction. Here we describe the development and initial performance evaluation of the developed HRM assays. The results demonstrate the potential use of HRM assays for rapid, and relatively inexpensive, screening of biological evidence.

DHB Task Force Review of DoD Biodefense Infrastructure and Biological Research Portfolio

DTIC Science & Technology

2009-04-29

Center and Biological Defense and Research Directorate (BORD) biodefense research portfolio includes vaccines, molecular diagnostics , genomics, and...human monoclonal antibodies, and innate immune agonists); molecular diagnostics (including recombinant reagents, assay development, reagent production

Overview of an internationally-harmonized program for adverse outcome pathway development

EPA Science Inventory

Adverse outcome pathways (AOPs) are critical frameworks for organizing knowledge concerning the scientifically-credible predictive linkages between toxicological observations made at molecular and cellular levels (e.g., via molecular screening assays, biomarker responses, or chem...

Differentiating high priority pathway-based toxicity from non ...

EPA Pesticide Factsheets

The ToxCast chemical screening approach enables the rapid assessment of large numbers of chemicals for biological effects, primarily at the molecular level. Adverse outcome pathways (AOPs) offer a means to link biomolecular effects with potential adverse outcomes at the level of the individual or population, thus enhancing the utility of the ToxCast effort for hazard assessment. Thus, efforts are underway to develop AOPs relevant to the pathway perturbations detected in ToxCast assays. However, activity (?‘hits’) determined for chemical-assay pairs may reflect target-specific activity relevant to a molecular initiating event of an AOP, or more generalized cell stress and cytotoxicity-mediated effects. Previous work identified a ?‘cytotoxic burst’ phenomenon wherein large numbers of assays begin to respond at or near concentrations that elicit cytotoxicity. The concentration range at which the “burst” occurs is definable, statistically. Consequently, in order to focus AOP development on the ToxCast assay targetswhich are most sensitive and relevant to pathway-specific effects, we conducted a meta-analysis to identify which assays were frequently responding at concentrations well below the cytotoxic burst. Assays were ranked by the fraction of chemical hits below the burst concentration range compared to the number of chemicals tested, resulting in a preliminary list of potentially important, target-specific assays. After eliminating cytotoxicity a

Targeted DNA sequencing and in situ mutation analysis using mobile phone microscopy

PubMed Central

Kühnemund, Malte; Wei, Qingshan; Darai, Evangelia; Wang, Yingjie; Hernández-Neuta, Iván; Yang, Zhao; Tseng, Derek; Ahlford, Annika; Mathot, Lucy; Sjöblom, Tobias; Ozcan, Aydogan; Nilsson, Mats

2017-01-01

Molecular diagnostics is typically outsourced to well-equipped centralized laboratories, often far from the patient. We developed molecular assays and portable optical imaging designs that permit on-site diagnostics with a cost-effective mobile-phone-based multimodal microscope. We demonstrate that targeted next-generation DNA sequencing reactions and in situ point mutation detection assays in preserved tumour samples can be imaged and analysed using mobile phone microscopy, achieving a new milestone for tele-medicine technologies. PMID:28094784

A Systems Biology Approach to Toxicology Research with Small Fish Models

EPA Science Inventory

Increasing use of mechanistically-based molecular and biochemical endpoints and in vitro assays is being advocated as a more efficient and cost-effective approach for generating chemical hazard data. However, development of effective assays and application of the resulting data i...

Competitive fluorescent pseudo-immunoassay exploiting molecularly imprinted polymers for the detection of biogenic amines in fish matrix.

PubMed

Mattsson, Leena; Xu, Jingjing; Preininger, Claudia; Tse Sum Bui, Bernadette; Haupt, Karsten

2018-05-01

We developed a competitive fluorescent molecularly imprinted polymer (MIP) assay to detect biogenic amines in fish samples. MIPs synthesized by precipitation polymerization using histamine as template were used in a batch binding assay analogous to competitive fluoroimmunoassays. Introducing a complex sample matrix, such as fish extract, into the assay changes the environment and the binding conditions, therefore the importance of the sample preparation is extensively discussed. Several extraction and purification methods for fish were comprehensively studied, and an optimal clean-up procedure for fish samples using liquid-liquid extraction was developed. The feasibility of the competitive MIP assay was shown in the purified fish extract over a broad histamine range (1 - 430µM). The MIP had the highest affinity towards histamine, but recognized also the structurally similar biogenic amines tyramine and tryptamine, as well as spermine and spermidine, providing simultaneous analysis and assessment of the total amount of biogenic amines. Copyright © 2018 Elsevier B.V. All rights reserved.

Pediatric tuberculosis-human immunodeficiency virus co-infection in the United Kingdom highlights the need for better therapy monitoring tools: a case report.

PubMed

Evangelopoulos, Dimitrios; Whittaker, Elizabeth; Honeyborne, Isobella; McHugh, Timothy D; Klein, Nigel; Shingadia, Delane

2017-02-26

Tuberculosis is an infection that requires at least 6 months of chemotherapy in order to clear the bacteria from the patient's lungs. Usually, therapeutic monitoring is dependent on smear microscopy where a decline in acid-fast bacilli is observed. However, this might not be indicative of the actual decline of bacterial load and thus other tools such as culture and molecular assays are required for patient management. Here, we report the case of a 12-year-old Black African boy co-infected with tuberculosis and human immunodeficiency virus who remained smear culture positive and liquid culture negative for a prolonged period of time following chemotherapy. In order to determine whether there was any live bacteria present in his specimens, we applied the newly developed molecular bacterial load assay that detects the presence of 16S ribosomal ribonucleic acid derived from the bacteria. Using this methodology, we were able to quantify his bacterial load and inform the management of his treatment in order to reduce the disease burden. Following this intervention he went on to make a complete recovery. This case report highlights the value of improved biomarkers for monitoring the treatment of tuberculosis and the role of molecular assays such as the molecular bacterial load assay applied here. The molecular bacterial load assay detects bacterial ribonucleic acid which corresponds closely with the number of live bacilli as compared with polymerase chain reaction that detects deoxyribonucleic acid and may include dead bacteria.

Development of a fluorescent probe-based recombinase polymerase amplification assay for rapid detection of Orf virus.

PubMed

Yang, Yang; Qin, Xiaodong; Wang, Guangxiang; Zhang, Yuen; Shang, Youjun; Zhang, Zhidong

2015-12-02

Orf virus (ORFV) is the causative agent of Orf (also known as contagious ecthyma or contagious papular dermatitis), a severe infectious skin disease in goats, sheep and other ruminants. The rapid detection of ORFV is of great importance in disease control and highly needed. A isothermal molecular diagnostic approach, termed recombinase polymerase amplification (RPA), is considered as an novel and rapid alternative techonology to PCR assay. In the present study, a novel fluorescent probe based on RPA assay (ORFV exo RPA assay) was developed. The developed ORFV exo RPA assay was capable of as low as 100 copies of ORFV DNA /reaction and was highly specific, with no cross-reaction with closely related viruses (capripox virus, foot-and-mouth disease virus or peste des petits ruminants virus). Further assessment with clinical samples showed that the developed ORFV exo RPA assay has good correlation with qPCR assays for detection of ORFV. These results suggest that the developed ORFV exo RPA assay is suitable for rapid detection of ORFV.

Surveyor assay to diagnose persistent Müllerian duct syndrome in Miniature Schnauzers.

PubMed

Kim, Young June; Kwon, Hyuk Jin; Byun, Hyuk Soo; Yeom, Donguk; Choi, Jea-Hong; Kim, Joong-Hyun; Shim, Hosup

2017-12-31

Persistent Müllerian duct syndrome (PMDS) is a pseudohermaphroditism in males characterized by the presence of Müllerian duct derivatives. As PMDS dogs often lack clinical symptoms, a molecular diagnosis is essential to identify the syndrome in these animals. In this study, a new molecular method using DNA mismatch-specific Surveyor nuclease was developed. The Surveyor nuclease assay identified the AMHR2 mutation that produced PMDS in a Miniature Schnauzer as accurately as that obtained by using the conventional method based on restriction digestion. As an alternative to the current molecular diagnostic method, the new method may result in increased accuracy when detecting PMDS.

Surveyor assay to diagnose persistent Müllerian duct syndrome in Miniature Schnauzers

PubMed Central

Kim, Young June; Kwon, Hyuk Jin; Byun, Hyuk Soo; Yeom, Donguk; Choi, Jea-Hong; Kim, Joong-Hyun

2017-01-01

Persistent Müllerian duct syndrome (PMDS) is a pseudohermaphroditism in males characterized by the presence of Müllerian duct derivatives. As PMDS dogs often lack clinical symptoms, a molecular diagnosis is essential to identify the syndrome in these animals. In this study, a new molecular method using DNA mismatch-specific Surveyor nuclease was developed. The Surveyor nuclease assay identified the AMHR2 mutation that produced PMDS in a Miniature Schnauzer as accurately as that obtained by using the conventional method based on restriction digestion. As an alternative to the current molecular diagnostic method, the new method may result in increased accuracy when detecting PMDS. PMID:27515263

Molecular methods for the detection of mutations.

PubMed

Monteiro, C; Marcelino, L A; Conde, A R; Saraiva, C; Giphart-Gassler, M; De Nooij-van Dalen, A G; Van Buuren-van Seggelen, V; Van der Keur, M; May, C A; Cole, J; Lehmann, A R; Steinsgrimsdottir, H; Beare, D; Capulas, E; Armour, J A

2000-01-01

We report the results of a collaborative study aimed at developing reliable, direct assays for mutation in human cells. The project used common lymphoblastoid cell lines, both with and without mutagen treatment, as a shared resource to validate the development of new molecular methods for the detection of low-level mutations in the presence of a large excess of normal alleles. As the "gold standard, " hprt mutation frequencies were also measured on the same samples. The methods under development included i) the restriction site mutation (RSM) assay, in which mutations lead to the destruction of a restriction site; ii) minisatellite length-change mutation, in which mutations lead to alleles containing new numbers of tandem repeat units; iii) loss of heterozygosity for HLA epitopes, in which antibodies can be used to direct selection for mutant cells; iv) multiple fluorescence-based long linker arm nucleotides assay (mf-LLA) technology, for the detection of substitutional mutations; v) detection of alterations in the TP53 locus using a (CA) array as the target for the screening; and vi) PCR analysis of lymphocytes for the presence of the BCL2 t(14:18) translocation. The relative merits of these molecular methods are discussed, and a comparison made with more "traditional" methods.

Evaluation of Two Lyophilized Molecular Assays to Rapidly Detect Foot-and-Mouth Disease Virus Directly from Clinical Samples in Field Settings.

PubMed

Howson, E L A; Armson, B; Madi, M; Kasanga, C J; Kandusi, S; Sallu, R; Chepkwony, E; Siddle, A; Martin, P; Wood, J; Mioulet, V; King, D P; Lembo, T; Cleaveland, S; Fowler, V L

2017-06-01

Accurate, timely diagnosis is essential for the control, monitoring and eradication of foot-and-mouth disease (FMD). Clinical samples from suspect cases are normally tested at reference laboratories. However, transport of samples to these centralized facilities can be a lengthy process that can impose delays on critical decision making. These concerns have motivated work to evaluate simple-to-use technologies, including molecular-based diagnostic platforms, that can be deployed closer to suspect cases of FMD. In this context, FMD virus (FMDV)-specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) and real-time RT-PCR (rRT-PCR) assays, compatible with simple sample preparation methods and in situ visualization, have been developed which share equivalent analytical sensitivity with laboratory-based rRT-PCR. However, the lack of robust 'ready-to-use kits' that utilize stabilized reagents limits the deployment of these tests into field settings. To address this gap, this study describes the performance of lyophilized rRT-PCR and RT-LAMP assays to detect FMDV. Both of these assays are compatible with the use of fluorescence to monitor amplification in real-time, and for the RT-LAMP assays end point detection could also be achieved using molecular lateral flow devices. Lyophilization of reagents did not adversely affect the performance of the assays. Importantly, when these assays were deployed into challenging laboratory and field settings within East Africa they proved to be reliable in their ability to detect FMDV in a range of clinical samples from acutely infected as well as convalescent cattle. These data support the use of highly sensitive molecular assays into field settings for simple and rapid detection of FMDV. © 2015 The Authors. Transboundary and Emerging Diseases Published by Blackwell Verlag GmbH.

Development of field-based real-time reverse transcription-polymerase chain reaction assays for detection of Chikungunya and O'nyong-nyong viruses in mosquitoes.

PubMed

Smith, Darci R; Lee, John S; Jahrling, Jordan; Kulesh, David A; Turell, Michael J; Groebner, Jennifer L; O'Guinn, Monica L

2009-10-01

Chikungunya (CHIK) and O'nyong-nyong (ONN) are important emerging arthropod-borne diseases. Molecular diagnosis of these two viruses in mosquitoes has not been evaluated, and the effects of extraneous mosquito tissue on assay performance have not been tested. Additionally, no real-time reverse transcription-polymerase chain reaction (RT-PCR) assay exists for detecting ONN virus (ONNV) RNA. We describe the development of sensitive and specific real-time RT-PCR assays for detecting CHIK and ONN viral RNA in mosquitoes, which have application for field use. In addition, we compared three methods for primer/probe design for assay development by evaluating their sensitivity and specificity. This comparison resulted in development of virus-specific assays that could detect less than one plaque-forming unit equivalent of each of the viruses in mosquitoes. The use of these assays will aid in arthropod-borne disease surveillance and in the control of the associated diseases.

Use of real-time quantitative PCR to detect Chlamydophila felis infection.

PubMed

Helps, C; Reeves, N; Tasker, S; Harbour, D

2001-07-01

A real-time PCR assay was developed to detect and quantify Chlamydophila felis infection of cats. The assay uses a molecular beacon to specifically identify the major outer membrane protein gene, is highly reproducible, and is able to detect fewer than 10 genomic copies.

Specific detection of common pathogens of acute bacterial meningitis using an internally controlled tetraplex-PCR assay.

PubMed

Farahani, Hamidreza; Ghaznavi-Rad, Ehsanollah; Mondanizadeh, Mahdieh; MirabSamiee, Siamak; Khansarinejad, Behzad

2016-08-01

Accurate and timely diagnosis of acute bacterial meningitis is critical for antimicrobial treatment of patients. Although PCR-based methods have been widely used for the diagnosis of acute meningitis caused by bacterial pathogens, the main disadvantage of these methods is their high cost. This disadvantage has hampered the widespread use of molecular assays in many developing countries. The application of multiplex assays and "in-house" protocols are two main approaches that can reduce the overall cost of a molecular test. In the present study, an internally controlled tetraplex-PCR was developed and validated for the specific detection of Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae in cerebrospinal fluid (CSF) samples. The analysis of a panel of other human pathogens showed no cross-reactivity in the assay. The analytical sensitivity of the in-house assay was 792.3 copies/ml, when all three bacteria were presentin the specimens. This value was calculated as 444.5, 283.7, 127.8 copies/ml when only S. pneumoniae, N. meningitidis and H. influenzae, respectively, were present. To demonstrate the diagnostic performance of the assay, a total of 150 archival CSF samples were tested and compared with a commercial multiplex real-time PCR kit. A diagnostic sensitivity of 92.8% and a specificity of 95.1% were determined for the present tetraplex-PCR assay. The results indicate that the established method is sensitive, specific and cost-effective, and can be used particularly in situations where the high cost of commercial kits prevents the use of molecular methods for the diagnosis of bacterial meningitis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Pseudoperonospora cubensis and P. humuli detection using species-specific probes and high definition melt curve analysis

USDA-ARS?s Scientific Manuscript database

Three assays were developed for molecular differentiation of Pseudoperonospora cubensis and P. humuli, causal agents of cucurbit and hop downy mildew, respectively, for detection of airborne sporangia and diagnosis of symptomatic leaf tissue. The assays were based on previously identified single nuc...

Next-Generation Molecular Histology Using Highly Multiplexed Ion Beam Imaging (MIBI) of Breast Cancer Tissue Specimens for Enhanced Clinical Guidance

DTIC Science & Technology

2015-07-01

SLC7A5, NRDG1, HTF9C, CEACAM5). Gene-expression assays using qRT-PCR, array hybridization, and RNA sequence assays have also been developed. The...and RNA sequence assays have also been developed. The OncotypeDX, for example, uses a panel of 21 genes (16 analytical, 5 controls: Ki67, STK15...Provide a brief list of keywords (limit to 20 words). Breast Cancer Diagnosis Pathology Immunophenotype Multiplex Morphology RNA In Situ

Molecular profiling and commercial predication assays in ovarian cancer: still not ready for prime time?

PubMed

Kohn, Elise C

2014-01-01

Short of early detection to allow curative primary intervention, the other major barrier to further success in treatment of ovarian cancers is matching the best treatment to the proper ovarian cancer type and to the individual patient. There are several decades of experience applying in vitro chemoresponse testing for solid tumors including ovarian cancer. This concept, first described in 1979, has yet to receive level one evidence supporting its application, despite the testing of numerous assays commercially as well as in academic centers and its use for tens of thousands of patients at a significant cost. The approach-rather than undergoing rigorous scientific examination-is now being muddied by the development of commercial molecular profiling assays from which treatment suggestions are provided. Molecular profiling as a research tool has added value to our understanding and treatment of patients with ovarian cancer. Morphologic and histochemical characterizations coupled now with increasing knowledge of ovarian cancer type-specific molecular patterns is improving our ability to properly diagnosis ovarian cancer type and thus guide therapy. With the exception of the role of germ-line and possibly somatic BRCA1 and BRCA2 mutations and their true predictiveness for probable response to poly(ADP-ribose) polymerase inhibition, molecular typing and profiling has yet to identify druggable molecular targets in ovarian cancer. Its use should be continued as a research and learning tool, and its results should be subjected to clinical trial validation. For very different reasons, neither chemoresponse assays nor molecular profiling are ready for prime time, yet.

Current and proposed molecular diagnostics in a genitourinary service line laboratory at a tertiary clinical institution.

PubMed

Udager, Aaron M; Alva, Ajjai; Mehra, Rohit

2014-01-01

The idea that detailed knowledge of molecular oncogenesis will drive diagnostic, prognostic, and therapeutic clinical decision making in an increasingly multidisciplinary practice of oncologic care has been anticipated for many years. With the recent rapid advancement in our understanding of the molecular underpinnings of genitourinary malignancies, this concept is now starting to take shape in the fields of prostate, kidney, bladder, testicular, and penile cancer. Such breakthroughs necessitate the development of robust clinical-grade assays that can be quickly made available for patients to facilitate diagnosis in challenging cases, risk-stratify patients for subsequent clinical management, select the appropriate targeted therapy from among increasingly diverse and numerous options, and enroll patients in advanced clinical trials. This rapid translation of basic and clinical cancer research requires a streamlined, multidisciplinary approach to clinical assay development, termed here the molecular diagnostics service line laboratory. In this review, we summarize the current state and explore the future of molecular diagnostics in genitourinary oncology to conceptualize a genitourinary service line laboratory at a tertiary clinical institution.

Imaging and Force Recognition of Single Molecular Behaviors Using Atomic Force Microscopy

PubMed Central

Li, Mi; Dang, Dan; Liu, Lianqing; Xi, Ning; Wang, Yuechao

2017-01-01

The advent of atomic force microscopy (AFM) has provided a powerful tool for investigating the behaviors of single native biological molecules under physiological conditions. AFM can not only image the conformational changes of single biological molecules at work with sub-nanometer resolution, but also sense the specific interactions of individual molecular pair with piconewton force sensitivity. In the past decade, the performance of AFM has been greatly improved, which makes it widely used in biology to address diverse biomedical issues. Characterizing the behaviors of single molecules by AFM provides considerable novel insights into the underlying mechanisms guiding life activities, contributing much to cell and molecular biology. In this article, we review the recent developments of AFM studies in single-molecule assay. The related techniques involved in AFM single-molecule assay were firstly presented, and then the progress in several aspects (including molecular imaging, molecular mechanics, molecular recognition, and molecular activities on cell surface) was summarized. The challenges and future directions were also discussed. PMID:28117741

A common standard is inappropriate for determining the potency of ultra low molecular weight heparins such as semuloparin and bemiparin.

PubMed

Jeske, Walter P; Hoppensteadt, Debra; Gray, Angel; Walenga, Jeanine M; Cunanan, Josephine; Myers, Lauren; Fareed, Jawed; Bayol, Alain; Rigal, Hélène; Viskov, Christian

2011-10-01

Lower low-molecular-weight heparins are being developed to improve on the safety and efficacy of antithrombotic therapy. Semuloparin and bemiparin are two depolymerized heparins produced by distinct manufacturing processes. The objective of this investigation was to determine whether a common standard could be used to define their potency. Activities were compared using typical clinical coagulation assays and pharmacological assays required for potency assessment. The activity of semuloparin and bemiparin was comparable in FXa-based assays (anti-FXa, Heptest). However, bemiparin produced a stronger effect in the aPTT, ACT and anti-thrombin assays. Assessment of the parallelism of the concentration-response curves indicated that bemiparin and semuloparin are not equivalent in terms of anti-FIIa activity. Bemiparin had a stronger inhibitory effect on thrombin induced platelet aggregation, and a stronger interaction with HIT antibodies. These data demonstrate that depolymerized heparins can exhibit a range of biologic activities making them unique agents. Pharmacopoeial parameters such as anti-IIa and anti-Xa potency and molecular weight are insufficient to characterize such agents. Copyright © 2011 Elsevier Ltd. All rights reserved.

A Simple, Inexpensive Device for Nucleic Acid Amplification without Electricity—Toward Instrument-Free Molecular Diagnostics in Low-Resource Settings

PubMed Central

LaBarre, Paul; Hawkins, Kenneth R.; Gerlach, Jay; Wilmoth, Jared; Beddoe, Andrew; Singleton, Jered; Boyle, David; Weigl, Bernhard

2011-01-01

Background Molecular assays targeted to nucleic acid (NA) markers are becoming increasingly important to medical diagnostics. However, these are typically confined to wealthy, developed countries; or, to the national reference laboratories of developing-world countries. There are many infectious diseases that are endemic in low-resource settings (LRS) where the lack of simple, instrument-free, NA diagnostic tests is a critical barrier to timely treatment. One of the primary barriers to the practicality and availability of NA assays in LRS has been the complexity and power requirements of polymerase chain reaction (PCR) instrumentation (another is sample preparation). Methodology/Principal Findings In this article, we investigate the hypothesis that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays. We assess the heater's equivalence to commercially available PCR instruments through the characterization of the temperature profiles produced, and a minimal method comparison. Versions of the prototype for several different isothermal techniques are presented. Conclusions/Significance We demonstrate that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays, and that the results of those assays are not significantly different from ones incubated in parallel in commercially available PCR instruments. These results clearly suggest the potential of the non-instrumented nucleic acid amplification (NINA) heater for molecular diagnostics in LRS. When combined with other innovations in development that eliminate power requirements for sample preparation, cold reagent storage, and readout, the NINA heater will comprise part of a kit that should enable electricity-free NA testing for many important analytes. PMID:21573065

Improved PCR-Based Detection of Soil Transmitted Helminth Infections Using a Next-Generation Sequencing Approach to Assay Design.

PubMed

Pilotte, Nils; Papaiakovou, Marina; Grant, Jessica R; Bierwert, Lou Ann; Llewellyn, Stacey; McCarthy, James S; Williams, Steven A

2016-03-01

The soil transmitted helminths are a group of parasitic worms responsible for extensive morbidity in many of the world's most economically depressed locations. With growing emphasis on disease mapping and eradication, the availability of accurate and cost-effective diagnostic measures is of paramount importance to global control and elimination efforts. While real-time PCR-based molecular detection assays have shown great promise, to date, these assays have utilized sub-optimal targets. By performing next-generation sequencing-based repeat analyses, we have identified high copy-number, non-coding DNA sequences from a series of soil transmitted pathogens. We have used these repetitive DNA elements as targets in the development of novel, multi-parallel, PCR-based diagnostic assays. Utilizing next-generation sequencing and the Galaxy-based RepeatExplorer web server, we performed repeat DNA analysis on five species of soil transmitted helminths (Necator americanus, Ancylostoma duodenale, Trichuris trichiura, Ascaris lumbricoides, and Strongyloides stercoralis). Employing high copy-number, non-coding repeat DNA sequences as targets, novel real-time PCR assays were designed, and assays were tested against established molecular detection methods. Each assay provided consistent detection of genomic DNA at quantities of 2 fg or less, demonstrated species-specificity, and showed an improved limit of detection over the existing, proven PCR-based assay. The utilization of next-generation sequencing-based repeat DNA analysis methodologies for the identification of molecular diagnostic targets has the ability to improve assay species-specificity and limits of detection. By exploiting such high copy-number repeat sequences, the assays described here will facilitate soil transmitted helminth diagnostic efforts. We recommend similar analyses when designing PCR-based diagnostic tests for the detection of other eukaryotic pathogens.

Assessment of sperm nucleus integrity in infertile men: a novel research field for anthropology in the molecular era.

PubMed

Lavranos, Giagkos; Manolakou, Panagiota; Katsiki, Evangelia; Angelopoulou, Roxani

2013-12-01

Anthropology has always been particularly interested in the origin of human life and the development towards adulthood. Although originally working with skeletal measurements and bio-morphological markers in modern populations, it has now entered the growing field of applied molecular biology. This relatively recent advance allows the detailed study of major events in human development and senescence. For instance, sperm DNA integrity and chromatin re-organization are crucial factors for fertilization and embryo development. Clinical researchers have developed improved methods for the evaluation of DNA integrity and protaminosis in sperm nuclei, such as the TUNEL and the CMA3 assays. DNA damage in spermatozoal nuclei is detected using the TUNEL assay which depends on the specific enzymatic reaction of TdT with the end strand breaks of DNA. Protaminosis in spermatozoal nucleus is evaluated using CMA3 assay, which is based on the in situ competition between CMA3 and protamines. Such measurements may provide useful data on human reproductive health, aiding the explanation of demographic differences across the world.

Molecular detection of Phytophthora ramorum by real-time PCR using Taqman, SYBR Green and molecular beacons with three genes

Treesearch

G.J. Bilodeau; C.A. Lévesque; A.W.A.M. De Cock; C. Duchaine; G. Kristjansson; R.C. Hamelin

2006-01-01

Sudden oak death, caused by Phytophthora ramorum, is a severe disease that can affect numerous species of trees and shrubs. This pathogen has been spread via nursery stock, and quarantine measures are currently in place to prevent further spread. Molecular assays have been developed to rapidly detect and identify P. ramorum, but...

The applicability of TaqMan-based quantitative real-time PCR assays for detecting and enumeratIng Cryptosporidium spp. oocysts in the environment

EPA Science Inventory

Molecular detection methods such as PCR have been extensively used to type Cryptosporidium oocysts detected in the environment. More recently, studies have developed quantitative real-time PCR assays for detection and quantification of microbial contaminants in water as well as ...

Molecular diversity of seed-borne Fusarium species associated with maize in India

USDA-ARS?s Scientific Manuscript database

A total of 62 Fusarium isolates were recovered from 106 maize seeds sampled across 13 states in India, 90% of which were identified as F. verticillioides. Our study included (1) species confirmation through PCR assay using the tef-1a gene, (2) a fumonisin cluster genotype assay using developed multi...

Building an Adverse Outcome Pathway Framework through HTS Data and Literature Mining Integration (FutureToxII)

EPA Science Inventory

Interpreting EPA’s ToxCast in vitro assay data in the context of Adverse Outcome Pathway (AOP) development is a significant challenge. While chemical activation in these assays may shed light on the molecular initiating event, the downstream effect of these activities at higher ...

Laser cutting eliminates nucleic acid cross-contamination in dried-blood-spot processing.

PubMed

Murphy, Sean C; Daza, Glenda; Chang, Ming; Coombs, Robert

2012-12-01

Dried blood spots (DBS) are useful for molecular assays but are prone to false positives from cross-contamination. In our malaria DBS assay, cross-contamination was encountered despite cleaning techniques suitable for HIV-1. We therefore developed a contact-free laser cutting system that effectively eliminated cross-contamination during DBS processing.

The impact of commercial rapid respiratory virus diagnostic tests on patient outcomes and health system utilization.

PubMed

Ko, Fiona; Drews, Steven J

2017-10-01

Acute respiratory tract infections due to influenza A/B and respiratory syncytial virus (RSV) are major causes of morbidity and mortality globally. Rapid tests for detection of these pathogens include antigen detection point of care tests (POC) and newer easy to use molecular tests. From experience, these assays improve both laboratory workflow and assay interpretation issues. However, the question of the benefits of using rapid test technology compared to routine laboratory testing for respiratory viral pathogens is still often asked. Areas covered: Specifically, this review aims to; 1) identify clinical/patient indicators that can be measured prior to and following the implementation of rapid diagnostic test for influenza and RSV, 2) provide multiple perspectives on the extent of impact of a rapid diagnostic test, including direct and indirect outcomes, and 3) identify the technological advancements in the development of rapid testing, demonstrating a timeline that transitions from antigen-based assays to molecular assays. Expert commentary: Key benefits to the use of either antigen-based or molecular rapid tests for patient care, patient flow within institutions, as well as laboratory utilization are identified. Due to improved test characteristics, the authors feel that rapid molecular tests have greater benefits than antigen-based detection methods.

Molecular Identification and Genetic Analysis of Norovirus Genogroups I and II in Water Environments: Comparative Analysis of Different Reverse Transcription-PCR Assays▿

PubMed Central

La Rosa, G.; Fontana, S.; Di Grazia, A.; Iaconelli, M.; Pourshaban, M.; Muscillo, M.

2007-01-01

Noroviruses have received increased attention in recent years because their role as etiologic agents in acute gastroenteritis outbreaks is now clearly established. Our inability to grow them in cell culture and the lack of an animal model hinder the characterization of these viruses. More recently, molecular approaches have been used to study the genetic relationships that exist among them. In the present study, environmental samples from seawater, estuarine water, and effluents of sewage treatment plants were analyzed in order to evaluate the role of environmental surface contamination as a possible vehicle for transmission of norovirus genogroups I and II. Novel broad-range reverse transcription-PCR/nested assays targeting the region coding for the RNA-dependent RNA polymerase were developed, amplifying fragments of 516 bp and 687 bp in the nested reactions for genogroups II and I, respectively. The assays were evaluated and compared against widely used published assays. The newly designed assays provide long regions for high-confidence BLAST searches in public databases and therefore are useful diagnostic tools for molecular diagnosis and typing of human noroviruses in clinical and environmental samples, as well as for the study of molecular epidemiology and the evolution of these viruses. PMID:17483265

Development of a PCR assay to detect papillomavirus infection in the snow leopard.

PubMed

Mitsouras, Katherine; Faulhaber, Erica A; Hui, Gordon; Joslin, Janis O; Eng, Curtis; Barr, Margaret C; Irizarry, Kristopher Jl

2011-07-18

Papillomaviruses (PVs) are a group of small, non-encapsulated, species-specific DNA viruses that have been detected in a variety of mammalian and avian species including humans, canines and felines. PVs cause lesions in the skin and mucous membranes of the host and after persistent infection, a subset of PVs can cause tumors such as cervical malignancies and head and neck squamous cell carcinoma in humans. PVs from several species have been isolated and their genomes have been sequenced, thereby increasing our understanding of the mechanism of viral oncogenesis and allowing for the development of molecular assays for the detection of PV infection. In humans, molecular testing for PV DNA is used to identify patients with persistent infections at risk for developing cervical cancer. In felids, PVs have been isolated and sequenced from oral papillomatous lesions of several wild species including bobcats, Asian lions and snow leopards. Since a number of wild felids are endangered, PV associated disease is a concern and there is a need for molecular tools that can be used to further study papillomavirus in these species. We used the sequence of the snow leopard papillomavirus UuPV1 to develop a PCR strategy to amplify viral DNA from samples obtained from captive animals. We designed primer pairs that flank the E6 and E7 viral oncogenes and amplify two DNA fragments encompassing these genes. We detected viral DNA for E6 and E7 in genomic DNA isolated from saliva, but not in paired blood samples from snow leopards. We verified the identity of these PCR products by restriction digest and DNA sequencing. The sequences of the PCR products were 100% identical to the published UuPV1 genome sequence. We developed a PCR assay to detect papillomavirus in snow leopards and amplified viral DNA encompassing the E6 and E7 oncogenes specifically in the saliva of animals. This assay could be utilized for the molecular investigation of papillomavirus in snow leopards using saliva, thereby allowing the detection of the virus in the anatomical site where oral papillomatous lesions develop during later stages of infection and disease development.

Development of a PCR Assay to detect Papillomavirus Infection in the Snow Leopard

PubMed Central

2011-01-01

Background Papillomaviruses (PVs) are a group of small, non-encapsulated, species-specific DNA viruses that have been detected in a variety of mammalian and avian species including humans, canines and felines. PVs cause lesions in the skin and mucous membranes of the host and after persistent infection, a subset of PVs can cause tumors such as cervical malignancies and head and neck squamous cell carcinoma in humans. PVs from several species have been isolated and their genomes have been sequenced, thereby increasing our understanding of the mechanism of viral oncogenesis and allowing for the development of molecular assays for the detection of PV infection. In humans, molecular testing for PV DNA is used to identify patients with persistent infections at risk for developing cervical cancer. In felids, PVs have been isolated and sequenced from oral papillomatous lesions of several wild species including bobcats, Asian lions and snow leopards. Since a number of wild felids are endangered, PV associated disease is a concern and there is a need for molecular tools that can be used to further study papillomavirus in these species. Results We used the sequence of the snow leopard papillomavirus UuPV1 to develop a PCR strategy to amplify viral DNA from samples obtained from captive animals. We designed primer pairs that flank the E6 and E7 viral oncogenes and amplify two DNA fragments encompassing these genes. We detected viral DNA for E6 and E7 in genomic DNA isolated from saliva, but not in paired blood samples from snow leopards. We verified the identity of these PCR products by restriction digest and DNA sequencing. The sequences of the PCR products were 100% identical to the published UuPV1 genome sequence. Conclusions We developed a PCR assay to detect papillomavirus in snow leopards and amplified viral DNA encompassing the E6 and E7 oncogenes specifically in the saliva of animals. This assay could be utilized for the molecular investigation of papillomavirus in snow leopards using saliva, thereby allowing the detection of the virus in the anatomical site where oral papillomatous lesions develop during later stages of infection and disease development. PMID:21767399

The development, evaluation and performance of molecular diagnostics for detection of Mycobacterium tuberculosis.

PubMed

Bates, Matthew; Zumla, Alimuddin

2016-01-01

The unique pathogenesis of tuberculosis (TB) poses several barriers to the development of accurate diagnostics: a) the establishment of life-long latency by Mycobacterium tuberculosis (M.tb) after primary infection confounds the development of classical antibody or antigen based assays; b) our poor understanding of the molecular pathways that influence progression from latent to active disease; c) the intracellular nature of M.tb infection in tissues means that M.tb and/or its components, are not readily detectable in peripheral specimens; and d) the variable presence of M.tb bacilli in specimens from patients with extrapulmonary TB or children. The literature on the current portfolio of molecular diagnostics tests for TB is reviewed here and the developmental pipeline is summarized. Also reviewed are data from recently published operational research on the GeneXpert MTB/RIF assay and discussed are the lessons that can be taken forward for the design of studies to evaluate the impact of TB diagnostics.

Nanoscale methods for single-molecule electrochemistry.

PubMed

Mathwig, Klaus; Aartsma, Thijs J; Canters, Gerard W; Lemay, Serge G

2014-01-01

The development of experiments capable of probing individual molecules has led to major breakthroughs in fields ranging from molecular electronics to biophysics, allowing direct tests of knowledge derived from macroscopic measurements and enabling new assays that probe population heterogeneities and internal molecular dynamics. Although still somewhat in their infancy, such methods are also being developed for probing molecular systems in solution using electrochemical transduction mechanisms. Here we outline the present status of this emerging field, concentrating in particular on optical methods, metal-molecule-metal junctions, and electrochemical nanofluidic devices.

Quantitative image analysis of cellular heterogeneity in breast tumors complements genomic profiling.

PubMed

Yuan, Yinyin; Failmezger, Henrik; Rueda, Oscar M; Ali, H Raza; Gräf, Stefan; Chin, Suet-Feung; Schwarz, Roland F; Curtis, Christina; Dunning, Mark J; Bardwell, Helen; Johnson, Nicola; Doyle, Sarah; Turashvili, Gulisa; Provenzano, Elena; Aparicio, Sam; Caldas, Carlos; Markowetz, Florian

2012-10-24

Solid tumors are heterogeneous tissues composed of a mixture of cancer and normal cells, which complicates the interpretation of their molecular profiles. Furthermore, tissue architecture is generally not reflected in molecular assays, rendering this rich information underused. To address these challenges, we developed a computational approach based on standard hematoxylin and eosin-stained tissue sections and demonstrated its power in a discovery and validation cohort of 323 and 241 breast tumors, respectively. To deconvolute cellular heterogeneity and detect subtle genomic aberrations, we introduced an algorithm based on tumor cellularity to increase the comparability of copy number profiles between samples. We next devised a predictor for survival in estrogen receptor-negative breast cancer that integrated both image-based and gene expression analyses and significantly outperformed classifiers that use single data types, such as microarray expression signatures. Image processing also allowed us to describe and validate an independent prognostic factor based on quantitative analysis of spatial patterns between stromal cells, which are not detectable by molecular assays. Our quantitative, image-based method could benefit any large-scale cancer study by refining and complementing molecular assays of tumor samples.

Multicentric comparative assessment of the bio-evolution Toxoplasma gondii detection kit with eight laboratory-developed PCR assays for molecular diagnosis of congenital toxoplasmosis.

PubMed

Filisetti, Denis; Sterkers, Yvon; Brenier-Pinchart, Marie-Pierre; Cassaing, Sophie; Dalle, Frédéric; Delhaes, Laurence; Pelloux, Hervé; Touafek, Fériel; Varlet-Marie, Emmanuelle; Yera, Hélène; Candolfi, Ermano; Bastien, Patrick

2015-01-01

The detection of Toxoplasma gondii in amniotic fluid is an essential tool for the prenatal diagnosis of congenital toxoplasmosis and is currently essentially based on the use of PCR. Although some consensus is emerging, this molecular diagnosis suffers from a lack of standardization and an extreme diversity of laboratory-developed methods. Commercial kits for the detection of T. gondii by PCR were recently developed and offer certain advantages; however, they must be assessed in comparison with optimized reference PCR assays. The present multicentric study aimed to compare the performances of the Bio-Evolution T. gondii detection kit and laboratory-developed PCR assays set up in eight proficient centers in France. The study compared 157 amniotic fluid samples and found concordances of 99% and 100% using 76 T. gondii-infected samples and 81 uninfected samples, respectively. Moreover, taking into account the classification of the European Research Network on Congenital Toxoplasmosis, the overall diagnostic sensitivity of all assays was identical and calculated to be 86% (54/63); specificity was 100% for all assays. Finally, the relative quantification results were in good agreement between the kit and the laboratory-developed assays. The good performances of this commercial kit are probably in part linked to the use of a number of good practices: detection in multiplicate, amplification of the repetitive DNA target rep529, and the use of an internal control for the detection of PCR inhibitors. The only drawbacks noted at the time of the study were the absence of uracil-N-glycosylase and small defects in the reliability of the production of different reagents. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Multicentric Comparative Assessment of the Bio-Evolution Toxoplasma gondii Detection Kit with Eight Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis

PubMed Central

Filisetti, Denis; Sterkers, Yvon; Brenier-Pinchart, Marie-Pierre; Cassaing, Sophie; Dalle, Frédéric; Delhaes, Laurence; Pelloux, Hervé; Touafek, Fériel; Varlet-Marie, Emmanuelle; Yera, Hélène; Candolfi, Ermano

2014-01-01

The detection of Toxoplasma gondii in amniotic fluid is an essential tool for the prenatal diagnosis of congenital toxoplasmosis and is currently essentially based on the use of PCR. Although some consensus is emerging, this molecular diagnosis suffers from a lack of standardization and an extreme diversity of laboratory-developed methods. Commercial kits for the detection of T. gondii by PCR were recently developed and offer certain advantages; however, they must be assessed in comparison with optimized reference PCR assays. The present multicentric study aimed to compare the performances of the Bio-Evolution T. gondii detection kit and laboratory-developed PCR assays set up in eight proficient centers in France. The study compared 157 amniotic fluid samples and found concordances of 99% and 100% using 76 T. gondii-infected samples and 81 uninfected samples, respectively. Moreover, taking into account the classification of the European Research Network on Congenital Toxoplasmosis, the overall diagnostic sensitivity of all assays was identical and calculated to be 86% (54/63); specificity was 100% for all assays. Finally, the relative quantification results were in good agreement between the kit and the laboratory-developed assays. The good performances of this commercial kit are probably in part linked to the use of a number of good practices: detection in multiplicate, amplification of the repetitive DNA target rep529, and the use of an internal control for the detection of PCR inhibitors. The only drawbacks noted at the time of the study were the absence of uracil-N-glycosylase and small defects in the reliability of the production of different reagents. PMID:25339393

Avirulent Bacillus anthracis Strain with Molecular Assay Targets as Surrogate for Irradiation-Inactivated Virulent Spores.

PubMed

Plaut, Roger D; Staab, Andrea B; Munson, Mark A; Gebhardt, Joan S; Klimko, Christopher P; Quirk, Avery V; Cote, Christopher K; Buhr, Tony L; Rossmaier, Rebecca D; Bernhards, Robert C; Love, Courtney E; Berk, Kimberly L; Abshire, Teresa G; Rozak, David A; Beck, Linda C; Stibitz, Scott; Goodwin, Bruce G; Smith, Michael A; Sozhamannan, Shanmuga

2018-04-01

The revelation in May 2015 of the shipment of γ irradiation-inactivated wild-type Bacillus anthracis spore preparations containing a small number of live spores raised concern about the safety and security of these materials. The finding also raised doubts about the validity of the protocols and procedures used to prepare them. Such inactivated reference materials were used as positive controls in assays to detect suspected B. anthracis in samples because live agent cannot be shipped for use in field settings, in improvement of currently deployed detection methods or development of new methods, or for quality assurance and training activities. Hence, risk-mitigated B. anthracis strains are needed to fulfill these requirements. We constructed a genetically inactivated or attenuated strain containing relevant molecular assay targets and tested to compare assay performance using this strain to the historical data obtained using irradiation-inactivated virulent spores.

A recombinase polymerase amplification assay for rapid detection of Crimean-Congo Haemorrhagic fever Virus infection.

PubMed

Bonney, Laura C; Watson, Robert J; Afrough, Babak; Mullojonova, Manija; Dzhuraeva, Viktoriya; Tishkova, Farida; Hewson, Roger

2017-10-01

Crimean-Congo Haemorrhagic fever Virus (CCHFV) is a rapidly emerging vector-borne pathogen and the cause of a virulent haemorrhagic fever affecting large parts of Europe, Africa, the Middle East and Asia. An isothermal recombinase polymerase amplification (RPA) assay was successfully developed for molecular detection of CCHFV. The assay showed rapid (under 10 minutes) detection of viral extracts/synthetic virus RNA of all 7 S-segment clades of CCHFV, with high target specificity. The assay was shown to tolerate the presence of inhibitors in crude preparations of mock field samples, indicating that this assay may be suitable for use in the field with minimal sample preparation. The CCHFV RPA was successfully used to screen and detect CCHFV positives from a panel of clinical samples from Tajikistan. The assay is a rapid, isothermal, simple-to-perform molecular diagnostic, which can be performed on a light, portable real-time detection device. It is ideally placed therefore for use as a field-diagnostic or in-low resource laboratories, for monitoring of CCHF outbreaks at the point-of-need, such as in remote rural regions in affected countries.

Bimolecular fluorescence complementation: visualization of molecular interactions in living cells.

PubMed

Kerppola, Tom K

2008-01-01

A variety of experimental methods have been developed for the analysis of protein interactions. The majority of these methods either require disruption of the cells to detect molecular interactions or rely on indirect detection of the protein interaction. The bimolecular fluorescence complementation (BiFC) assay provides a direct approach for the visualization of molecular interactions in living cells and organisms. The BiFC approach is based on the facilitated association between two fragments of a fluorescent protein when the fragments are brought together by an interaction between proteins fused to the fragments. The BiFC approach has been used for visualization of interactions among a variety of structurally diverse interaction partners in many different cell types. It enables detection of transient complexes as well as complexes formed by a subpopulation of the interaction partners. It is essential to include negative controls in each experiment in which the interface between the interaction partners has been mutated or deleted. The BiFC assay has been adapted for simultaneous visualization of multiple protein complexes in the same cell and the competition for shared interaction partners. A ubiquitin-mediated fluorescence complementation assay has also been developed for visualization of the covalent modification of proteins by ubiquitin family peptides. These fluorescence complementation assays have a great potential to illuminate a variety of biological interactions in the future.

Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification.

PubMed

Poon, Leo L M; Wong, Bonnie W Y; Ma, Edmund H T; Chan, Kwok H; Chow, Larry M C; Abeyewickreme, Wimal; Tangpukdee, Noppadon; Yuen, Kwok Y; Guan, Yi; Looareesuwan, Sornchai; Peiris, J S Malik

2006-02-01

Malaria is one of the most important parasitic infections in humans. A sensitive diagnostic test for malaria that could be applied at the community level could be useful in programs to control the disease. The aim of the present work was to develop a simple, inexpensive molecular test for Plasmodium falciparum. Blood was collected from controls (n = 100) and from patients diagnosed with falciparum malaria infection (n = 102), who were recruited to the study. Heat-treated blood samples were tested by a loop-mediated isothermal amplification (LAMP) assay for P. falciparum. Results were interpreted by a turbidity meter in real time or visually at the end of the assay. To evaluate the assay, DNA from these samples was purified and tested by PCR. Results from the LAMP and PCR assays were compared. The LAMP assay detected P. falciparum directly from heat-treated blood. The quantitative data from the assay correlated to the parasite counts obtained by blood-film microscopic analyses. When we used the PCR assay as the comparison method, the sensitivity and specificity of the LAMP assay were 95% and 99%, respectively. Unlike PCR, the LAMP assay does not require purified DNA for efficient DNA amplification, thereby reducing the cost and turnaround time for P. falciparum diagnosis. The assay requires only basic instruments, and assay positivity can be verified by visual inspection.

Improved Safety for Molecular Diagnosis of Classical Rabies Viruses by Use of a TaqMan Real-Time Reverse Transcription-PCR “Double Check” Strategy▿ †

PubMed Central

Hoffmann, B.; Freuling, C. M.; Wakeley, P. R.; Rasmussen, T. B.; Leech, S.; Fooks, A. R.; Beer, M.; Müller, T.

2010-01-01

To improve the diagnosis of classical rabies virus with molecular methods, a validated, ready-to-use, real-time reverse transcription-PCR (RT-PCR) assay was developed. In a first step, primers and 6-carboxyfluorescien-labeled TaqMan probes specific for rabies virus were selected from the consensus sequence of the nucleoprotein gene of 203 different rabies virus sequences derived from GenBank. The selected primer-probe combination was highly specific and sensitive. During validation using a sample set of rabies virus strains from the virus archives of the Friedrich-Loeffler-Institut (FLI; Germany), the Veterinary Laboratories Agency (VLA; United Kingdom), and the DTU National Veterinary Institute (Lindholm, Denmark), covering the global diversity of rabies virus lineages, it was shown that both the newly developed assay and a previously described one had some detection failures. This was overcome by a combined assay that detected all samples as positive. In addition, the introduction of labeled positive controls (LPC) increased the diagnostic safety of the single as well as the combined assay. Based on the newly developed, alternative assay for the detection of rabies virus and the application of LPCs, an improved diagnostic sensitivity and reliability can be ascertained for postmortem and intra vitam real-time RT-PCR analyses in rabies reference laboratories. PMID:20739489

Improved safety for molecular diagnosis of classical rabies viruses by use of a TaqMan real-time reverse transcription-PCR "double check" strategy.

PubMed

Hoffmann, B; Freuling, C M; Wakeley, P R; Rasmussen, T B; Leech, S; Fooks, A R; Beer, M; Müller, T

2010-11-01

To improve the diagnosis of classical rabies virus with molecular methods, a validated, ready-to-use, real-time reverse transcription-PCR (RT-PCR) assay was developed. In a first step, primers and 6-carboxyfluorescien-labeled TaqMan probes specific for rabies virus were selected from the consensus sequence of the nucleoprotein gene of 203 different rabies virus sequences derived from GenBank. The selected primer-probe combination was highly specific and sensitive. During validation using a sample set of rabies virus strains from the virus archives of the Friedrich-Loeffler-Institut (FLI; Germany), the Veterinary Laboratories Agency (VLA; United Kingdom), and the DTU National Veterinary Institute (Lindholm, Denmark), covering the global diversity of rabies virus lineages, it was shown that both the newly developed assay and a previously described one had some detection failures. This was overcome by a combined assay that detected all samples as positive. In addition, the introduction of labeled positive controls (LPC) increased the diagnostic safety of the single as well as the combined assay. Based on the newly developed, alternative assay for the detection of rabies virus and the application of LPCs, an improved diagnostic sensitivity and reliability can be ascertained for postmortem and intra vitam real-time RT-PCR analyses in rabies reference laboratories.

Pathogen Inactivating Properties and Increased Sensitivity in Molecular Diagnostics by PAXgene, a Novel Non-Crosslinking Tissue Fixative.

PubMed

Loibner, Martina; Buzina, Walter; Viertler, Christian; Groelz, Daniel; Hausleitner, Anja; Siaulyte, Gintare; Kufferath, Iris; Kölli, Bettina; Zatloukal, Kurt

2016-01-01

Requirements on tissue fixatives are getting more demanding as molecular analysis becomes increasingly relevant for routine diagnostics. Buffered formaldehyde in pathology laboratories for tissue fixation is known to cause chemical modifications of biomolecules which affect molecular testing. A novel non-crosslinking tissue preservation technology, PAXgene Tissue (PAXgene), was developed to preserve the integrity of nucleic acids in a comparable way to cryopreservation and also to preserve morphological features comparable to those of formalin fixed samples. Because of the excellent preservation of biomolecules by PAXgene we investigated its pathogen inactivation ability and biosafety in comparison to formalin by in-vitro testing of bacteria, human relevant fungi and human cytomegalovirus (CMV). Guidelines for testing disinfectants served as reference for inactivation assays. Furthermore, we tested the properties of PAXgene for detection of pathogens by PCR based assays. All microorganisms tested were similarly inactivated by PAXgene and formalin except Clostridium sporogenes, which remained viable in seven out of ten assays after PAXgene treatment and in three out of ten assays after formalin fixation. The findings suggest that similar biosafety measures can be applied for PAXgene and formalin fixed samples. Detection of pathogens in PCR-based diagnostics using two CMV assays resulted in a reduction of four to ten quantification cycles of PAXgene treated samples which is a remarkable increase of sensitivity. PAXgene fixation might be superior to formalin fixation when molecular diagnostics and highly sensitive detection of pathogens is required in parallel to morphology assessment.

MOLECULAR METHODS USED TO ASSESS THE RISKS OF TRANSGENE FLOW; BENEFITS AND LIMITATIONS

EPA Science Inventory

The US EPA WED has initiated a gene flow project to characterize ecological risks of gene flow from GM plants to native species. Development of molecular assays for risk characterization down to gene expression level is of high interest to the EPA. Phylogenetic analyses of ampl...

Potential biological targets for bioassay development in drug discovery of Sturge-Weber syndrome.

PubMed

Mohammadipanah, Fatemeh; Salimi, Fatemeh

2018-02-01

Sturge-Weber Syndrome (SWS) is a neurocutaneous disease with clinical manifestations including ocular (glaucoma), cutaneous (port-wine birthmark), neurologic (seizures), and vascular problems. Molecular mechanisms of SWS pathogenesis are initiated by the somatic mutation in GNAQ. Therefore, no definite treatments exist for SWS and treatment options only mitigate the intensity of its clinical manifestations. Biological assay design for drug discovery against this syndrome demands comprehensive knowledge on mechanisms which are involved in its pathogenesis. By analysis of the interrelated molecular targets of SWS, some in vitro bioassay systems can be allotted for drug screening against its progression. Development of such platforms of bioassay can bring along the implementation of high-throughput screening of natural or synthetic compounds in drug discovery programs. Regarding the fact that study of molecular targets and their integration in biological assay design can facilitate the process of effective drug discovery; some potential biological targets and their respective biological assay for SWS drug discovery are propounded in this review. For this purpose, some biological targets for SWS drug discovery such as acetylcholinesterase, alkaline phosphatase, GABAergic receptors, Hypoxia-Inducible Factor (HIF)-1α and 2α are suggested. © 2017 John Wiley & Sons A/S.

Progress in Life Marker Chip Technology for Detection of Life on Mars

NASA Astrophysics Data System (ADS)

Sims, M. R.; Cullen, D. C.; Laan, E.; Borst, G.; Prak, A.; Richter, L.; Gaubert, F.; Steele, A.; Parnell, J.; Sephton, M.

2007-12-01

Detection of Life on Mars will rely on detection of biomarkers, physical or chemical structures that can be associated with Life. As a possible payload for the ESA ExoMars rover mission planned in 2013 and other future missions a Life Marker Chip instrument is being developed. This instrument uses immuno-assay techniques to detect the relevant biomarkers. This paper describes the typical targets it will search for, its operating principle and the status of development. 63 biomarker targets have been identified and assays have been developed for a limited subset. Assay development includes use of recombinant DNA techniques to generate the molecular receptors (antibodies). This type of instrument has applications in terrestrial research e.g. sub-glacial lakes as well as planetary exploration. Breadboard demonstrators have been built of the assay system and key components of the micro-fluidics. Results from these breadboards will be presented, along with plans for future development.

The pivotal role of multimodality reporter sensors in drug discovery: from cell based assays to real time molecular imaging.

PubMed

Ray, Pritha

2011-04-01

Development and marketing of new drugs require stringent validation that are expensive and time consuming. Non-invasive multimodality molecular imaging using reporter genes holds great potential to expedite these processes at reduced cost. New generations of smarter molecular imaging strategies such as Split reporter, Bioluminescence resonance energy transfer, Multimodality fusion reporter technologies will further assist to streamline and shorten the drug discovery and developmental process. This review illustrates the importance and potential of molecular imaging using multimodality reporter genes in drug development at preclinical phases.

Leveraging a high resolution microfluidic assay reveals insights into pathogenic fungal spore germination

PubMed Central

Barkal, Layla J.; Walsh, Naomi M.; Botts, Michael R.; Beebe, David J.; Hull, Christina M.

2016-01-01

Germination of spores into actively growing cells is a process essential for survival and pathogenesis of many microbes. Molecular mechanisms governing germination, however, are poorly understood in part because few tools exist for evaluating and interrogating the process. Here, we introduce an assay that leverages developments in microfluidic technology and image processing to quantitatively measure germination with unprecedented resolution, assessing both individual cells and the population as a whole. Using spores from Cryptococcus neoformans, a leading cause of fatal fungal disease in humans, we developed a platform to evaluate spores as they undergo morphological changes during differentiation into vegetatively growing yeast. The assay uses pipet-accessible microdevices that can be arrayed for efficient testing of diverse microenvironmental variables, including temperature and nutrients. We discovered that temperature influences germination rate, a carbon source alone is sufficient to induce germination, and the addition of a nitrogen source sustains it. Using this information, we optimized the assay for use with fungal growth inhibitors to pinpoint stages of germination inhibition. Unexpectedly, the clinical antifungal drugs amphotericin B and fluconazole did not significantly alter the process or timing of the transition from spore to yeast, indicating that vegetative growth and germination are distinct processes in C. neoformans. Finally, we used the high temporal resolution of the assay to determine the precise defect in a slow-germination mutant. Combining advances in microfluidics with a robust fungal molecular genetic system allowed us to identify and alter key temporal, morphological, and molecular events that occur during fungal germination. PMID:27026574

Rapid detection of rifampin resistance in Mycobacterium tuberculosis isolates from India and Mexico by a molecular beacon assay.

PubMed

Varma-Basil, Mandira; El-Hajj, Hiyam; Colangeli, Roberto; Hazbón, Manzour Hernando; Kumar, Sujeet; Bose, Mridula; Bobadilla-del-Valle, Miriam; García, Lourdes García; Hernández, Araceli; Kramer, Fred Russell; Osornio, Jose Sifuentes; Ponce-de-León, Alfredo; Alland, David

2004-12-01

We assessed the performance of a rapid, single-well, real-time PCR assay for the detection of rifampin-resistant Mycobacterium tuberculosis by using clinical isolates from north India and Mexico, regions with a high incidence of tuberculosis. The assay uses five differently colored molecular beacons to determine if a short region of the M. tuberculosis rpoB gene contains mutations that predict rifampin resistance in most isolates. Until now, the assay had not been sufficiently tested on samples from countries with a high incidence of tuberculosis. In the present study, the assay detected mutations in 16 out of 16 rifampin-resistant isolates from north India (100%) and in 55 of 64 rifampin-resistant isolates from Mexico (86%) compared to results with standard susceptibility testing. The assay did not detect mutations (a finding predictive of rifampin susceptibility) in 37 out of 37 rifampin-susceptible isolates from India (100%) and 125 out of 126 rifampin-susceptible isolates from Mexico (99%). DNA sequencing revealed that none of the nine rifampin-resistant isolates from Mexico, which were misidentified as rifampin susceptible by the molecular beacon assay, contained a mutation in the region targeted by the molecular beacons. The one rifampin-susceptible isolate from Mexico that appeared to be rifampin resistant by the molecular beacon assay contained an S531W mutation, which is usually associated with rifampin resistance. Of the rifampin-resistant isolates that were correctly identified in the molecular beacon assay, one contained a novel L530A mutation and another contained a novel deletion between codons 511 and 514. Overall, the molecular beacon assay appears to have sufficient sensitivity (89%) and specificity (99%) for use in countries with a high prevalence of tuberculosis.

Laser Cutting Eliminates Nucleic Acid Cross-Contamination in Dried-Blood-Spot Processing

PubMed Central

Daza, Glenda; Chang, Ming; Coombs, Robert

2012-01-01

Dried blood spots (DBS) are useful for molecular assays but are prone to false positives from cross-contamination. In our malaria DBS assay, cross-contamination was encountered despite cleaning techniques suitable for HIV-1. We therefore developed a contact-free laser cutting system that effectively eliminated cross-contamination during DBS processing. PMID:23052309

A Fluorescence Quenching Assay Based on Molecular Beacon Formation through a Ligase Detection Reaction for Facile and Rapid Detection of Point Mutations.

PubMed

Sawamura, Kensuke; Hashimoto, Masahiko

2017-01-01

A fluorescence quenching assay based on a ligase detection reaction was developed for facile and rapid detection of point mutations present in a mixed population of non-variant DNA. If the test DNA carried a targeted mutation, then the two allele-specific primers were ligated to form a molecular beacon resulting in the expected fluorescence quenching signatures. Using this method, we successfully detected as low as 5% mutant DNA in a mixture of wild-type DNA (t test at 99% confidence level).

Insight into the novel inhibition mechanism of apigenin to Pneumolysin by molecular modeling

NASA Astrophysics Data System (ADS)

Niu, Xiaodi; Yang, Yanan; Song, Meng; Wang, Guizhen; Sun, Lin; Gao, Yawen; Wang, Hongsu

2017-11-01

In this study, the mechanism of apigenin inhibition was explored using molecular modelling, binding energy calculation, and mutagenesis assays. Energy decomposition analysis indicated that apigenin binds in the gap between domains 3 and 4 of PLY. Using principal component analysis, we found that binding of apigenin to PLY weakens the motion of domains 3 and 4. Consequently, these domains cannot complete the transition from monomer to oligomer, thereby blocking oligomerisation of PLY and counteracting its haemolytic activity. This inhibitory mechanism was confirmed by haemolysis assays, and these findings will promote the future development of an antimicrobial agent.

Development of an RT-qPCR assay for the specific detection of a distinct genetic lineage of the infectious bursal disease virus.

PubMed

Tomás, Gonzalo; Hernández, Martín; Marandino, Ana; Techera, Claudia; Grecco, Sofia; Hernández, Diego; Banda, Alejandro; Panzera, Yanina; Pérez, Ruben

2017-04-01

The infectious bursal disease virus (IBDV) is a major health threat to the world's poultry industry despite intensive controls including proper biosafety practices and vaccination. IBDV (Avibirnavirus, Birnaviridae) is a non-enveloped virus with a bisegmented double-stranded RNA genome. The virus is traditionally classified into classic, variant and very virulent strains, each with different epidemiological relevance and clinical implications. Recently, a novel worldwide spread genetic lineage was described and denoted as distinct (d) IBDV. Here, we report the development and validation of a reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay for the specific detection of dIBDVs in the global poultry industry. The assay employs a TaqMan-MGB probe that hybridizes with a unique molecular signature of dIBDV. The assay successfully detected all the assessed strains belonging to the dIBDV genetic lineage, showing high specificity and absence of cross-reactivity with non-dIBDVs, IBDV-negative samples and other common avian viruses. Using serial dilutions of in vitro-transcribed RNA we obtained acceptable PCR efficiencies and determination coefficients, and relatively small intra- and inter-assay variability. The assay demonstrated a wide dynamic range between 10 3 and 10 8 RNA copies/reaction. This rapid, specific and quantitative assay is expected to improve IBDV surveillance and control worldwide and to increase our understanding of the molecular epidemiology of this economically detrimental poultry pathogen.

Direct detection of microRNAs using isothermal amplification and molecular beacon with excellent sensitivity and specificity

NASA Astrophysics Data System (ADS)

Zhang, Wancun; Zhang, Qi; Qian, Zhiyu; Gu, Yueqing

2017-02-01

MicroRNAs (miRNAs) play important roles in a wide range of biological processes, including proliferation, development, metabolism, immunological response, tumorigenesis, and viral infection. The detection of miRNAs is imperative for gaining a better understanding of the functions of these biomolecules and has great potential for the early diagnosis of human disease as well as the discovery of new drugs through the use of miRNAs as targets. In this article, we develop a highly sensitive, and specific miRNA assay based on the two-stage isothermal amplification reactions and molecular beacon. The two-stage isothermal amplification reactions involves two templates and two-stage amplification reactions under isothermal conditions. The first template enables the amplification of miRNA, and the second template enables the conversion of miRNA to the reporter oligonucleotide(Y). Importantly, different miRNAs can be converted to the same Y seperately, which can hybridize with the same set of molecular beacon to generate fluorescent signals. This assay is highly sensitive and specific with a detection limit of 1 fM and can even discriminate single-nucleotide differences. Moreover, in combination with the specific templates, this method can be applied for multiplex miRNA assay by simply using the same molecular beacon. This method has potential to become a promising miRNA quantification method in biomedical research and clinical diagnosis.

G-Protein Coupled Receptors: Surface Display and Biosensor Technology

NASA Astrophysics Data System (ADS)

McMurchie, Edward; Leifert, Wayne

Signal transduction by G-protein coupled receptors (GPCRs) underpins a multitude of physiological processes. Ligand recognition by the receptor leads to the activation of a generic molecular switch involving heterotrimeric G-proteins and guanine nucleotides. With growing interest and commercial investment in GPCRs in areas such as drug targets, orphan receptors, high-throughput screening of drugs and biosensors, greater attention will focus on assay development to allow for miniaturization, ultrahigh-throughput and, eventually, microarray/biochip assay formats that will require nanotechnology-based approaches. Stable, robust, cell-free signaling assemblies comprising receptor and appropriate molecular switching components will form the basis of future GPCR/G-protein platforms, which should be able to be adapted to such applications as microarrays and biosensors. This chapter focuses on cell-free GPCR assay nanotechnologies and describes some molecular biological approaches for the construction of more sophisticated, surface-immobilized, homogeneous, functional GPCR sensors. The latter points should greatly extend the range of applications to which technologies based on GPCRs could be applied.

Direct replacement of antibodies with molecularly imprinted polymer nanoparticles in ELISA--development of a novel assay for vancomycin.

PubMed

Chianella, Iva; Guerreiro, Antonio; Moczko, Ewa; Caygill, J Sarah; Piletska, Elena V; De Vargas Sansalvador, Isabel M Perez; Whitcombe, Michael J; Piletsky, Sergey A

2013-09-03

A simple and straightforward technique for coating microplate wells with molecularly imprinted polymer nanoparticles (nanoMIPs) to develop assays similar to the enzyme-linked immunosorbent assay (ELISA) is presented here for the first time. NanoMIPs were synthesized by a solid-phase approach with an immobilized vancomycin (template) and characterized using Biacore 3000, dynamic light scattering, and electron microscopy. Immobilization, blocking, and washing conditions were optimized in microplate format. The detection of vancomycin was achieved in competitive binding experiments with a horseradish peroxidase-vancomycin conjugate. The assay was capable of measuring vancomycin in buffer and in blood plasma within the range of 0.001-70 nM with a detection limit of 0.0025 nM (2.5 pM). The sensitivity of the assay was 3 orders of magnitude better than a previously described ELISA based on antibodies. In these experiments, nanoMIPs have shown high affinity and minimal interference from blood plasma components. Immobilized nanoMIPs were stored for 1 month at room temperature without any detrimental effects to their binding properties. The high affinity of nanoMIPs and the lack of a requirement for cold chain logistics make them an attractive alternative to traditional antibodies used in ELISA.

A duplex PCR-based assay for measuring the amount of bacterial contamination in a nucleic acid extract from a culture of free-living protists.

PubMed

Marron, Alan O; Akam, Michael; Walker, Giselle

2013-01-01

Cultures of heterotrophic protists often require co-culturing with bacteria to act as a source of nutrition. Such cultures will contain varying levels of intrinsic bacterial contamination that can interfere with molecular research and cause problems with the collection of sufficient material for sequencing. Measuring the levels of bacterial contamination for the purposes of molecular biology research is non-trivial, and can be complicated by the presence of a diverse bacterial flora, or by differences in the relative nucleic acid yield per bacterial or eukaryotic cell. Here we describe a duplex PCR-based assay that can be used to measure the levels of contamination from marine bacteria in a culture of loricate choanoflagellates. By comparison to a standard culture of known target sequence content, the assay can be used to quantify the relative proportions of bacterial and choanoflagellate material in DNA or RNA samples extracted from a culture. We apply the assay to compare methods of purifying choanoflagellate cultures prior to DNA extraction, to determine their effectiveness in reducing bacterial contamination. Together with measurements of the total nucleic acid concentration, the assay can then be used as the basis for determining the absolute amounts of choanoflagellate DNA or RNA present in a sample. The assay protocol we describe here is a simple and relatively inexpensive method of measuring contamination levels in nucleic acid samples. This provides a new way to establish quantification and purification protocols for molecular biology and genomics in novel heterotrophic protist species. Guidelines are provided to develop a similar protocol for use with any protistan culture. This assay method is recommended where qPCR equipment is unavailable, where qPCR is not viable because of the nature of the bacterial contamination or starting material, or where prior sequence information is insufficient to develop qPCR protocols.

An optimized one-tube, semi-nested PCR assay for Paracoccidioides brasiliensis detection.

PubMed

Pitz, Amanda de Faveri; Koishi, Andrea Cristine; Tavares, Eliandro Reis; Andrade, Fábio Goulart de; Loth, Eduardo Alexandre; Gandra, Rinaldo Ferreira; Venancio, Emerson José

2013-01-01

Herein, we report a one-tube, semi-nested-polymerase chain reaction (OTsn-PCR) assay for the detection of Paracoccidioides brasiliensis. We developed the OTsn-PCR assay for the detection of P. brasiliensis in clinical specimens and compared it with other PCR methods. The OTsn-PCR assay was positive for all clinical samples, and the detection limit was better or equivalent to the other nested or semi-nested PCR methods for P. brasiliensis detection. The OTsn-PCR assay described in this paper has a detection limit similar to other reactions for the molecular detection of P. brasiliensis, but this approach is faster and less prone to contamination than other conventional nested or semi-nested PCR assays.

Development of an immunomagnetic separation-polymerase chain reaction (IMS-PCR) assay specific for Enterocytozoon bieneusi in water samples.

PubMed

Sorel, N; Guillot, E; Thellier, M; Accoceberry, I; Datry, A; Mesnard-Rouiller, L; Miégeville, M

2003-01-01

Microsporidia have become widely recognized as important human pathogens. Among Microsporidia, Enterocytozoon bieneusi is responsible for severe gastrointestinal disease. To date, no current therapy has been proven effective. Their mode of transmission and environmental occurrence are poorly documented because of the lack of detection methods that are both species-specific and sensitive. In this study, we developed a sensitive and specific molecular method to detect E. bieneusi spores in water samples. The molecular assay combined immunomagnetic separation (IMS) and polymerase chain reaction (PCR) amplification to detect E. bieneusi spores. A comparison was made of IMS magnetic beads coated with two different monoclonal antibodies, one specific for the Encephalitozoon genus that cross-reacts with E. bieneusi and the other specific only for the E. bieneusi species itself. Immunotech beads coated with the antibody specific for E. bieneusi were found to be the most effective combination. The highly specific IMS-PCR assay developed in this study provides a rapid and sensitive means of screening water samples for the presence of E. bieneusi spores.

Development of a biomimetic enzyme-linked immunosorbent assay based on molecularly imprinted polymers on paper for the detection of carbaryl.

PubMed

Zhang, Can; Cui, Hanyu; Han, Yufeng; Yu, Fangfang; Shi, Xiaoman

2018-02-01

A biomimetic enzyme-linked immunosorbent assay (BELISA) which was based on molecularly imprinted polymers on paper (MIPs-paper) with specific recognition was developed. As a detector, the surface of paper was modified with γ-MAPS by hydrolytic action and anchored the MIP layer on γ-MAPS modified-paper by copolymerization to construct the artificial antibody Through a series of experimentation and verification, we successful got the MIPs-paper and established BELISA for the detection of carbaryl. The development of MIPs-paper based on BELISA was applied to detect carbaryl in real samples and validated by an enzyme-linked immunosorbent assay (ELISA) based on anti-carbaryl biological antibody. The results of these two methods (BELISA and ELISA) were well correlated (R 2 =0.944). The established method of MIPs-paper BELISA exhibits the advantages of low cost, higher stability and being re-generable, which can be applied as a convenient tool for the fast and efficient detection of carbaryl. Copyright © 2017. Published by Elsevier Ltd.

Development of a pan-rickettsial molecular diagnostic test based on recombinase polymerase amplification assay.

PubMed

Kissenkötter, Jonas; Hansen, Sören; Böhlken-Fascher, Susanne; Ademowo, Olusegun George; Oyinloye, Oladapo Elijah; Bakarey, Adeleye Solomon; Dobler, Gerhard; Tappe, Dennis; Patel, Pranav; Czerny, Claus-Peter; Abd El Wahed, Ahmed

2018-03-01

Rickettsioses are zoonotic vector-transmitted bacterial infections leading to flu-like symptoms and can progress to severe illness in humans. The gold standard for diagnosis of rickettsial infections is the indirect immunofluorescence assay, a serological method which is not suitable for pathogen identification during the acute phase of the disease. Therefore, several real-time PCR assays were developed. These assays are very sensitive, but require high-equipped laboratories and well-trained personnel. Hence, in this study, a rapid point-of-need detection method was developed to detect all Rickettsia species. The 23S and 16S rRNA genes were targeted to develop a recombinase polymerase amplification (RPA) assay. Both 23S and 16S_RPA assays required between seven to ten minutes to amplify and detect one or ten DNA molecules/reaction, respectively. The 16S_RPA assay detected all tested species, whereas the 23S_RPA assay identified only species of the spotted fever and transitional rickettsial groups. All results were compared with real-time PCR assays directed against the same rickettsial genes. The RPA assays are easy to handle and produced quicker results in comparison to real-time PCRs. Both RPA assays were implemented in a mobile suitcase laboratory to ease the use in rural areas. This method can help to provide rapid management of rickettsial infections. Copyright © 2017 Elsevier Inc. All rights reserved.

Development of Short-term Molecular Thresholds to Predict Long-term Mouse Liver Tumor Outcomes: Phthalate Case Study

EPA Science Inventory

Short-term molecular profiles are a central component of strategies to model health effects of environmental chemicals. In this study, a 7 day mouse assay was used to evaluate transcriptomic and proliferative responses in the liver for a hepatocarcinogenic phthalate, di (2-ethylh...

Fibroblast Growth Factor Receptors: From the Oncogenic Pathway to Targeted Therapy.

PubMed

Saichaemchan, S; Ariyawutyakorn, W; Varella-Garcia, M

2016-01-01

The family of fibroblast growth factor (FGFs) and their receptors (FGFRs) regulates vital roles in many biological processes affecting cell proliferation, migration, differentiation and survival. Deregulation of the FGF/FGFR signaling pathway in cancers has been better understood and the main molecular mechanisms responsible for the activation of this pathway are gene mutations, gene fusions and gene amplification. DNA and RNA-based technologies have been used to detect these abnormalities, especially in FGFR1, FGFR2 and FGFR3 and tests have been developed for their detection, but no assay has been proved ideal for molecular diagnosis. Interestingly, the increase in the molecular biology knowledge has supported and assisted the development of therapeutic drugs targeting the most important components of this pathway. Multi- and selective tyrosine kinase inhibitors (TKIs) as well as monoclonal antibodies anti-FGFR are under investigation in preclinical and clinical trials. In this article, we reviewed those aspects with special emphasis on the pathway genomic alterations related to solid tumors, and the molecular diagnostic assays potentially able to stratify patients for the treatment with FGFR TKIs.

Isolation of RNA From Peripheral Blood Cells: A Validation Study for Molecular Diagnostics by Microarray and Kinetic RT-PCR Assays - Application in Aerospace Medicine

DTIC Science & Technology

2004-01-01

of RNA From Peripheral Blood Cells: A Validation Study for Molecular Diagnostics by Microarray and Kinetic RT-PCR Assays  Application in...VALIDATION STUDY FOR MOLECULAR DIAGNOSTICS BY MICROARRAY AND KINETIC RT-PCR ASSAYS  APPLICATION IN AEROSPACE MEDICINE INTRODUCTION Extraction of cellular

Development of a Computational Assay for the Estrogen Receptor

DTIC Science & Technology

2006-07-01

University Ashley Deline, Senior Thesis in chemistry, " Molecular Dynamic Simulations of a Glycoform and its Constituent Parts Related to Rheumatoid Arthritis...involves running a long molecular dynamics (MD) simulation of the uncoupled receptor in order to sample the protein’s unique conformations. The second...Receptor binding domain. * Performed several long molecular dynamics simulations (800 ps - 3 ns) on the ligand-ER system using ligands with known

Rapid Detection of Pathogens

DOE Office of Scientific and Technical Information (OSTI.GOV)

David Perlin

2005-08-14

Pathogen identification is a crucial first defense against bioterrorism. A major emphasis of our national biodefense strategy is to establish fast, accurate and sensitive assays for diagnosis of infectious diseases agents. Such assays will ensure early and appropriate treatment of infected patients. Rapid diagnostics can also support infection control measures, which monitor and limit the spread of infectious diseases agents. Many select agents are highly transmissible in the early stages of disease, and it is critical to identify infected patients and limit the risk to the remainder of the population and to stem potential panic in the general population. Nucleicmore » acid-based molecular approaches for identification overcome many of the deficiencies associated with conventional culture methods by exploiting both large- and small-scale genomic differences between organisms. PCR-based amplification of highly conserved ribosomal RNA (rRNA) genes, intergenic sequences, and specific toxin genes is currently the most reliable approach for bacterial, fungal and many viral pathogenic agents. When combined with fluorescence-based oligonucleotide detection systems, this approach provides real-time, quantitative, high fidelity analysis capable of single nucleotide allelic discrimination (4). These probe systems offer rapid turn around time (<2 h) and are suitable for high throughput, automated multiplex operations that are critical for clinical diagnostic laboratories. In this pilot program, we have used molecular beacon technology invented at the Public health Research Institute to develop a new generation of molecular probes to rapidly detect important agents of infectious diseases. We have also developed protocols to rapidly extract nucleic acids from a variety of clinical specimen including and blood and tissue to for detection in the molecular assays. This work represented a cooperative research development program between the Kramer-Tyagi/Perlin labs on probe development and the Perlin lab in sample preparation and testing in animal models.« less

Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria, Cylindrospermopsis and Microcystis

PubMed Central

Te, Shu Harn; Chen, Enid Yingru

2015-01-01

The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques—qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples. PMID:26025892

Real-time assays with molecular beacons and other fluorescent nucleic acid hybridization probes.

PubMed

Marras, Salvatore A E; Tyagi, Sanjay; Kramer, Fred Russell

2006-01-01

A number of formats for nucleic acid hybridization have been developed to identify DNA and RNA sequences that are involved in cellular processes and that aid in the diagnosis of genetic and infectious diseases. The introduction of hybridization probes with interactive fluorophore pairs has enabled the development of homogeneous hybridization assays for the direct identification of nucleic acids. A change in the fluorescence of these probes indicates the presence of a target nucleic acid, and there is no need to separate unbound probes from hybridized probes. The advantages of homogeneous hybridization assays are their speed and simplicity. In addition, homogeneous assays can be combined with nucleic acid amplification, enabling the detection of rare target nucleic acids. These assays can be followed in real time, providing quantitative determination of target nucleic acids over a broad range of concentrations.

The SULSA Assay Development Fund: accelerating translation of new biology from academia to pharma.

PubMed

McElroy, Stuart P; Jones, Philip S; Barrault, Denise V

2017-02-01

With industry increasingly sourcing preclinical drug discovery projects from academia it is important that new academic discoveries are enabled through translation with HTS-ready assays. However, many scientifically interesting, novel molecular targets lack associated high-quality, robust assays suitable for hit finding and development. To bridge this gap, the Scottish Universities Life Sciences Alliance (SULSA) established a fund to develop assays to meet quality criteria such as those of the European Lead Factory. A diverse project portfolio was quickly assembled, and a review of the learnings and successful outcomes showed this fund as a new highly cost-effective model for leveraging significant follow-on resources, training early-career scientists and establishing a culture of translational drug discovery in the academic community. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Long-lasting, experience-dependent alcohol preference in Drosophila

PubMed Central

Peru y Colón de Portugal, Raniero L.; Ojelade, Shamsideen A.; Penninti, Pranav S.; Dove, Rachel J.; Nye, Matthew J.; Acevedo, Summer F.; Lopez, Antonio; Rodan, Aylin R.; Rothenfluh, Adrian

2013-01-01

To understand the molecular and neural mechanisms underlying alcohol addiction, many models ranging from vertebrates to invertebrates have been developed. In Drosophila melanogaster, behavioral paradigms from assaying acute responses to alcohol, to behaviors more closely modeling addiction, have emerged in recent years. However, both the CAFÉ assay, similar to a 2-bottle choice consumption assay, as well as conditioned odor preference, where ethanol is used as the reinforcer, are labor intensive and have low throughput. To address this limitation, we have established a novel ethanol consumption preference assay, called FRAPPÉ, which allows for fast, high throughput measurement of consumption in individual flies, using a fluorescence plate reader. We show that naïve flies do not prefer to consume ethanol, but various pre-exposures, such as ethanol vapor or voluntary ethanol consumption, induce ethanol preference. This ethanol-primed preference is long lasting and is not driven by calories contained in ethanol during the consumption choice. Our novel experience-dependent model of ethanol preference in Drosophila – a highly genetically tractable organism – therefore recapitulates salient features of human alcohol abuse and will facilitate the molecular understanding of the development of alcohol preference. PMID:24164972

Direct replacement of antibodies with molecularly imprinted polymer (MIP) nanoparticles in ELISA – development of a novel assay for vancomycin

PubMed Central

Chianella, Iva; Guerreiro, Antonio; Moczko, Ewa; Caygill, J. Sarah; Piletska, Elena V.; Perez De Vargas Sansalvador, Isabel M.; Whitcombe, Michael J.; Piletsky, Sergey A.

2016-01-01

A simple and straightforward technique for coating microplate wells with molecularly imprinted polymer nanoparticles (nanoMIPs) to develop ELISA type assays is presented here for the first time. NanoMIPs were synthesized by a solid phase approach with immobilized vancomycin (template) and characterized using Biacore 3000, dynamic light scattering and electron microscopy. Immobilization, blocking and washing conditions were optimized in microplate format. The detection of vancomycin was achieved in competitive binding experiments with a HRP-vancomycin conjugate. The assay was capable of measuring vancomycin in buffer and in blood plasma within the range 0.001-70 nM with a detection limit of 0.0025 nM (2.5 pM). The sensitivity of the assay was three orders of magnitude better than a previously described ELISA based on antibodies. In these experiments nanoMIPs have shown high affinity and minimal interference from blood plasma components. Immobilized nanoMIPs were stored for 1 month at room temperature without any detrimental effects to their binding properties. The high affinity of nanoMIPs and the lack of a requirement for cold chain logistics make them an attractive alternative to traditional antibodies used in ELISA. PMID:23947402

Development and validation of the JAX Cancer Treatment Profile™ for detection of clinically actionable mutations in solid tumors

PubMed Central

Ananda, Guruprasad; Mockus, Susan; Lundquist, Micaela; Spotlow, Vanessa; Simons, Al; Mitchell, Talia; Stafford, Grace; Philip, Vivek; Stearns, Timothy; Srivastava, Anuj; Barter, Mary; Rowe, Lucy; Malcolm, Joan; Bult, Carol; Karuturi, Radha Krishna Murthy; Rasmussen, Karen; Hinerfeld, Douglas

2015-01-01

Background The continued development of targeted therapeutics for cancer treatment has required the concomitant development of more expansive methods for the molecular profiling of the patient’s tumor. We describe the validation of the JAX Cancer Treatment Profile™ (JAX-CTP™), a next generation sequencing (NGS)-based molecular diagnostic assay that detects actionable mutations in solid tumors to inform the selection of targeted therapeutics for cancer treatment. Methods NGS libraries are generated from DNA extracted from formalin fixed paraffin embedded tumors. Using hybrid capture, the genes of interest are enriched and sequenced on the Illumina HiSeq 2500 or MiSeq sequencers followed by variant detection and functional and clinical annotation for the generation of a clinical report. Results The JAX-CTP™ detects actionable variants, in the form of single nucleotide variations and small insertions and deletions (≤50bp) in 190 genes in specimens with a neoplastic cell content of ≥10%. The JAX-CTP™ is also validated for the detection of clinically actionable gene amplifications. Conclusions There is a lack of consensus in the molecular diagnostics field on the best method for the validation of NGS-based assays in oncology, thus the importance of communicating methods, as contained in this report. The growing number of targeted therapeutics and the complexity of the tumor genome necessitates continued development and refinement of advanced assays for tumor profiling to enable precision cancer treatment. PMID:25562415

A recombinase polymerase amplification assay for rapid detection of Crimean-Congo Haemorrhagic fever Virus infection

PubMed Central

Afrough, Babak; Mullojonova, Manija; Dzhuraeva, Viktoriya; Tishkova, Farida; Hewson, Roger

2017-01-01

Background Crimean-Congo Haemorrhagic fever Virus (CCHFV) is a rapidly emerging vector-borne pathogen and the cause of a virulent haemorrhagic fever affecting large parts of Europe, Africa, the Middle East and Asia. Methodology/principle findings An isothermal recombinase polymerase amplification (RPA) assay was successfully developed for molecular detection of CCHFV. The assay showed rapid (under 10 minutes) detection of viral extracts/synthetic virus RNA of all 7 S-segment clades of CCHFV, with high target specificity. The assay was shown to tolerate the presence of inhibitors in crude preparations of mock field samples, indicating that this assay may be suitable for use in the field with minimal sample preparation. The CCHFV RPA was successfully used to screen and detect CCHFV positives from a panel of clinical samples from Tajikistan. Conclusions/significance The assay is a rapid, isothermal, simple-to-perform molecular diagnostic, which can be performed on a light, portable real-time detection device. It is ideally placed therefore for use as a field-diagnostic or in-low resource laboratories, for monitoring of CCHF outbreaks at the point-of-need, such as in remote rural regions in affected countries. PMID:29028804

Subtype and pathway specific responses to anticancer compounds in breast cancer.

PubMed

Heiser, Laura M; Sadanandam, Anguraj; Kuo, Wen-Lin; Benz, Stephen C; Goldstein, Theodore C; Ng, Sam; Gibb, William J; Wang, Nicholas J; Ziyad, Safiyyah; Tong, Frances; Bayani, Nora; Hu, Zhi; Billig, Jessica I; Dueregger, Andrea; Lewis, Sophia; Jakkula, Lakshmi; Korkola, James E; Durinck, Steffen; Pepin, François; Guan, Yinghui; Purdom, Elizabeth; Neuvial, Pierre; Bengtsson, Henrik; Wood, Kenneth W; Smith, Peter G; Vassilev, Lyubomir T; Hennessy, Bryan T; Greshock, Joel; Bachman, Kurtis E; Hardwicke, Mary Ann; Park, John W; Marton, Laurence J; Wolf, Denise M; Collisson, Eric A; Neve, Richard M; Mills, Gordon B; Speed, Terence P; Feiler, Heidi S; Wooster, Richard F; Haussler, David; Stuart, Joshua M; Gray, Joe W; Spellman, Paul T

2012-02-21

Breast cancers are comprised of molecularly distinct subtypes that may respond differently to pathway-targeted therapies now under development. Collections of breast cancer cell lines mirror many of the molecular subtypes and pathways found in tumors, suggesting that treatment of cell lines with candidate therapeutic compounds can guide identification of associations between molecular subtypes, pathways, and drug response. In a test of 77 therapeutic compounds, nearly all drugs showed differential responses across these cell lines, and approximately one third showed subtype-, pathway-, and/or genomic aberration-specific responses. These observations suggest mechanisms of response and resistance and may inform efforts to develop molecular assays that predict clinical response.

Peptide-based biosensors: From self-assembled interfaces to molecular probes in electrochemical assays.

PubMed

Puiu, Mihaela; Bala, Camelia

2018-04-01

Redox-tagged peptides have emerged as functional materials with multiple applications in the area of sensing and biosensing applications due to their high stability, excellent redox properties and versatility of biomolecular interactions. They allow direct observation of molecular interactions in a wide range of affinity and enzymatic assays and act as electron mediators. Short helical peptides possess the ability to self-assemble in specific configurations with the possibility to develop in highly-ordered, stable 1D, 2D and 3D architectures in a hierarchical controlled manner. We provide here a brief overview of the electrochemical techniques available to study the electron transfer in peptide films with particular interest in developing biosensors with immobilized peptide motifs, for biological and clinical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Isolation of circulating tumor cells by immunomagnetic enrichment and fluorescence-activated cell sorting (IE/FACS) for molecular profiling.

PubMed

Magbanua, Mark Jesus M; Park, John W

2013-12-01

Circulating tumor cells (CTCs) are cells shed by the primary tumor into the blood stream capable of initiating distant metastasis. In the past decade, numerous assays have been developed to reliably detect these extremely rare cells. However, methods for purification of CTCs with little or no contamination of normal blood cells for molecular profiling are limited. We have developed a novel protocol to isolate CTCs by combining immunomagnetic enrichment and fluorescence-activated cell sorting (IE/FACS). The two-part assay includes (1) immunomagnetic capture using magnetic beads conjugated to monoclonal antibody against an epithelial cell adhesion marker (EpCAM) to enrich for tumor cells; and (2) FACS analysis using EpCAM to purify tumor cells away from mononuclear cells of hematopoietic lineage. Downstream molecular analyses of single and pooled cells confirmed the isolation of highly pure CTCs with characteristics typical that of malignant cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Forensic interlaboratory evaluation of the ForFLUID kit for vaginal fluids identification.

PubMed

Giampaoli, Saverio; Alessandrini, Federica; Berti, Andrea; Ripani, Luigi; Choi, Ajin; Crab, Roselien; De Vittori, Elisabetta; Egyed, Balazs; Haas, Cordula; Lee, Hwan Young; Korabecná, Marie; Noel, Fabrice; Podini, Daniele; Tagliabracci, Adriano; Valentini, Alessio; Romano Spica, Vincenzo

2014-01-01

Identification of vaginal fluids is an important step in the process of sexual assaults confirmation. Advances in both microbiology and molecular biology defined technical approaches allowing the discrimination of body fluids. These protocols are based on the identification of specific bacterial communities by microfloraDNA (mfDNA) amplification. A multiplex real time-PCR assay (ForFLUID kit) has been developed for identifying biological fluids and for discrimination among vaginal, oral and fecal samples. In order to test its efficacy and reliability of the assay in the identification of vaginal fluids, an interlaboratory evaluation has been performed on homogeneous vaginal swabs. All the involved laboratories were able to correctly recognize all the vaginal swabs, and no false positives were identified when the assay was applied on non-vaginal samples. The assay represents an useful molecular tool that can be easily adopted by forensic geneticists involved in vaginal fluid identification. Copyright © 2013 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

A Pan-GTPase Inhibitor as a Molecular Probe

PubMed Central

Hong, Lin; Guo, Yuna; BasuRay, Soumik; Agola, Jacob O.; Romero, Elsa; Simpson, Denise S.; Schroeder, Chad E.; Simons, Peter; Waller, Anna; Garcia, Matthew; Carter, Mark; Ursu, Oleg; Gouveia, Kristine; Golden, Jennifer E.; Aubé, Jeffrey; Wandinger-Ness, Angela; Sklar, Larry A.

2015-01-01

Overactive GTPases have often been linked to human diseases. The available inhibitors are limited and have not progressed far in clinical trials. We report here a first-in-class small molecule pan-GTPase inhibitor discovered from a high throughput screening campaign. The compound CID1067700 inhibits multiple GTPases in biochemical, cellular protein and protein interaction, as well as cellular functional assays. In the biochemical and protein interaction assays, representative GTPases from Rho, Ras, and Rab, the three most generic subfamilies of the GTPases, were probed, while in the functional assays, physiological processes regulated by each of the three subfamilies of the GTPases were examined. The chemical functionalities essential for the activity of the compound were identified through structural derivatization. The compound is validated as a useful molecular probe upon which GTPase-targeting inhibitors with drug potentials might be developed. PMID:26247207

Molecular blood grouping of donors.

PubMed

St-Louis, Maryse

2014-04-01

For many decades, hemagglutination has been the sole means to type blood donors. Since the first blood group gene cloning in the early 1990s, knowledge on the molecular basis of most red blood cell, platelet and neutrophil antigens brought the possibility of using nucleotide-based techniques to predict phenotype. This review will summarized methodologies available to genotype blood groups from laboratory developed assays to commercially available platforms, and how proficiency assays become more present. The author will also share her vision of the transfusion medicine future. The field is presently at the crossroads, bringing new perspectives to a century old practice. Copyright © 2014 Elsevier Ltd. All rights reserved.

Phenotypic and genotypic characterisation of drug-resistant Plasmodium vivax

PubMed Central

Price, Ric N.; Auburn, Sarah; Marfurt, Jutta; Cheng, Qin

2015-01-01

In this review we present recent developments in the analysis of Plasmodium vivax clinical trials and ex vivo drug-susceptibility assays, as well approaches currently being used to identify molecular markers of drug resistance. Clinical trials incorporating the measurement of in vivo drug concentrations and parasite clearance times are needed to detect early signs of resistance. Analysis of P. vivax growth dynamics ex vivo have defined the criteria for acceptable assay thresholds for drug susceptibility testing, and their subsequent interpretation. Genotyping and next-generation sequencing studies in P. vivax field isolates are set to transform our understanding of the molecular mechanisms of drug resistance. PMID:23044287

Evaluation of Various Campylobacter-Specific Quantitative PCR (qPCR) Assays for Detection and Enumeration of Campylobacteraceae in Irrigation Water and Wastewater via a Miniaturized Most-Probable-Number–qPCR Assay

PubMed Central

Banting, Graham S.; Braithwaite, Shannon; Scott, Candis; Kim, Jinyong; Jeon, Byeonghwa; Ashbolt, Nicholas; Ruecker, Norma; Tymensen, Lisa; Charest, Jollin; Pintar, Katarina; Checkley, Sylvia

2016-01-01

ABSTRACT Campylobacter spp. are the leading cause of bacterial gastroenteritis worldwide, and water is increasingly seen as a risk factor in transmission. Here we describe a most-probable-number (MPN)–quantitative PCR (qPCR) assay in which water samples are centrifuged and aliquoted into microtiter plates and the bacteria are enumerated by qPCR. We observed that commonly used Campylobacter molecular assays produced vastly different detection rates. In irrigation water samples, detection rates varied depending upon the PCR assay and culture method used, as follows: 0% by the de Boer Lv1-16S qPCR assay, 2.5% by the Van Dyke 16S and Jensen glyA qPCR assays, and 75% by the Linton 16S endpoint PCR when cultured at 37°C. Primer/probe specificity was the major confounder, with Arcobacter spp. routinely yielding false-positive results. The primers and PCR conditions described by Van Dyke et al. (M. I. Van Dyke, V. K. Morton, N. L. McLellan, and P. M. Huck, J Appl Microbiol 109:1053–1066, 2010, http://dx.doi.org/10.1111/j.1365-2672.2010.04730.x) proved to be the most sensitive and specific for Campylobacter detection in water. Campylobacter occurrence in irrigation water was found to be very low (<2 MPN/300 ml) when this Campylobacter-specific qPCR was used, with the most commonly detected species being C. jejuni, C. coli, and C. lari. Campylobacters in raw sewage were present at ∼102/100 ml, with incubation at 42°C required for reducing microbial growth competition from arcobacters. Overall, when Campylobacter prevalence and/or concentration in water is reported using molecular methods, considerable validation is recommended when adapting methods largely developed for clinical applications. Furthermore, combining MPN methods with molecular biology-based detection algorithms allows for the detection and quantification of Campylobacter spp. in environmental samples and is potentially suited to quantitative microbial risk assessment for improved public health disease prevention related to food and water exposures. IMPORTANCE The results of this study demonstrate the importance of assay validation upon data interpretation of environmental monitoring for Campylobacter when using molecular biology-based assays. Previous studies describing Campylobacter prevalence in Canada utilized primers that we have determined to be nonspecific due to their cross-amplification of Arcobacter spp. As such, Campylobacter prevalence may have been vastly overestimated in other studies. Additionally, the development of a quantitative assay described in this study will allow accurate determination of Campylobacter concentrations in environmental water samples, allowing more informed decisions to be made about water usage based on quantitative microbial risk assessment. PMID:27235434

Benefits and challenges of molecular diagnostics for childhood tuberculosis.

PubMed

Gutierrez, Cristina

2016-12-01

Expanding tuberculosis (TB)-diagnostic services, including access to rapid tests, is a World Health Organization (WHO) strategy to accelerate progress toward ending TB. Faster and more sensitive molecular tests capable of diagnosing TB and drug-resistant TB have the technical capacity to address limitations associated with smears and cultures by increasing accuracy and shortening turnaround times as compared with those of these conventional laboratory methods. Nucleic acid amplification assays used to detect and analyze Mycobacterium tuberculosis (MTB)-complex nucleic acids can be used directly on specimens from patients suspected of having TB. Recently, several commercial molecular tests were developed to detect MTB and determine the drug resistance (DR) based on detection of specific genetic mutations conferring resistance. The first to be endorsed by the WHO was molecular line-probe assay technology. This test uses polymerase chain reaction (PCR) and reverse-hybridization methods to rapidly identify MTB and DR-related mutations simultaneously. More recently, the WHO endorsed Xpert MTB/RIF, Cepheid Inc, CA, USA, a fully automated assay used for TB diagnosis that relies upon PCR techniques for detection of TB and rifampicin resistance-related mutations. Other promising molecular TB assays for simplifying PCR-based testing protocols and increasing their accuracy are under development and evaluation. Although we lack a practical gold standard for the diagnosis of childhood TB, its bacteriological confirmation is always recommended to be sought whenever possible prior to a diagnostic decision being made. Conventional diagnostic laboratory TB tests are less efficient for children as compared with adults, because sufficient sputum samples are more difficult to collect from infants and young children, and their disease is often paucibacillary, resulting in smear-negative disease. These inherent challenges associated with childhood TB are due to immunological- and pathophysiological-response differences relative to those observed in adults. Several recent meta-analyses showed low sensitivity estimates of PCR-based TB assays for paucibacillary forms of TB (extrapulmonary TB and smear-negative pulmonary disease), which represent the vast majority of childhood TB cases. Despite the lack of evidence regarding use of the rapid molecular assays to identify TB and detect DR in children, and due to the clinical nature of childhood TB, TB-expert groups recommend including rapid methods for TB identification and DR detection in diagnostic algorithms for children suspected of both smear-positive and -negative pulmonary or extrapulmonary TB, both with or without human immunodeficiency virus (HIV)-coinfection, when combined with standard methods (including clinical, microbiological, and radiological assessment) for diagnosing active TB and conventional DR. Since 2011, the WHO has specifically recommended use of the Xpert MTB/RIF test as an initial diagnostic tool for children with suspected HIV-associated TB or multidrug-resistant TB based on successful treatment data related to adults. Implementation of the rapid molecular assays for rapid detection of TB and DR should occur in laboratories with proven capability to run molecular tests and where quality control systems are implemented. Molecular approaches should be more largely tested in children, given their status as the group in whom the diagnostic dilemma is most pronounced. These tests should also be included in specific childhood TB diagnostic algorithms adapted to the local/national context in combination with other strategies for improving diagnostics, including more effective specimen collection. Copyright © 2016.

Development and evaluation of tailored specific real-time RT-PCR assays for detection of foot-and-mouth disease virus serotypes circulating in East Africa.

PubMed

Bachanek-Bankowska, Katarzyna; Mero, Herieth R; Wadsworth, Jemma; Mioulet, Valerie; Sallu, Raphael; Belsham, Graham J; Kasanga, Christopher J; Knowles, Nick J; King, Donald P

2016-11-01

Rapid, reliable and accurate diagnostic methods provide essential support to programmes that monitor and control foot-and-mouth disease (FMD). While pan-specific molecular tests for FMD virus (FMDV) detection are well established and widely used in endemic and FMD-free countries, current serotyping methods mainly rely either on antigen detection ELISAs or nucleotide sequencing approaches. This report describes the development of a panel of serotype-specific real-time RT-PCR assays (rRT-PCR) tailored to detect FMDV lineages currently circulating in East Africa. These assays target sequences within the VP1-coding region that share high intra-lineage identity, but do not cross-react with FMD viruses from other serotypes that circulate in the region. These serotype-specific assays operate with the same thermal profile as the pan-diagnostic tests making it possible to run them in parallel to produce C T values comparable to the pan-diagnostic test detecting the 3D-coding region. These assays were evaluated alongside the established pan-specific molecular test using field samples and virus isolates collected from Tanzania, Kenya and Ethiopia that had been previously characterised by nucleotide sequencing. Samples (n=71) representing serotype A (topotype AFRICA, lineage G-I), serotype O (topotypes EA-2 and EA-4), serotype SAT 1 (topotype I (NWZ)) and serotype SAT2 (topotype IV) were correctly identified with these rRT-PCR assays. Furthermore, FMDV RNA from samples that did not contain infectious virus could still be serotyped using these assays. These serotype-specific real-time RT-PCR assays can detect and characterise FMDVs currently circulating in East Africa and hence improve disease control in this region. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Fluorescence lifetime assays: current advances and applications in drug discovery.

PubMed

Pritz, Stephan; Doering, Klaus; Woelcke, Julian; Hassiepen, Ulrich

2011-06-01

Fluorescence lifetime assays complement the portfolio of established assay formats available in drug discovery, particularly with the recent advances in microplate readers and the commercial availability of novel fluorescent labels. Fluorescence lifetime assists in lowering complexity of compound screening assays, affording a modular, toolbox-like approach to assay development and yielding robust homogeneous assays. To date, materials and procedures have been reported for biochemical assays on proteases, as well as on protein kinases and phosphatases. This article gives an overview of two assay families, distinguished by the origin of the fluorescence signal modulation. The pharmaceutical industry demands techniques with a robust, integrated compound profiling process and short turnaround times. Fluorescence lifetime assays have already helped the drug discovery field, in this sense, by enhancing productivity during the hit-to-lead and lead optimization phases. Future work will focus on covering other biochemical molecular modifications by investigating the detailed photo-physical mechanisms underlying the fluorescence signal.

Rapid genetic typing of diarrheagenic Escherichia coli using a two-tube modified molecular beacon based multiplex real-time PCR assay and its clinical application

PubMed Central

2014-01-01

Background Diarrheagenic Escherichia coli (DEC), including Enterotoxigenic E.coli (ETEC), Enteroaggregative E.coli (EAEC), Enteropathogenic E.coli (EPEC), Enterohemolysin E.coli (EHEC) and Enteroinvasive E.coli (EIEC) causes diarrhea or hemolytic uremic syndromes among infants and travelers around the world. A rapid, reliable and repeatable method is urgent for identifying DEC so as to provide the reference for responding to diarrheal disease outbreak and the treatment of the diarrheal patients associated with DEC. Methods In this study, specific primers and modified molecular beacon probes of nine specific virulence genes, whose 5′end were added with homo tail sequence, were designed; and a two-tube modified molecular beacon based multiplex real–time PCR (rtPCR) assay for the identification of five Escherichia coli pathotypes, including ETEC, EAEC, EPEC, EHEC and EIEC was developed and optimized. Totally 102 bacterial strains, including 52 reference bacterial strains and 50 clinical strains were detected to confirm whether the target genes selected were specific. Then detection limits of the assay were tested. Lastly, the assay was applied to the detection of 11860 clinical samples to evaluate the specificity and sensitivity of the developed assay compared with the conventional PCR. Results The target genes were 100% specific as assessed on 102 bacterial strains since no cross-reactions were observed. The detection limits ranged from 88 CFU/mL (EHEC) to 880 CFU/mL (EPEC). Compared with the conventional PCR, the specificity and sensitivity of the multiplex rtPCR was 100% and over 99%, respectively. The coefficient of variation (CV) for each target gene ranged from 0.45% to 1.53%. 171 positive clinical samples were mostly identified as ETEC (n = 111, 64.9%) and EPEC (n = 38, 22.2%), which were the dominating pathotypes of DEC strains. Conclusion The developed multiplex rtPCR assay for the identification of DEC was high sensitive and specific and could be applied to the rapid identification of DEC in clinical and public health laboratories. PMID:25023669

Multiplex PCR for rapid diagnosis and differentiation of pox and pox-like diseases in dromedary Camels.

PubMed

Khalafalla, Abdelmalik I; Al-Busada, Khalid A; El-Sabagh, Ibrahim M

2015-07-07

Pox and pox-like diseases of camels are a group of exanthematous skin conditions that have become increasingly important economically. Three distinct viruses may cause them: camelpox virus (CMLV), camel parapox virus (CPPV) and camelus dromedary papilloma virus (CdPV). These diseases are often difficult to differentiate based on clinical presentation in disease outbreaks. Molecular methods such as PCR targeting species-specific genes have been developed and used to identify these diseases, but not simultaneously in a single tube. Recently, multiplex PCR has gained reputation as a convenient diagnostic method with cost-and timesaving benefits. In the present communication, we describe the development, optimization and validation of a multiplex PCR assay able to detect simultaneously the genome of the three viruses in one single test allowing for rapid and efficient molecular diagnosis. The assay was developed based on the evaluation and combination of published and new primer sets and was validated with viral genomic DNA extracted from known virus strains (n = 14) and DNA extracted from homogenized clinical skin specimens (n = 86). The assay detects correctly the target pathogens by amplification of targeted genes, even in case of co-infection. The method showed high sensitivity, and the specificity was confirmed by PCR-product sequencing. This assay provide rapid, sensitive and specific method for identifying three important viruses in specimens collected from dromedary camels with varying clinical presentations.

Improving validation methods for molecular diagnostics: application of Bland-Altman, Deming and simple linear regression analyses in assay comparison and evaluation for next-generation sequencing

PubMed Central

Misyura, Maksym; Sukhai, Mahadeo A; Kulasignam, Vathany; Zhang, Tong; Kamel-Reid, Suzanne; Stockley, Tracy L

2018-01-01

Aims A standard approach in test evaluation is to compare results of the assay in validation to results from previously validated methods. For quantitative molecular diagnostic assays, comparison of test values is often performed using simple linear regression and the coefficient of determination (R2), using R2 as the primary metric of assay agreement. However, the use of R2 alone does not adequately quantify constant or proportional errors required for optimal test evaluation. More extensive statistical approaches, such as Bland-Altman and expanded interpretation of linear regression methods, can be used to more thoroughly compare data from quantitative molecular assays. Methods We present the application of Bland-Altman and linear regression statistical methods to evaluate quantitative outputs from next-generation sequencing assays (NGS). NGS-derived data sets from assay validation experiments were used to demonstrate the utility of the statistical methods. Results Both Bland-Altman and linear regression were able to detect the presence and magnitude of constant and proportional error in quantitative values of NGS data. Deming linear regression was used in the context of assay comparison studies, while simple linear regression was used to analyse serial dilution data. Bland-Altman statistical approach was also adapted to quantify assay accuracy, including constant and proportional errors, and precision where theoretical and empirical values were known. Conclusions The complementary application of the statistical methods described in this manuscript enables more extensive evaluation of performance characteristics of quantitative molecular assays, prior to implementation in the clinical molecular laboratory. PMID:28747393

The analytical validation of the Oncotype DX Recurrence Score assay

PubMed Central

Baehner, Frederick L

2016-01-01

In vitro diagnostic multivariate index assays are highly complex molecular assays that can provide clinically actionable information regarding the underlying tumour biology and facilitate personalised treatment. These assays are only useful in clinical practice if all of the following are established: analytical validation (i.e., how accurately/reliably the assay measures the molecular characteristics), clinical validation (i.e., how consistently/accurately the test detects/predicts the outcomes of interest), and clinical utility (i.e., how likely the test is to significantly improve patient outcomes). In considering the use of these assays, clinicians often focus primarily on the clinical validity/utility; however, the analytical validity of an assay (e.g., its accuracy, reproducibility, and standardisation) should also be evaluated and carefully considered. This review focuses on the rigorous analytical validation and performance of the Oncotype DX® Breast Cancer Assay, which is performed at the Central Clinical Reference Laboratory of Genomic Health, Inc. The assay process includes tumour tissue enrichment (if needed), RNA extraction, gene expression quantitation (using a gene panel consisting of 16 cancer genes plus 5 reference genes and quantitative real-time RT-PCR), and an automated computer algorithm to produce a Recurrence Score® result (scale: 0–100). This review presents evidence showing that the Recurrence Score result reported for each patient falls within a tight clinically relevant confidence interval. Specifically, the review discusses how the development of the assay was designed to optimise assay performance, presents data supporting its analytical validity, and describes the quality control and assurance programmes that ensure optimal test performance over time. PMID:27729940

The analytical validation of the Oncotype DX Recurrence Score assay.

PubMed

Baehner, Frederick L

2016-01-01

In vitro diagnostic multivariate index assays are highly complex molecular assays that can provide clinically actionable information regarding the underlying tumour biology and facilitate personalised treatment. These assays are only useful in clinical practice if all of the following are established: analytical validation (i.e., how accurately/reliably the assay measures the molecular characteristics), clinical validation (i.e., how consistently/accurately the test detects/predicts the outcomes of interest), and clinical utility (i.e., how likely the test is to significantly improve patient outcomes). In considering the use of these assays, clinicians often focus primarily on the clinical validity/utility; however, the analytical validity of an assay (e.g., its accuracy, reproducibility, and standardisation) should also be evaluated and carefully considered. This review focuses on the rigorous analytical validation and performance of the Oncotype DX ® Breast Cancer Assay, which is performed at the Central Clinical Reference Laboratory of Genomic Health, Inc. The assay process includes tumour tissue enrichment (if needed), RNA extraction, gene expression quantitation (using a gene panel consisting of 16 cancer genes plus 5 reference genes and quantitative real-time RT-PCR), and an automated computer algorithm to produce a Recurrence Score ® result (scale: 0-100). This review presents evidence showing that the Recurrence Score result reported for each patient falls within a tight clinically relevant confidence interval. Specifically, the review discusses how the development of the assay was designed to optimise assay performance, presents data supporting its analytical validity, and describes the quality control and assurance programmes that ensure optimal test performance over time.

Advances in serological, imaging techniques and molecular diagnosis of Toxoplasma gondii infection.

PubMed

Rostami, Ali; Karanis, Panagiotis; Fallahi, Shirzad

2018-06-01

Toxoplasmosis is worldwide distributed zoonotic infection disease with medical importance in immunocompromised patients, pregnant women and congenitally infected newborns. Having basic information on the traditional and new developed methods is essential for general physicians and infectious disease specialists for choosing a suitable diagnostic approach for rapid and accurate diagnosis of the disease and, consequently, timely and effective treatment. We conducted English literature searches in PubMed from 1989 to 2016 using relevant keywords and summarized the recent advances in diagnosis of toxoplasmosis. Enzyme-linked immunosorbent assay (ELISA) was most used method in past century. Recently advanced ELISA-based methods including chemiluminescence assays (CLIA), enzyme-linked fluorescence assay (ELFA), immunochromatographic test (ICT), serum IgG avidity test and immunosorbent agglutination assays (ISAGA) have shown high sensitivity and specificity. Recent studies using recombinant or chimeric antigens and multiepitope peptides method demonstrated very promising results to development of new strategies capable of discriminating recently acquired infections from chronic infection. Real-time PCR and loop-mediated isothermal amplification (LAMP) are two recently developed PCR-based methods with high sensitivity and specificity and could be useful to early diagnosis of infection. Computed tomography, magnetic resonance imaging, nuclear imaging and ultrasonography could be useful, although their results might be not specific alone. This review provides a summary of recent developed methods and also attempts to improve their sensitivity for diagnosis of toxoplasmosis. Serology, molecular and imaging technologies each has their own advantages and limitations which can certainly achieve definitive diagnosis of toxoplasmosis by combining these diagnostic techniques.

Development of companion diagnostics

DOE PAGES

Mankoff, David A.; Edmonds, Christine E.; Farwell, Michael D.; ...

2015-12-12

The goal of individualized and targeted treatment and precision medicine requires the assessment of potential therapeutic targets to direct treatment selection. The biomarkers used to direct precision medicine, often termed companion diagnostics, for highly targeted drugs have thus far been almost entirely based on in vitro assay of biopsy material. Molecular imaging companion diagnostics offer a number of features complementary to those from in vitro assay, including the ability to measure the heterogeneity of each patient’s cancer across the entire disease burden and to measure early changes in response to treatment. We discuss the use of molecular imaging methods asmore » companion diagnostics for cancer therapy with the goal of predicting response to targeted therapy and measuring early (pharmacodynamic) response as an indication of whether the treatment has “hit” the target. We also discuss considerations for probe development for molecular imaging companion diagnostics, including both small-molecule probes and larger molecules such as labeled antibodies and related constructs. We then describe two examples where both predictive and pharmacodynamic molecular imaging markers have been tested in humans: endocrine therapy for breast cancer and human epidermal growth factor receptor type 2–targeted therapy. Lastly, the review closes with a summary of the items needed to move molecular imaging companion diagnostics from early studies into multicenter trials and into the clinic.« less

Development of companion diagnostics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mankoff, David A.; Edmonds, Christine E.; Farwell, Michael D.

The goal of individualized and targeted treatment and precision medicine requires the assessment of potential therapeutic targets to direct treatment selection. The biomarkers used to direct precision medicine, often termed companion diagnostics, for highly targeted drugs have thus far been almost entirely based on in vitro assay of biopsy material. Molecular imaging companion diagnostics offer a number of features complementary to those from in vitro assay, including the ability to measure the heterogeneity of each patient’s cancer across the entire disease burden and to measure early changes in response to treatment. We discuss the use of molecular imaging methods asmore » companion diagnostics for cancer therapy with the goal of predicting response to targeted therapy and measuring early (pharmacodynamic) response as an indication of whether the treatment has “hit” the target. We also discuss considerations for probe development for molecular imaging companion diagnostics, including both small-molecule probes and larger molecules such as labeled antibodies and related constructs. We then describe two examples where both predictive and pharmacodynamic molecular imaging markers have been tested in humans: endocrine therapy for breast cancer and human epidermal growth factor receptor type 2–targeted therapy. Lastly, the review closes with a summary of the items needed to move molecular imaging companion diagnostics from early studies into multicenter trials and into the clinic.« less

Development of Companion Diagnostics

PubMed Central

Mankoff, David A.; Edmonds, Christine E.; Farwell, Michael D.; Pryma, Daniel A.

2016-01-01

The goal of individualized and targeted treatment and precision medicine requires the assessment of potential therapeutic targets to direct treatment selection. The biomarkers used to direct precision medicine, often termed companion diagnostics, for highly targeted drugs have thus far been almost entirely based on in vitro assay of biopsy material. Molecular imaging companion diagnostics offer a number of features complementary to those from in vitro assay, including the ability to measure the heterogeneity of each patient’s cancer across the entire disease burden and to measure early changes in response to treatment. We discuss the use of molecular imaging methods as companion diagnostics for cancer therapy with the goal of predicting response to targeted therapy and measuring early (pharmacodynamic) response as an indication of whether the treatment has “hit” the target. We also discuss considerations for probe development for molecular imaging companion diagnostics, including both small-molecule probes and larger molecules such as labeled antibodies and related constructs. We then describe two examples where both predictive and pharmacodynamic molecular imaging markers have been tested in humans: endocrine therapy for breast cancer and human epidermal growth factor receptor type 2–targeted therapy. The review closes with a summary of the items needed to move molecular imaging companion diagnostics from early studies into multicenter trials and into the clinic. PMID:26687857

Adenovirus 2, Bordetella bronchiseptica, and Parainfluenza Molecular Diagnostic Assay Results in Puppies After vaccination with Modified Live Vaccines.

PubMed

Ruch-Gallie, R; Moroff, S; Lappin, M R

2016-01-01

Canine adenovirus 2, parainfluenza, and Bordetella bronchiseptica cause respiratory disease in dogs, and each has a modified live intranasal vaccine available. Molecular diagnostic assays to amplify specific nucleic acids are available for each of these agents. If positive molecular diagnostic assay results are common after vaccination, the positive predictive value of the diagnostic assays for disease would be decreased. To determine the impact of administration of commercially available modified live topical adenovirus 2, B. bronchiseptica, and parainfluenza vaccine has on the results of a commercially available PCR panel. Eight puppies from a research breeding facility negative for these pathogens. Blinded prospective pilot study. Puppies were vaccinated with a single dose of modified live topical adenovirus 2, B. bronchiseptica, and parainfluenza and parenteral dose of adenovirus 2, canine distemper virus, and parvovirus. Nasal and pharyngeal swabs were collected on multiple days and submitted for PCR assay. Nucleic acids of all 3 organisms contained in the topical vaccine were detected from both samples multiple times through 28 days after vaccination with higher numbers of positive samples detected between days 3 and 10 after vaccination. Vaccine status should be considered when interpreting respiratory agent PCR results if modified live vaccines have been used. Development of quantitative PCR and wild-type sequencing are necessary to improve positive predictive value of these assays by distinguishing vaccinate from natural infection. Copyright © 2015 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria, Cylindrospermopsis and Microcystis.

PubMed

Te, Shu Harn; Chen, Enid Yingru; Gin, Karina Yew-Hoong

2015-08-01

The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques-qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Rapid Detection of Cell-Free Mycobacterium tuberculosis DNA in Tuberculous Pleural Effusion.

PubMed

Che, Nanying; Yang, Xinting; Liu, Zichen; Li, Kun; Chen, Xiaoyou

2017-05-01

Tuberculous pleurisy is one of the most common types of extrapulmonary tuberculosis, but its diagnosis remains difficult. In this study, we report for the first time on the detection of cell-free Mycobacterium tuberculosis DNA in pleural effusion and an evaluation of a newly developed molecular assay for the detection of cell-free Mycobacterium tuberculosis DNA. A total of 78 patients with pleural effusion, 60 patients with tuberculous pleurisy, and 18 patients with alternative diseases were included in this study. Mycobacterial culture, the Xpert MTB/RIF assay, the adenosine deaminase assay, the T-SPOT.TB assay, and the cell-free Mycobacterium tuberculosis DNA assay were performed on all the pleural effusion samples. The cell-free Mycobacterium tuberculosis DNA assay and adenosine deaminase assay showed significantly higher sensitivities of 75.0% and 68.3%, respectively, than mycobacterial culture and the Xpert MTB/RIF assay, which had sensitivities of 26.7% and 20.0%, respectively ( P < 0.01). All four of these tests showed good specificities: 88.9% for the adenosine deaminase assay and 100% for the remaining three assays. The T-SPOT.TB assay with pleural effusion showed the highest sensitivity of 95.0% but the lowest specificity of 38.9%. The cell-free Mycobacterium tuberculosis DNA assay detected as few as 1.25 copies of IS 6110 per ml of pleural effusion and showed good accordance of the results between repeated tests ( r = 0.978, P = 2.84 × 10 -10 ). These data suggest that the cell-free Mycobacterium tuberculosis DNA assay is a rapid and accurate molecular test which provides direct evidence of Mycobacterium tuberculosis etiology. Copyright © 2017 American Society for Microbiology.

Rapid Detection of Cell-Free Mycobacterium tuberculosis DNA in Tuberculous Pleural Effusion

PubMed Central

Yang, Xinting; Liu, Zichen; Li, Kun

2017-01-01

ABSTRACT Tuberculous pleurisy is one of the most common types of extrapulmonary tuberculosis, but its diagnosis remains difficult. In this study, we report for the first time on the detection of cell-free Mycobacterium tuberculosis DNA in pleural effusion and an evaluation of a newly developed molecular assay for the detection of cell-free Mycobacterium tuberculosis DNA. A total of 78 patients with pleural effusion, 60 patients with tuberculous pleurisy, and 18 patients with alternative diseases were included in this study. Mycobacterial culture, the Xpert MTB/RIF assay, the adenosine deaminase assay, the T-SPOT.TB assay, and the cell-free Mycobacterium tuberculosis DNA assay were performed on all the pleural effusion samples. The cell-free Mycobacterium tuberculosis DNA assay and adenosine deaminase assay showed significantly higher sensitivities of 75.0% and 68.3%, respectively, than mycobacterial culture and the Xpert MTB/RIF assay, which had sensitivities of 26.7% and 20.0%, respectively (P < 0.01). All four of these tests showed good specificities: 88.9% for the adenosine deaminase assay and 100% for the remaining three assays. The T-SPOT.TB assay with pleural effusion showed the highest sensitivity of 95.0% but the lowest specificity of 38.9%. The cell-free Mycobacterium tuberculosis DNA assay detected as few as 1.25 copies of IS6110 per ml of pleural effusion and showed good accordance of the results between repeated tests (r = 0.978, P = 2.84 × 10−10). These data suggest that the cell-free Mycobacterium tuberculosis DNA assay is a rapid and accurate molecular test which provides direct evidence of Mycobacterium tuberculosis etiology. PMID:28275073

A universal and reliable assay for molecular sex identification of three-spined sticklebacks (Gasterosteus aculeatus).

PubMed

Toli, E-A; Calboli, F C F; Shikano, T; Merilä, J

2016-11-01

In heterogametic species, biological differences between the two sexes are ubiquitous, and hence, errors in sex identification can be a significant source of noise and bias in studies where sex-related sources of variation are of interest or need to be controlled for. We developed and validated a universal multimarker assay for reliable sex identification of three-spined sticklebacks (Gasterosteus aculeatus). The assay makes use of genotype scores from three sex-linked loci and utilizes Bayesian probabilistic inference to identify sex of the genotyped individuals. The results, validated with 286 phenotypically sexed individuals from six populations of sticklebacks representing all major genetic lineages (cf. Pacific, Atlantic and Japan Sea), indicate that in contrast to commonly used single-marker-based sex identification assays, the developed multimarker assay should be 100% accurate. As the markers in the assay can be scored from agarose gels, it provides a quick and cost-efficient tool for universal sex identification of three-spined sticklebacks. The general principle of combining information from multiple markers to improve the reliability of sex identification is transferable and can be utilized to develop and validate similar assays for other species. © 2016 John Wiley & Sons Ltd.

Plant genotoxicity: a molecular cytogenetic approach in plant bioassays.

PubMed

Maluszynska, Jolanta; Juchimiuk, Jolanta

2005-06-01

It is important for the prevention of DNA changes caused by environment to understand the biological consequences of DNA damages and their molecular modes of action that lead to repair or alterations of the genetic material. Numerous genotoxicity assay systems have been developed to identify DNA reactive compounds. The available data show that plant bioassays are important tests in the detection of genotoxic contamination in the environment and the establishment of controlling systems. Plant system can detect a wide range of genetic damage, including gene mutations and chromosome aberrations. Recently introduced molecular cytogenetic methods allow analysis of genotoxicity, both at the chromosomal and DNA level. FISH gives a new possibility of the detection and analysis of chromosomal rearrangements in a great detail. DNA fragmentation can be estimated using the TUNEL test and the single cell gel electrophoresis (Comet assay).

System for Informatics in the Molecular Pathology Laboratory: An Open-Source End-to-End Solution for Next-Generation Sequencing Clinical Data Management.

PubMed

Kang, Wenjun; Kadri, Sabah; Puranik, Rutika; Wurst, Michelle N; Patil, Sushant A; Mujacic, Ibro; Benhamed, Sonia; Niu, Nifang; Zhen, Chao Jie; Ameti, Bekim; Long, Bradley C; Galbo, Filipo; Montes, David; Iracheta, Crystal; Gamboa, Venessa L; Lopez, Daisy; Yourshaw, Michael; Lawrence, Carolyn A; Aisner, Dara L; Fitzpatrick, Carrie; McNerney, Megan E; Wang, Y Lynn; Andrade, Jorge; Volchenboum, Samuel L; Furtado, Larissa V; Ritterhouse, Lauren L; Segal, Jeremy P

2018-04-24

Next-generation sequencing (NGS) diagnostic assays increasingly are becoming the standard of care in oncology practice. As the scale of an NGS laboratory grows, management of these assays requires organizing large amounts of information, including patient data, laboratory processes, genomic data, as well as variant interpretation and reporting. Although several Laboratory Information Systems and/or Laboratory Information Management Systems are commercially available, they may not meet all of the needs of a given laboratory, in addition to being frequently cost-prohibitive. Herein, we present the System for Informatics in the Molecular Pathology Laboratory, a free and open-source Laboratory Information System/Laboratory Information Management System for academic and nonprofit molecular pathology NGS laboratories, developed at the Genomic and Molecular Pathology Division at the University of Chicago Medicine. The System for Informatics in the Molecular Pathology Laboratory was designed as a modular end-to-end information system to handle all stages of the NGS laboratory workload from test order to reporting. We describe the features of the system, its clinical validation at the Genomic and Molecular Pathology Division at the University of Chicago Medicine, and its installation and testing within a different academic center laboratory (University of Colorado), and we propose a platform for future community co-development and interlaboratory data sharing. Copyright © 2018. Published by Elsevier Inc.

Democratizing molecular diagnostics for the developing world.

PubMed

Abou Tayoun, Ahmad N; Burchard, Paul R; Malik, Imran; Scherer, Axel; Tsongalis, Gregory J

2014-01-01

Infectious diseases that are largely treatable continue to pose a tremendous burden on the developing world despite the availability of highly potent drugs. The high mortality and morbidity rates of these diseases are largely due to a lack of affordable diagnostics that are accessible to resource-limited areas and that can deliver high-quality results. In fact, modified molecular diagnostics for infectious diseases were rated as the top biotechnology to improve health in developing countries. In this review, we describe the characteristics of accessible molecular diagnostic tools and discuss the challenges associated with implementing such tools at low infrastructure sites. We highlight our experience as part of the "Grand Challenge" project supported by the Gates Foundation for addressing global health inequities and describe issues and solutions associated with developing adequate technologies or molecular assays needed for broad access in the developing world. We believe that sharing this knowledge will facilitate the development of new molecular technologies that are extremely valuable for improving global health.

Real-time PCR detection of Didemnum perlucidum (Monniot, 1983) and Didemnum vexillum (Kott, 2002) in an applied routine marine biosecurity context.

PubMed

Simpson, Tiffany J S; Dias, P Joana; Snow, Michael; Muñoz, Julieta; Berry, Tina

2017-05-01

Prevention and early detection are well recognized as the best strategies for minimizing the risks posed by nonindigenous species (NIS) that have the potential to become marine pests. Central to this is the ability to rapidly and accurately identify the presence of NIS, often from complex environmental samples like biofouling and ballast water. Molecular tools have been increasingly applied to assist with the identification of NIS and can prove particularly useful for taxonomically difficult groups like ascidians. In this study, we have developed real-time PCR assays suited to the specific identification of the ascidians Didemnum perlucidum and Didemnum vexillum. Despite being recognized as important global pests, this is the first time specific molecular detection methods have been developed that can support the early identification and detection of these species from a broad range of environmental sample types. These fast, robust and high-throughput assays represent powerful tools for routine marine biosecurity surveillance, as detection and confirmation of the early presence of species could assist in the timely establishment of emergency responses and control strategies. This study applied the developed assays to confirm the ability to detect Didemnid eDNA in water samples. While previous work has focused on detection of marine larvae from water samples, the development of real-time PCR assays specifically aimed at detecting eDNA of sessile invertebrate species in the marine environment represents a world first and a significant step forwards in applied marine biosecurity surveillance. Demonstrated success in the detection of D. perlucidum eDNA from water samples at sites where it could not be visually identified suggests value in incorporating such assays into biosecurity survey designs targeting Didemnid species. © 2016 John Wiley & Sons Ltd.

DEVELOPMENT OF A MOLECULAR METHOD TO DETECT ASTROVIRUS

EPA Science Inventory

Astrovirus is a common cause of gastroenteritis that has been determined to be responsible for several outbreaks. Since astrovirus can be waterborne, there is interest in testing environmental water for astrovirus and we have developed a sensitive RT-PCR assay that is designed t...

Development and characterization of a synthetic DNA, NUversa, to be used as a standard in quantitative polymerase chain reactions for molecular pneumococcal serotyping.

PubMed

Sakai, Fuminori; Sonaty, Griffin; Watson, David; Klugman, Keith P; Vidal, Jorge E

2017-09-15

Identification of Streptococcus pneumoniae and its more than 90 serotypes is routinely conducted by culture and Quellung reactions. Quantitative polymerase chain reactions (qPCRs) have been developed for molecular detection, including a pan-pneumococcus lytA assay, and assays targeting 79 serotypes. Reactions require genomic DNA from every target to prepare standards, which can be time consuming. In this study, we have developed a synthetic DNA molecule as a surrogate for genomic DNA and present new single-plex qPCR reactions to increase molecular detection to 94 pneumococcal serotypes. Specificity of these new reactions was confirmed with a limit of detection between 2 and 20 genome equivalents/reaction. A synthetic DNA (NUversa, ∼8.2 kb) was then engineered to contain all available qPCR targets for serotyping and lytA. NUversa was cloned into pUC57-Amp-modified to generate pNUversa (∼10.2 kb). Standards prepared from pNUversa and NUversa were compared against standards made out of genomic DNA. Linearity [NUversa (R2 > 0.982); pNUversa (R2 > 0.991)] and efficiency of qPCR reactions were similar to those utilizing chromosomal DNA (R2 > 0.981). Quantification with plasmid pNUversa was affected, however, whereas quantification with synthetic NUversa was comparable to that of genomic DNA. Therefore, NUversa may be utilized as DNA standard in single-plex assays of the currently known 94 pneumococcal serotypes. © FEMS 2017.

Development of molecular methods for the rapid detection of antibiotic susceptibility of Mycoplasma bovis.

PubMed

Sulyok, Kinga M; Bekő, Katinka; Kreizinger, Zsuzsa; Wehmann, Enikő; Jerzsele, Ákos; Rónai, Zsuzsanna; Turcsányi, Ibolya; Makrai, László; Szeredi, Levente; Jánosi, Szilárd; Nagy, Sára Ágnes; Gyuranecz, Miklós

2018-01-01

Determining the antibiotic susceptibility profile of Mycoplasma bovis isolates in vitro provides the basis for the appropriate choice of antibiotics in the therapy. Traditionally, the antibiotic susceptibility examination of mycoplasmas is technically demanding, time-consuming and rarely performed in diagnostic laboratories. The aim of the present study was to develop rapid molecular assays to determine mutations responsible for elevated minimal inhibitory concentrations (MICs) to fluoroquinolones, tetracyclines, aminocyclitols, macrolides, lincosamides, phenicols and pleuromutilins in M. bovis. The nine mismatch amplification mutation assays (MAMA) and seven high resolution melt (HRM) tests designed in the present study enable the simultaneous detection of these genetic markers. The sensitivity of the assays varied between 10 2 -10 5 copy numbers/reaction. Cross-reactions with other mycoplasmas occurring in cattle were detected in assays targeting universal regions (e.g. 16S rRNA). Nevertheless, results of the novel method were in accordance with sequence and MICs data of the M. bovis pure cultures. Also, the tests of clinical samples containing high amount of M. bovis DNA were congruent even in the presence of other Mycoplasma spp. The presented method is highly cost-effective and can provide an antibiogram to 12 antibiotics in approximately 3-4 days when previous isolation of M. bovis is applied. In order to assure the proper identification of the genetic markers at issue, the regions examined by the MAMA and HRM tests are overlapping. In conclusion, the developed assays have potential to be used in routine diagnostics for the detection of antibiotic susceptibility in M. bovis. Copyright © 2017 Elsevier B.V. All rights reserved.

A Field-Tailored Reverse Transcription Loop-Mediated Isothermal Assay for High Sensitivity Detection of Plasmodium falciparum Infections

PubMed Central

Kemleu, Sylvie; Guelig, Dylan; Eboumbou Moukoko, Carole; Essangui, Estelle; Diesburg, Steven; Mouliom, Abas; Melingui, Bernard; Manga, Jeanne; Donkeu, Christiane; Epote, Annie; Texier, Gaëtan; LaBarre, Paul; Burton, Robert

2016-01-01

Highly sensitive and field deployable molecular diagnostic tools are critically needed for detecting submicroscopic, yet transmissible levels of malaria parasites prevalent in malaria endemic countries worldwide. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and evaluated in comparison with thick blood smear microscopy, an antigen-based rapid diagnostic test (RDT), and an in-house RT-PCR targeting the same RT-LAMP transcript. The optimized assay detected Plasmodium falciparum infections in as little as 0.25ng of total parasite RNA, and exhibited a detection limit of 0.08 parasites/ μL when tested directly on infected whole blood lysates, or ~0.0008 parasites/ μL when using RNA extracts. Assay positivity was observed as early as eight minutes from initiation of the RT-LAMP and in most cases the reaction was complete before twenty minutes. Clinical evaluation of the assay on 132 suspected malaria cases resulted in a positivity rate of 90% for RT-LAMP using extracted RNA, and 85% when using whole blood lysates. The positivity rates were 70% for P. falciparum-specific RDT, 83% for RT-PCR, and 74% for thick blood smear microscopy (Mean parasite density = 36,986 parasites/ μL). Concordance rates between the developed RT-LAMP and comparator tests were greater than 75%, the lowest being with light microscopy (78%, McNemar’s test: P = 0.0002), and the highest was with RT-PCR (87%, McNemar’s test: P = 0.0523). Compared to reference RT-PCR, assay sensitivity was 90% for RT-LAMP on whole blood, and 96% for RT-LAMP using corresponding RNA extracts. Electricity-free heaters were further developed and evaluated in comparison with a battery-operated isothermal amplification machine for use with the developed test in resource-limited settings. Taken together, the data highlight the benefits of targeting high abundant RNA transcripts in molecular diagnosis, as well as the potential usefulness of the developed RT-LAMP-assay in malaria diagnosis in low to high parasite density settings. PMID:27824866

Advances in Diagnosis, Surveillance, and Monitoring of Zika Virus: An Update

PubMed Central

Singh, Raj K.; Dhama, Kuldeep; Karthik, Kumaragurubaran; Tiwari, Ruchi; Khandia, Rekha; Munjal, Ashok; Iqbal, Hafiz M. N.; Malik, Yashpal S.; Bueno-Marí, Rubén

2018-01-01

Zika virus (ZIKV) is associated with numerous human health-related disorders, including fetal microcephaly, neurological signs, and autoimmune disorders such as Guillain-Barré syndrome (GBS). Perceiving the ZIKA associated losses, in 2016, the World Health Organization (WHO) declared it as a global public health emergency. In consequence, an upsurge in the research on ZIKV was seen around the globe, with significant attainments over developing several effective diagnostics, drugs, therapies, and vaccines countering this life-threatening virus at an early step. State-of-art tools developed led the researchers to explore virus at the molecular level, and in-depth epidemiological investigations to understand the reason for increased pathogenicity and different clinical manifestations. These days, ZIKV infection is diagnosed based on clinical manifestations, along with serological and molecular detection tools. As, isolation of ZIKV is a tedious task; molecular assays such as reverse transcription-polymerase chain reaction (RT-PCR), real-time qRT-PCR, loop-mediated isothermal amplification (LAMP), lateral flow assays (LFAs), biosensors, nucleic acid sequence-based amplification (NASBA) tests, strand invasion-based amplification tests and immune assays like enzyme-linked immunosorbent assay (ELISA) are in-use to ascertain the ZIKV infection or Zika fever. Herein, this review highlights the recent advances in the diagnosis, surveillance, and monitoring of ZIKV. These new insights gained from the recent advances can aid in the rapid and definitive detection of this virus and/or Zika fever. The summarized information will aid the strategies to design and adopt effective prevention and control strategies to counter this viral pathogen of great public health concern. PMID:29403448

PCR-based karyotyping of Anopheles gambiae inversion 2Rj identifies the BAMAKO chromosomal form.

PubMed

Coulibaly, Mamadou B; Pombi, Marco; Caputo, Beniamino; Nwakanma, Davis; Jawara, Musa; Konate, Lassana; Dia, Ibrahima; Fofana, Abdrahamane; Kern, Marcia; Simard, Frédéric; Conway, David J; Petrarca, Vincenzo; della Torre, Alessandra; Traoré, Sékou; Besansky, Nora J

2007-10-01

The malaria vector Anopheles gambiae is polymorphic for chromosomal inversions on the right arm of chromosome 2 that segregate nonrandomly between assortatively mating populations in West Africa. One such inversion, 2Rj, is associated with the BAMAKO chromosomal form endemic to southern Mali and northern Guinea Conakry near the Niger River. Although it exploits a unique ecology and both molecular and chromosomal data suggest reduced gene flow between BAMAKO and other A. gambiae populations, no molecular markers exist to identify this form. To facilitate study of the BAMAKO form, a PCR assay for molecular karyotyping of 2Rj was developed based on sequences at the breakpoint junctions. The assay was extensively validated using more than 700 field specimens whose karyotypes were determined in parallel by cytogenetic and molecular methods. As inversion 2Rj also occurs in SAVANNA populations outside the geographic range of BAMAKO, samples were tested from Senegal, Cameroon and western Guinea Conakry as well as from Mali. In southern Mali, where 2Rj polymorphism in SAVANNA populations was very low and most of the 2Rj homozygotes were found in BAMAKO karyotypes, the molecular and cytogenetic methods were almost perfectly congruent. Elsewhere agreement between the methods was much poorer, as the molecular assay frequently misclassified 2Rj heterozygotes as 2R+j standard homozygotes. Molecular karyotyping of 2Rj is robust and accurate on 2R+j standard and 2Rj inverted homozygotes. Therefore, the proposed approach overcomes the lack of a rapid tool for identifying the BAMAKO form across developmental stages and sexes, and opens new perspectives for the study of BAMAKO ecology and behaviour. On the other hand, the method should not be applied for molecular karyotyping of j-carriers within the SAVANNA chromosomal form.

Development of a reverse transcription loop-mediated isothermal amplification assay for the detection of vesicular stomatitis New Jersey virus: use of rapid molecular assay to differentiate between vesicular disease viruses

USDA-ARS?s Scientific Manuscript database

Vesicular stomatitis (VS) is endemic in Central America and northern regions of South America. Sporadic outbreaks of VS can occur in cattle and pigs where the clinical presentation can be similar to foot-and-mouth disease (FMD). There is therefore a pressing need for rapid, sensitive and specific d...

Fluorescent and Lanthanide Labeling for Ligand Screens, Assays, and Imaging

PubMed Central

Josan, Jatinder S.; De Silva, Channa R.; Yoo, Byunghee; Lynch, Ronald M.; Pagel, Mark D.; Vagner, Josef; Hruby, Victor J.

2012-01-01

The use of fluorescent (or luminescent) and metal contrast agents in high-throughput screens, in vitro assays, and molecular imaging procedures has rapidly expanded in recent years. Here we describe the development and utility of high-affinity ligands for cancer theranostics and other in vitro screening studies. In this context, we also illustrate the syntheses and use of heteromultivalent ligands as targeted imaging agents. PMID:21318902

Rapid identification of Enterobacter hormaechei and Enterobacter cloacae genetic cluster III.

PubMed

Ohad, S; Block, C; Kravitz, V; Farber, A; Pilo, S; Breuer, R; Rorman, E

2014-05-01

Enterobacter cloacae complex bacteria are of both clinical and environmental importance. Phenotypic methods are unable to distinguish between some of the species in this complex, which often renders their identification incomplete. The goal of this study was to develop molecular assays to identify Enterobacter hormaechei and Ent. cloacae genetic cluster III which are relatively frequently encountered in clinical material. The molecular assays developed in this study are qPCR technology based and served to identify both Ent. hormaechei and Ent. cloacae genetic cluster III. qPCR results were compared to hsp60 sequence analysis. Most clinical isolates were assigned to Ent. hormaechei subsp. steigerwaltii and Ent. cloacae genetic cluster III. The latter was proportionately more frequently isolated from bloodstream infections than from other material (P < 0·05). The qPCR assays detecting Ent. hormaechei and Ent. cloacae genetic cluster III demonstrated high sensitivity and specificity. The presented qPCR assays allow accurate and rapid identification of clinical isolates of the Ent. cloacae complex. The improved identifications obtained can specifically assist analysis of Ent. hormaechei and Ent. cloacae genetic cluster III in nosocomial outbreaks and can promote rapid environmental monitoring. An association was observed between Ent. cloacae cluster III and systemic infection that deserves further attention. © 2014 The Society for Applied Microbiology.

Cytomegalovirus infection management in solid organ transplant recipients across European centers in the time of molecular diagnostics: An ESGICH survey.

PubMed

Navarro, David; San-Juan, Rafael; Manuel, Oriol; Giménez, Estela; Fernández-Ruiz, Mario; Hirsch, Hans H; Grossi, Paolo Antonio; Aguado, José María

2017-12-01

Scant information is available about how transplant centers are managing their use of quantitative molecular testing (QNAT) assays for active cytomegalovirus (CMV) infection monitoring in solid organ transplant (SOT) recipients. The current study was aimed at gathering information on current practices in the management of CMV infection across European centers in the era of molecular testing assays. A questionnaire-based cross-sectional survey study was conducted by the European Study Group of Infections in Immunocompromised Hosts (ESGICH) of the Society of Clinical Microbiology and Infectious Diseases (ESCMID). The invitation and a weekly reminder with a personal link to an Internet service provider (https://es.surveymonkey.com/) was sent to transplant physicians, transplant infectious diseases specialists, and clinical virologists working at 340 European transplant centers. Of the 1181 specialists surveyed, a total of 173 responded (14.8%): 73 transplant physicians, 57 transplant infectious diseases specialists, and 43 virologists from 173 institutions located at 23 different countries. The majority of centers used QNAT assays for active CMV infection monitoring. Most centers preferred commercially available real-time polymerase chain reaction (RT-PCR) assays over laboratory-developed procedures for quantifying CMV DNA load in whole blood or plasma. Use of a wide variety of DNA extraction platforms and RT-PCR assays was reported. All programs used antiviral prophylaxis, preemptive therapy, or both, according to current guidelines. However, the centers used different criteria for starting preemptive antiviral treatment, for monitoring systemic CMV DNA load, and for requesting genotypic assays to detect emerging CMV-resistant variants. Significant variation in CMV infection management in SOT recipients still remains across European centers in the era of molecular testing. International multicenter studies are required to achieve commutability of CMV testing and antiviral management procedures. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Molecular Detection of 10 of the Most Unwanted Alien Forest Pathogens in Canada Using Real-Time PCR

PubMed Central

Lamarche, Josyanne; Potvin, Amélie; Pelletier, Gervais; Stewart, Don; Feau, Nicolas; Alayon, Dario I. O.; Dale, Angela L.; Coelho, Aaron; Uzunovic, Adnan; Bilodeau, Guillaume J.; Brière, Stephan C.; Hamelin, Richard C.; Tanguay, Philippe

2015-01-01

Invasive alien tree pathogens can cause significant economic losses as well as large-scale damage to natural ecosystems. Early detection to prevent their establishment and spread is an important approach used by several national plant protection organizations (NPPOs). Molecular detection tools targeting 10 of the most unwanted alien forest pathogens in Canada were developed as part of the TAIGA project (http://taigaforesthealth.com/). Forest pathogens were selected following an independent prioritization. Specific TaqMan real-time PCR detection assays were designed to function under homogeneous conditions so that they may be used in 96- or 384-well plate format arrays for high-throughput testing of large numbers of samples against multiple targets. Assays were validated for 1) specificity, 2) sensitivity, 3) precision, and 4) robustness on environmental samples. All assays were highly specific when evaluated against a panel of pure cultures of target and phylogenetically closely-related species. Sensitivity, evaluated by assessing the limit of detection (with a threshold of 95% of positive samples), was found to be between one and ten target gene region copies. Precision or repeatability of each assay revealed a mean coefficient of variation of 3.4%. All assays successfully allowed detection of target pathogen on positive environmental samples, without any non-specific amplification. These molecular detection tools will allow for rapid and reliable detection of 10 of the most unwanted alien forest pathogens in Canada. PMID:26274489

High sample throughput genotyping for estimating C-lineage introgression in the dark honeybee: an accurate and cost-effective SNP-based tool.

PubMed

Henriques, Dora; Browne, Keith A; Barnett, Mark W; Parejo, Melanie; Kryger, Per; Freeman, Tom C; Muñoz, Irene; Garnery, Lionel; Highet, Fiona; Jonhston, J Spencer; McCormack, Grace P; Pinto, M Alice

2018-06-04

The natural distribution of the honeybee (Apis mellifera L.) has been changed by humans in recent decades to such an extent that the formerly widest-spread European subspecies, Apis mellifera mellifera, is threatened by extinction through introgression from highly divergent commercial strains in large tracts of its range. Conservation efforts for A. m. mellifera are underway in multiple European countries requiring reliable and cost-efficient molecular tools to identify purebred colonies. Here, we developed four ancestry-informative SNP assays for high sample throughput genotyping using the iPLEX Mass Array system. Our customized assays were tested on DNA from individual and pooled, haploid and diploid honeybee samples extracted from different tissues using a diverse range of protocols. The assays had a high genotyping success rate and yielded accurate genotypes. Performance assessed against whole-genome data showed that individual assays behaved well, although the most accurate introgression estimates were obtained for the four assays combined (117 SNPs). The best compromise between accuracy and genotyping costs was achieved when combining two assays (62 SNPs). We provide a ready-to-use cost-effective tool for accurate molecular identification and estimation of introgression levels to more effectively monitor and manage A. m. mellifera conservatories.

Analysis of molecular chaperones using a Xenopus oocyte protein refolding assay.

PubMed

Heikkila, John J; Kaldis, Angelo; Abdulle, Rashid

2006-01-01

Heat shock proteins (Hsps) are molecular chaperones that aid in the folding and translocation of protein under normal conditions and protect cellular proteins during stressful situations. A family of Hsps, the small Hsps, can maintain denatured target proteins in a folding-competent state such that they can be refolded and regain biological activity in the presence of other molecular chaperones. Previous assays have employed cellular lysates as a source of molecular chaperones involved in folding. In this chapter, we describe the production and purification of a Xenopus laevis recombinant small Hsp, Hsp30C, and an in vivo luciferase (LUC) refolding assay employing microinjected Xenopus oocytes. This assay tests whether LUC can be maintained in a folding-competent state when heat denatured in the presence of a small Hsp or other molecular chaperone. For example, micro-injection of heat-denatured LUC alone into oocytes resulted in minimal reactivation of enzyme activity. However, LUC heat denatured in the presence of Hsp30C resulted in 100% recovery of enzyme activity after microinjection. The in vivo oocyte refolding system is more sensitive and requires less molecular chaperone than in vitro refolding assays. Also, this protocol is not limited to testing Xenopus molecular chaperones because small Hsps from other organisms have been used successfully.

Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection

PubMed Central

Abd El Wahed, Ahmed; Patel, Pranav; Faye, Oumar; Thaloengsok, Sasikanya; Heidenreich, Doris; Matangkasombut, Ponpan; Manopwisedjaroen, Khajohnpong; Sakuntabhai, Anavaj; Sall, Amadou A.; Hufert, Frank T.; Weidmann, Manfred

2015-01-01

Background Over 2.5 billion people are exposed to the risk of contracting dengue fever (DF). Early diagnosis of DF helps to diminish its burden on public health. Real-time reverse transcription polymerase amplification assays (RT-PCR) are the standard method for molecular detection of the dengue virus (DENV). Real-time RT-PCR analysis is not suitable for on-site screening since mobile devices are large, expensive, and complex. In this study, two RT-recombinase polymerase amplification (RT-RPA) assays were developed to detect DENV1-4. Methodology/Principal Findings Using two quantitative RNA molecular standards, the analytical sensitivity of a RT-RPA targeting the 3´non-translated region of DENV1-4 was found to range from 14 (DENV4) to 241 (DENV1-3) RNA molecules detected. The assay was specific and did not cross detect other Flaviviruses. The RT-RPA assay was tested in a mobile laboratory combining magnetic-bead based total nucleic acid extraction and a portable detection device in Kedougou (Senegal) and in Bangkok (Thailand). In Kedougou, the RT-RPA was operated at an ambient temperature of 38°C with auxiliary electricity tapped from a motor vehicle and yielded a clinical sensitivity and specificity of 98% (n=31) and 100% (n=23), respectively. While in the field trial in Bangkok, the clinical sensitivity and specificity were 72% (n=90) and 100%(n=41), respectively. Conclusions/Significance During the first 5 days of infection, the developed DENV1-4 RT-RPA assays constitute a suitable accurate and rapid assay for DENV diagnosis. Moreover, the use of a portable fluorescence-reading device broadens its application potential to the point-of-care for outbreak investigations. PMID:26075598

Development of a rapid diagnostic assay for the detection of tomato chlorotic dwarf viroid based on isothermal reverse-transcription-recombinase polymerase amplification.

PubMed

Hammond, Rosemarie W; Zhang, Shulu

2016-10-01

A molecular diagnostic assay utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 39°C and target-specific primers and probe were developed for the rapid, sensitive, and specific detection of tomato chlorotic dwarf viroid (TCDVd) in infected leaf and seed tissues. The performance of the AmplifyRP(®) Acceler8™ RT-RPA diagnostic assay, utilizing a lateral flow strip contained within an amplicon detection chamber, was evaluated and the results were compared with a standard RT-PCR assay. The AmplifyRP(®) Acceler8™ assay was specific for TCDVd in leaf and seed tissues, its sensitivity was comparable to conventional RT-PCR in leaf tissues, and it does not require extensive sample purification, specialized equipment, or technical expertise. This is the first report utilizing an RT-RPA assay to detect viroids and the assay can be used both in the laboratory and in the field for TCDVd detection. Published by Elsevier B.V.

Development of a High Throughput Assay for Rapid and Accurate 10-Plex Detection of Citrus Pathogens

USDA-ARS?s Scientific Manuscript database

The need to reliably detect and identify multiple plant pathogens simultaneously, especially in woody perennial hosts, has led to development of new molecular diagnostic approaches. In this study, a Luminex-based system was developed that provided a robust and sensitive test for simultaneous detect...

Improving validation methods for molecular diagnostics: application of Bland-Altman, Deming and simple linear regression analyses in assay comparison and evaluation for next-generation sequencing.

PubMed

Misyura, Maksym; Sukhai, Mahadeo A; Kulasignam, Vathany; Zhang, Tong; Kamel-Reid, Suzanne; Stockley, Tracy L

2018-02-01

A standard approach in test evaluation is to compare results of the assay in validation to results from previously validated methods. For quantitative molecular diagnostic assays, comparison of test values is often performed using simple linear regression and the coefficient of determination (R 2 ), using R 2 as the primary metric of assay agreement. However, the use of R 2 alone does not adequately quantify constant or proportional errors required for optimal test evaluation. More extensive statistical approaches, such as Bland-Altman and expanded interpretation of linear regression methods, can be used to more thoroughly compare data from quantitative molecular assays. We present the application of Bland-Altman and linear regression statistical methods to evaluate quantitative outputs from next-generation sequencing assays (NGS). NGS-derived data sets from assay validation experiments were used to demonstrate the utility of the statistical methods. Both Bland-Altman and linear regression were able to detect the presence and magnitude of constant and proportional error in quantitative values of NGS data. Deming linear regression was used in the context of assay comparison studies, while simple linear regression was used to analyse serial dilution data. Bland-Altman statistical approach was also adapted to quantify assay accuracy, including constant and proportional errors, and precision where theoretical and empirical values were known. The complementary application of the statistical methods described in this manuscript enables more extensive evaluation of performance characteristics of quantitative molecular assays, prior to implementation in the clinical molecular laboratory. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

DEVELOPMENT OF A MOLECULAR METHOD TO IDENTIFY ASTROVIRUS IN WATER.

EPA Science Inventory

Astrovirus is a common cause of gastroenteritis that has been determined to be responsible for several outbreaks. Since astrovirus can be waterborne, there is interest in testing environmental water for astrovirus and we have developed a sensitive RT-PCR assay that is designed t...

CPTAC Collaborates with Molecular & Cellular Proteomics to Address Reproducibility in Targeted Assay Development | Office of Cancer Clinical Proteomics Research

Cancer.gov

The journal Molecular & Cellular Proteomics (MCP), in collaboration with the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI), part of the National Institutes of Health, announce new guidelines and requirements for papers describing the development and application of targeted mass spectrometry measurements of peptides, modified peptides and proteins (Mol Cell Proteomics 2017; PMID: 28183812).  NCI’s participation is part of NIH’s overall effort to address the r

Comparison of five assays for detection of Clostridium difficile toxin.

PubMed

Chapin, Kimberle C; Dickenson, Roberta A; Wu, Fongman; Andrea, Sarah B

2011-07-01

Performance characteristics of five assays for detection of Clostridium difficile toxin were compared using fresh stool samples from patients with C. difficile infection (CDI). Assays were performed simultaneously and according to the manufacturers' instructions. Patients were included in the study if they exhibited clinical symptoms consistent with CDI. Nonmolecular assays included glutamate dehydrogenase antigen tests, with positive findings followed by the Premier Toxin A and B Enzyme Immunoassay (GDH/EIA), and the C. Diff Quik Chek Complete test. Molecular assays (PCR) included the BD GeneOhm Cdiff Assay, the Xpert C. difficile test, and the ProGastro Cd assay. Specimens were considered true positive if results were positive in two or more assays. For each method, the Youden index was calculated and cost-effectiveness was analyzed. Of 81 patients evaluated, 26 (32.1%) were positive for CDI. Sensitivity of the BD GeneOhm Cdiff assay, the Xpert C. difficile test, the ProGastro Cd assay, C. Diff Quik Chek Complete test, and two-step GDH/EIA was 96.2%, 96.2%, 88.5%, 61.5%, and 42.3%, respectively. Specificity of the Xpert C. difficile test was 96.4%, and for the other four assays was 100%. Compared with nonmolecular methods, molecular methods detected 34.7% more positive specimens. Assessment of performance characteristics and cost-effectiveness demonstrated that the BD GeneOhm Cdiff assay yielded the best results. While costly, the Xpert C. difficile test required limited processing and yielded rapid results. Because of discordant results, specimen processing, and extraction equipment requirements, the ProGastro Cd assay was the least favored molecular assay. The GDH/EIA method lacked sufficient sensitivity to be recommended. Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Introducing MINA--The Molecularly Imprinted Nanoparticle Assay.

PubMed

Shutov, Roman V; Guerreiro, Antonio; Moczko, Ewa; de Vargas-Sansalvador, Isabel Perez; Chianella, Iva; Whitcombe, Michael J; Piletsky, Sergey A

2014-03-26

A new ELISA- (enzyme-linked immunosorbent assay)-like assay is demonstrated in which no elements of biological origin are used for molecular recognition or signaling. Composite imprinted nanoparticles that contain a catalytic core and which are synthesized by using a solid-phase approach can simultaneously act as recognition/signaling elements, and be used with minimal modifications to standard assay protocols. This assay provides a new route towards replacement of unstable biomolecules in immunoassays. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ex vivo inhibition of Clostridium botulinum neurotoxin types B, C, E, and F by small molecular weight inhibitors.

PubMed

Montgomery, Vicki A; Ahmed, S Ashraf; Olson, Mark A; Mizanur, Rahman M; Stafford, Robert G; Roxas-Duncan, Virginia I; Smith, Leonard A

2015-05-01

Two small molecular weight inhibitors, compounds CB7969312 and CB7967495, that displayed inhibition of botulinum neurotoxin serotype A in a previous study, were evaluated for inhibition of botulinum neurotoxin serotypes B, C, E, and F. The small molecular weight inhibitors were assessed by molecular modeling, UPLC-based peptide cleavage assay; and an ex vivo assay, the mouse phrenic nerve - hemidiaphragm assay (MPNHDA). While both compounds were inhibitors of botulinum neurotoxin (BoNT) serotypes B, C, and F in the MPNHDA, compound CB7969312 was effective at lower molar concentrations than compound CB7967495. However, compound CB7967495 was significantly more effective at preventing BoNTE intoxication than compound CB7969312. In the UPLC-based peptide cleavage assay, CB7969312 was also more effective against LcC. Both compounds inhibited BoNTE, but not BoNTF, LcE, or LcF in the UPLC-based peptide cleavage assay. Molecular modeling studies predicted that both compounds would be effective inhibitors of BoNTs B, C, E, and F. But CB7967495 was predicted to be a more effective inhibitor of the four serotypes (B, C, E, and F) than CB7969312. This is the first report of a small molecular weight compound that inhibits serotypes B, C, E, and F in the ex vivo assay. Published by Elsevier Ltd.

Molecular markers for colorectal cancer screening

PubMed Central

Dickinson, Brandon T.; Kisiel, John; Ahlquist, David A.; Grady, William M.

2016-01-01

Colorectal cancer (CRC), although a significant cause of morbidity and mortality worldwide, has seen a declining incidence and mortality in countries with programmatic screening. Fecal occult blood testing (FOBT) and endoscopic approaches are the predominant screening methods currently. The discovery of the adenoma→carcinoma sequence and a greater understanding of the genetic and epigenetic changes that drive the formation of CRC have contributed to innovative research to identify molecular markers for highly accurate, non-invasive screening tests for CRC. DNA, proteins, messenger RNA, and micro-RNA have all been evaluated. The observation of tumor cell exfoliation into the mucocellular layer of the colonic epithelium and proven stability of DNA in a harsh stool environment make stool DNA a particularly promising marker. The development of a clinically useful stool DNA test has required numerous technical advances, including optimization in DNA stabilization, the development of assays with high analytical sensitivity, and the identification of specific and broadly informative molecular markers. A multi-target stool DNA (MT-sDNA) test, which combines both mutant and methylated DNA markers and a fecal immunochemical test (FIT), recently performed favorably in a large cross-sectional validation study and has been approved by the US Food and Drug Administration (FDA) for the screening of asymptomatic, average risk individuals. The ultimate way in which molecular marker screening assays will be used in clinical practice will require additional studies to determine optimal screening intervals, factors affecting compliance, management of false positive results, and the use of these assays in high-risk populations, as well as other considerations. PMID:25994221

A targeted next-generation sequencing assay for the molecular diagnosis of genetic disorders with orodental involvement

PubMed Central

Prasad, Megana K; Geoffroy, Véronique; Vicaire, Serge; Jost, Bernard; Dumas, Michael; Le Gras, Stéphanie; Switala, Marzena; Gasse, Barbara; Laugel-Haushalter, Virginie; Paschaki, Marie; Leheup, Bruno; Droz, Dominique; Dalstein, Amelie; Loing, Adeline; Grollemund, Bruno; Muller-Bolla, Michèle; Lopez-Cazaux, Séréna; Minoux, Maryline; Jung, Sophie; Obry, Frédéric; Vogt, Vincent; Davideau, Jean-Luc; Davit-Beal, Tiphaine; Kaiser, Anne-Sophie; Moog, Ute; Richard, Béatrice; Morrier, Jean-Jacques; Duprez, Jean-Pierre; Odent, Sylvie; Bailleul-Forestier, Isabelle; Rousset, Monique Marie; Merametdijan, Laure; Toutain, Annick; Joseph, Clara; Giuliano, Fabienne; Dahlet, Jean-Christophe; Courval, Aymeric; El Alloussi, Mustapha; Laouina, Samir; Soskin, Sylvie; Guffon, Nathalie; Dieux, Anne; Doray, Bérénice; Feierabend, Stephanie; Ginglinger, Emmanuelle; Fournier, Benjamin; de la Dure Molla, Muriel; Alembik, Yves; Tardieu, Corinne; Clauss, François; Berdal, Ariane; Stoetzel, Corinne; Manière, Marie Cécile; Dollfus, Hélène; Bloch-Zupan, Agnès

2016-01-01

Background Orodental diseases include several clinically and genetically heterogeneous disorders that can present in isolation or as part of a genetic syndrome. Due to the vast number of genes implicated in these disorders, establishing a molecular diagnosis can be challenging. We aimed to develop a targeted next-generation sequencing (NGS) assay to diagnose mutations and potentially identify novel genes mutated in this group of disorders. Methods We designed an NGS gene panel that targets 585 known and candidate genes in orodental disease. We screened a cohort of 101 unrelated patients without a molecular diagnosis referred to the Reference Centre for Oro-Dental Manifestations of Rare Diseases, Strasbourg, France, for a variety of orodental disorders including isolated and syndromic amelogenesis imperfecta (AI), isolated and syndromic selective tooth agenesis (STHAG), isolated and syndromic dentinogenesis imperfecta, isolated dentin dysplasia, otodental dysplasia and primary failure of tooth eruption. Results We discovered 21 novel pathogenic variants and identified the causative mutation in 39 unrelated patients in known genes (overall diagnostic rate: 39%). Among the largest subcohorts of patients with isolated AI (50 unrelated patients) and isolated STHAG (21 unrelated patients), we had a definitive diagnosis in 14 (27%) and 15 cases (71%), respectively. Surprisingly, COL17A1 mutations accounted for the majority of autosomal-dominant AI cases. Conclusions We have developed a novel targeted NGS assay for the efficient molecular diagnosis of a wide variety of orodental diseases. Furthermore, our panel will contribute to better understanding the contribution of these genes to orodental disease. Trial registration numbers NCT01746121 and NCT02397824. PMID:26502894

Emerging technologies in point-of-care molecular diagnostics for resource-limited settings.

PubMed

Peeling, Rosanna W; McNerney, Ruth

2014-06-01

Emerging molecular technologies to diagnose infectious diseases at the point at which care is delivered have the potential to save many lives in developing countries where access to laboratories is poor. Molecular tests are needed to improve the specificity of syndromic management, monitor progress towards disease elimination and screen for asymptomatic infections with the goal of interrupting disease transmission and preventing long-term sequelae. In simplifying laboratory-based molecular assays for use at point-of-care, there are inevitable compromises between cost, ease of use and test performance. Despite significant technological advances, many challenges remain for the development of molecular diagnostics for resource-limited settings. There needs to be more advocacy for these technologies to be applied to infectious diseases, increased efforts to lower the barriers to market entry through streamlined and harmonized regulatory approaches, faster policy development for adoption of new technologies and novel financing mechanisms to enable countries to scale up implementation.

How molecular epidemiology studies can support the National Malaria Control Program in Papua New Guinea.

PubMed

Koepfli, Cristian; Barry, Alyssa; Javati, Sarah; Timinao, Lincoln; Nate, Elma; Mueller, Ivo; Barnadas, Celine

2014-01-01

Papua New Guinea (PNG) is undertaking intensified efforts to control malaria. The National Malaria Control Program aims to reduce the burden of disease by large-scale distribution of insecticide-treated bednets, improved diagnosis and implementation of new treatments. A scientific program monitoring the effect of these interventions, including molecular epidemiology studies, closely accompanies the program. Laboratory assays have been developed in (or transferred to) PNG to measure prevalence of infection and intensity of transmission as well as potential resistance to currently used drugs. These assays help to assess the impact of the National Malaria Control Program, and they reveal a much clearer picture of malaria epidemiology in PNG. In addition, analysis of the geographical clustering of parasites aids in selecting areas where intensified control will be most successful. This paper gives an overview of current research and recently completed studies in the molecular epidemiology of malaria conducted in Papua New Guinea.

Correlation of crAssphage-based qPCR markers with culturable and molecular indicators of human fecal pollution in an impacted urban watershed.

PubMed

Stachler, Elyse; Akyon, Benay; Aquino de Carvalho, Nathalia; Ference, Christian; Bibby, Kyle

2018-06-06

Environmental waters are monitored for fecal pollution to protect public health. Many previously developed human-specific fecal pollution indicators lack adequate sensitivity to be reliably detected in environmental waters or do not correlate well with viral pathogens. Recently, two novel human sewage-associated source tracking qPCR markers were developed based on the bacteriophage crAssphage, CPQ_056 and CPQ_064. These assays are highly human specific, abundant in sewage, and are viral-based, suggesting great promise for environmental application as human fecal pollution indicators. A 30-day sampling study was conducted in an urban stream impacted by combined sewer overflows to evaluate the crAssphage markers' performance in an environmental system. The crAssphage markers were present at concentrations of 4.02-6.04 log10 copies/100 mL throughout the study period, indicating their high abundance and ease of detection in polluted environmental waters. In addition, the crAssphage assays were correlated with rain events, molecular markers for human polyomavirus and HF183, as well as culturable E. coli, enterococci, and somatic coliphage. The CPQ_064 assay correlated strongly to a greater number of biological indicators than the CPQ_056 assay. This study is the first to evaluate both crAssphage qPCR assays in an extended environmental application of crAssphage markers for monitoring of environmental waters. It is also the first study to compare crAssphage marker concentration with other viral-based indicators.

DNA barcoding and real-time PCR detection of Bactrocera xanthodes (Tephritidae: Diptera) complex.

PubMed

Li, D; Waite, D W; Gunawardana, D N; McCarthy, B; Anderson, D; Flynn, A; George, S

2018-05-06

Immature fruit fly stages of the family Tephritidae are commonly intercepted on breadfruit from Pacific countries at the New Zealand border but are unable to be identified to the species level using morphological characters. Subsequent molecular identification showed that they belong to Bactrocera xanthodes, which is part of a species complex that includes Bactrocera paraxanthodes, Bactrocera neoxanthodes and an undescribed species. To establish a more reliable molecular identification system for B. xanthodes, a reference database of DNA barcode sequences for the 5'-fragment of COI gene region was constructed for B. xanthodes from Fiji, Samoa and Tonga. To better understand the species complex, B. neoxanthodes from Vanuatu and B. paraxanthodes from New Caledonia were also barcoded. Using the results of this analysis, real-time TaqMan polymerase chain reaction (PCR) assays for the detection of B. xanthodes complex and for the three individual species of the complex were developed and validated. The assay showed high specificity for the target species, with no cross-reaction observed for closely related organisms. Each of the real-time PCR assays is sensitive, detecting the target sequences at concentrations as low as ten copies µl-1 and can be used as either singleplex or multiplex formats. This real-time PCR assay for B. xanthodes has been successfully applied at the borders in New Zealand, leading to the rapid identification of intercepted Tephritidae eggs and larvae. The developed assays will be useful biosecurity tools for rapid detection of species in the B. xanthodes complex worldwide.

Sandwich enzyme-linked immunosorbent assay for naringin.

PubMed

Qu, Huihua; Wang, Xueqian; Qu, Baoping; Kong, Hui; Zhang, Yue; Shan, Wenchao; Cheng, Jinjun; Wang, Qingguo; Zhao, Yan

2016-01-15

Among the currently used immunoassay techniques, sandwich ELISA exhibits higher specificity, lower cross-reactivity, and a wider working range compared to the corresponding competitive assays. However, it is difficult to obtain a pair of antibodies that can simultaneously bind to two epitopes of a molecule with a molecular weight of less than 1000 Da. Naringin (Nar) is a flavonoid with a molecular mass of 580 Da. The main aim of this study was to develop a sandwich ELISA for detecting Nar. Two hybridomas secreting anti-Nar monoclonal antibodies (mAbs) were produced by fusing splenocytes from a mouse immunised against Nar-bovine serum albumin (BSA) conjugated with a hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line; a sandwich ELISA for detecting Nar was developed using these two well-characterised anti-Nar mAbs. The performance of the sandwich assay was further evaluated by limit of detection (LOD), limit of quantification (LOQ), recovery, and interference analyses. A dose-response curve to Nar was obtained with an LOD of 6.78 ng mL(-1) and an LOQ of 13.47 ng mL(-1). The inter-assay and intra-assay coefficients of variation were 4.32% and 7.48%, respectively. The recovery rate of Nar from concentrated Fructus aurantii granules was 83.63%. A high correlation was obtained between HPLC and sandwich ELISA. These results demonstrate that the sandwich ELISA method has higher specificity for Nar than indirect competitive ELISA. Copyright © 2015 Elsevier B.V. All rights reserved.

The Molecular Biology of Nitroamine Degradation in Soils

DTIC Science & Technology

2015-07-26

analysis and activity assays .............................................................................. 28 Determination of a putative...81 Figure 52: Specific XplA activity in cells treated with different nitrogen sources. .......... 83 Figure 53: Effect of... activity . Our efforts to develop a functional screen for genes from the soil metagenome were unsuccessful. We developed efficient methods of

Analytical and clinical evaluation of the Abbott RealTime hepatitis B sequencing assay.

PubMed

Huh, Hee Jae; Kim, Ji-Youn; Lee, Myoung-Keun; Lee, Nam Yong; Kim, Jong-Won; Ki, Chang-Seok

2016-12-01

Long-term nucleoside analogue (NA) treatment leads to selection for drug-resistant mutations in patients undergoing hepatitis B virus (HBV) therapy. The Abbott RealTime HBV Sequencing assay (Abbott assay; Abbott Molecular Inc., Des Plaines, IL, USA) targets the reverse transcriptase region of the polymerase gene and as such has the ability to detect NA resistance-associated mutations in HBV. We evaluated the analytical performance of the Abbott assay and compared its diagnostic performance to that of a laboratory-developed nested-PCR and sequencing method. The analytical sensitivity of the Abbott assay was determined using a serially-diluted WHO International Standard. To validate the clinical performances of the Abbott assay and the laboratory-developed assay, 89 clinical plasma samples with various levels of HBV DNA were tested using both assays. The limit of detection of the Abbott assay, was 210IU/ml and it successfully detected mutations when the mutant types were present at levels ≥20%. Among 89 clinical specimens, 43 and 42 were amplification positive in the Abbott and laboratory-developed assays, respectively, with 87.6% overall agreement (78/89; 95% confidence interval [CI], 78.6-93.4). The Abbott assay failed to detect the minor mutant populations in two specimens, and therefore overall concordance was 85.3% (76/89), and the kappa value was 0.79 (95% CI, 0.67-0.90). The Abbott assay showed comparable diagnostic performance to laboratory-developed nested PCR followed by direct sequencing, and may be useful as a routine method for detecting HBV NA resistance-associated mutations in clinical laboratory settings. Copyright © 2016 Elsevier B.V. All rights reserved.

Comparison of quantitative cytomegalovirus (CMV) PCR in plasma and CMV antigenemia assay: clinical utility of the prototype AMPLICOR CMV MONITOR test in transplant recipients.

PubMed

Caliendo, A M; St George, K; Kao, S Y; Allega, J; Tan, B H; LaFontaine, R; Bui, L; Rinaldo, C R

2000-06-01

The correlation between the prototype AMPLICOR CMV MONITOR test (Roche Molecular Systems), a quantitative PCR assay, and the cytomegalovirus (CMV) pp65 antigenemia assay was evaluated in transplant recipients. Sequential blood specimens were collected on 29 patients (491 specimens), the leukocyte fraction was tested by CMV antigenemia, and quantitative PCR was performed on plasma specimens. None of the 15 patients (242 specimens) who were antigenemia negative were positive for CMV DNA by PCR, and none of these patients developed active CMV disease. There were 14 antigenemia-positive patients, 8 of whom developed active CMV disease. In all patients, there was a good association between the antigenemia and PCR assays. Ganciclovir-resistant virus was isolated from three patients with active CMV disease. These three patients had persistently elevated levels of antigenemia and CMV DNA by PCR when resistance to ganciclovir developed. This standardized, quantitative CMV PCR assay on plasma has clinical utility for the diagnosis of active disease and in monitoring the response to antiviral therapy in transplant recipients.

Comparison of Quantitative Cytomegalovirus (CMV) PCR in Plasma and CMV Antigenemia Assay: Clinical Utility of the Prototype AMPLICOR CMV MONITOR Test in Transplant Recipients

PubMed Central

Caliendo, Angela M.; St. George, Kirsten; Kao, Shaw-Yi; Allega, Jessica; Tan, Ban-Hock; LaFontaine, Robert; Bui, Larry; Rinaldo, Charles R.

2000-01-01

The correlation between the prototype AMPLICOR CMV MONITOR test (Roche Molecular Systems), a quantitative PCR assay, and the cytomegalovirus (CMV) pp65 antigenemia assay was evaluated in transplant recipients. Sequential blood specimens were collected on 29 patients (491 specimens), the leukocyte fraction was tested by CMV antigenemia, and quantitative PCR was performed on plasma specimens. None of the 15 patients (242 specimens) who were antigenemia negative were positive for CMV DNA by PCR, and none of these patients developed active CMV disease. There were 14 antigenemia-positive patients, 8 of whom developed active CMV disease. In all patients, there was a good association between the antigenemia and PCR assays. Ganciclovir-resistant virus was isolated from three patients with active CMV disease. These three patients had persistently elevated levels of antigenemia and CMV DNA by PCR when resistance to ganciclovir developed. This standardized, quantitative CMV PCR assay on plasma has clinical utility for the diagnosis of active disease and in monitoring the response to antiviral therapy in transplant recipients. PMID:10834964

Development of a high-throughput detection system for HIV-1 using real-time NASBA based on molecular beacons

NASA Astrophysics Data System (ADS)

van Beuningen, Rinie; Marras, Salvatore A.; Kramer, Fred R.; Oosterlaken, Tom; Weusten, Jos; Borst, G.; van de Wiel, Paul

2001-04-01

HIV-1 viral load assays require accuracy and sensitivity at low RNA levels with the capability to detect all subtypes. Furthermore, the assay should be easy to perform and fast to be useful for routine diagnostics. In order to meet these demands we have combined isothermal NASBA amplification with molecular beacon probes for real-time detection and quantitation of HIV-1 RNA. Quantitation is based on co-amplification of the HIV-1 RNA in the clinical sample and a synthetic calibrator RNA which is amplified by the same primer set but detected with a differently labeled molecular beacon. The entire procedure is simple and analysis of 48 samples requires less than 1» hours with minimal hands-on time. A fluorescent plate reader is used for real-time detection and isothermal amplification. The linearity and precision of the assay was determined with the VQC HIV-1 type B standard of the Central Laboratory of the Dutch Red Cross Blood Banks, The Netherlands. Sensitivity was shown to be 50 copies per ml (cps/ml). The average assay precision was 0,19 log10 over a range of 100-300,000 cps/ml tested at nine concentrations. The linearity of dilution series of 15 cultured HIV-1 gag clades A-H was shown. The specificity was 100% on non HIV-1 samples HIV-2, HTLV-1 and HTLV-2. The assay robustness in terms of valid results was 99%. In conclusion, the new real-time NASBA assay meets state-of-the-art HIV-1 viral load performance requirements combined with a high level of user convenience.

Development and performance assessment of a luminex xMAP® direct hybridization assay for the detection and identification of indoor air fungal contamination.

PubMed

Libert, Xavier; Packeu, Ann; Bureau, Fabrice; Roosens, Nancy H; De Keersmaecker, Sigrid C J

2017-01-01

Considered as a public health problem, indoor fungal contamination is generally monitored using classical protocols based on culturing. However, this culture dependency could influence the representativeness of the fungal population detected in an analyzed sample as this includes the dead and uncultivable fraction. Moreover, culture-based protocols are often time-consuming. In this context, molecular tools are a powerful alternative, especially those allowing multiplexing. In this study a Luminex xMAP® assay was developed for the simultaneous detection of 10 fungal species which are most frequently in indoor air and that may cause health problems. This xMAP® assay was found to be sensitive, i.e. its limit of detection is ranging between 0.05 and 0.01 ng of gDNA. The assay was subsequently tested with environmental air samples which were also analyzed with a classical protocol. All the species identified with the classical method were also detected with the xMAP® assay, however in a shorter time frame. These results demonstrate that the Luminex xMAP® fungal assay developed in this study could contribute to the improvement of public health and specifically to the indoor fungal contamination treatment.

Behavioral and Functional Assays for Investigating Mechanisms of Noxious Cold Detection and Multimodal Sensory Processing in Drosophila Larvae

PubMed Central

Patel, Atit A.; Cox, Daniel N.

2017-01-01

To investigate cellular, molecular and behavioral mechanisms of noxious cold detection, we developed cold plate behavioral assays and quantitative means for evaluating the predominant noxious cold-evoked contraction behavior. To characterize neural activity in response to noxious cold, we implemented a GCaMP6-based calcium imaging assay enabling in vivo studies of intracellular calcium dynamics in intact Drosophila larvae. We identified Drosophila class III multidendritic (md) sensory neurons as multimodal sensors of innocuous mechanical and noxious cold stimuli and to dissect the mechanistic bases of multimodal sensory processing we developed two independent functional assays. First, we developed an optogenetic dose response assay to assess whether levels of neural activation contributes to the multimodal aspects of cold sensitive sensory neurons. Second, we utilized CaMPARI, a photo-switchable calcium integrator that stably converts fluorescence from green to red in presence of high intracellular calcium and photo-converting light, to assess in vivo functional differences in neural activation levels between innocuous mechanical and noxious cold stimuli. These novel assays enable investigations of behavioral and functional roles of peripheral sensory neurons and multimodal sensory processing in Drosophila larvae. PMID:28835907

How Imaging Can Impact Clinical Trial Design: Molecular Imaging as a Biomarker for Targeted Cancer Therapy.

PubMed

Mankoff, David A; Farwell, Michael D; Clark, Amy S; Pryma, Daniel A

2015-01-01

The ability to measure biochemical and molecular processes to guide cancer treatment represents a potentially powerful tool for trials of targeted cancer therapy. These assays have traditionally been performed by analysis of tissue samples. However, more recently, functional and molecular imaging has been developed that is capable of in vivo assays of cancer biochemistry and molecular biology and is highly complementary to tissue-based assays. Cancer imaging biomarkers can play a key role in increasing the efficacy and efficiency of therapeutic clinical trials and also provide insight into the biologic mechanisms that bring about a therapeutic response. Future progress will depend on close collaboration between imaging scientists and cancer physicians and on public and commercial sponsors, to take full advantage of what imaging has to offer for clinical trials of targeted cancer therapy. This review will provide examples of how molecular imaging can inform targeted cancer clinical trials and clinical decision making by (1) measuring regional expression of the therapeutic target, (2) assessing early (pharmacodynamic) response to treatment, and (3) predicting therapeutic outcome. The review includes a discussion of basic principles of molecular imaging biomarkers in cancer, with an emphasis on those methods that have been tested in patients. We then review clinical trials designed to evaluate imaging tests as integrated markers embedded in a therapeutic clinical trial with the goal of validating the imaging tests as integral markers that can aid patient selection and direct response-adapted treatment strategies. Examples of recently completed multicenter trials using imaging biomarkers are highlighted.

The LLNA: A Brief Review of Recent Advances and Limitations

PubMed Central

Anderson, Stacey E.; Siegel, Paul D.; Meade, B. J.

2011-01-01

Allergic contact dermatitis is the second most commonly reported occupational illness, accounting for 10% to 15% of all occupational diseases. This highlights the importance of developing rapid and sensitive methods for hazard identification of chemical sensitizers. The murine local lymph node assay (LLNA) was developed and validated for the identification of low molecular weight sensitizing chemicals. It provides several benefits over other tests for sensitization because it provides a quantitative endpoint, dose-responsive data, and allows for prediction of potency. However, there are also several concerns with this assay including: levels of false positive responses, variability due to vehicle, and predictivity. This report serves as a concise review which briefly summarizes the progress, advances and limitations of the assay over the last decade. PMID:21747867

Recommendations for the generation, quantification, storage and handling of peptides used for mass spectrometry-based assays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hoofnagle, Andrew N.; Whiteaker, Jeffrey R.; Carr, Steven A.

2015-12-30

The Clinical Proteomic Tumor Analysis Consortium (1) (CPTAC) of the National Cancer Institute (NCI) is a comprehensive and coordinated effort to accelerate the understanding of the molecular basis of cancer through the application of robust technologies and workflows for the quantitative measurements of proteins. The Assay Development Working Group of the CPTAC Program aims to foster broad uptake of targeted mass spectrometry-based assays employing isotopically labeled peptides for confident assignment and quantification, including multiple reaction monitoring (MRM; also referred to as Selected Reaction Monitoring), parallel reaction monitoring (PRM), and other targeted methods.

Scientific Impact Recognition Award. Sentinel node staging for breast cancer: intraoperative molecular pathology overcomes conventional histologic sampling errors.

PubMed

Blumencranz, Peter; Whitworth, Pat W; Deck, Kenneth; Rosenberg, Anne; Reintgen, Douglas; Beitsch, Peter; Chagpar, Anees; Julian, Thomas; Saha, Sukamal; Mamounas, Eleftherios; Giuliano, Armando; Simmons, Rache

2007-10-01

When sentinel node dissection reveals breast cancer metastasis, completion axillary lymph node dissection is ideally performed during the same operation. Intraoperative histologic techniques have low and variable sensitivity. A new intraoperative molecular assay (GeneSearch BLN Assay; Veridex, LLC, Warren, NJ) was evaluated to determine its efficiency in identifying significant sentinel lymph node metastases (>.2 mm). Positive or negative BLN Assay results generated from fresh 2-mm node slabs were compared with results from conventional histologic evaluation of adjacent fixed tissue slabs. In a prospective study of 416 patients at 11 clinical sites, the assay detected 98% of metastases >2 mm and 88% of metastasis greater >.2 mm, results superior to frozen section. Micrometastases were less frequently detected (57%) and assay positive results in nodes found negative by histology were rare (4%). The BLN Assay is properly calibrated for use as a stand alone intraoperative molecular test.

A Combined Fabrication and Instrumentation Platform for Sample Preparation.

PubMed

Guckenberger, David J; Thomas, Peter C; Rothbauer, Jacob; LaVanway, Alex J; Anderson, Meghan; Gilson, Dan; Fawcett, Kevin; Berto, Tristan; Barrett, Kevin; Beebe, David J; Berry, Scott M

2014-06-01

While potentially powerful, access to molecular diagnostics is substantially limited in the developing world. Here we present an approach to reduced cost molecular diagnostic instrumentation that has the potential to empower developing world communities by reducing costs through streamlining the sample preparation process. In addition, this instrument is capable of producing its own consumable devices on demand, reducing reliance on assay suppliers. Furthermore, this instrument is designed with an "open" architecture, allowing users to visually observe the assay process and make modifications as necessary (as opposed to traditional "black box" systems). This open environment enables integration of microfluidic fabrication and viral RNA purification onto an easy-to-use modular system via the use of interchangeable trays. Here we employ this system to develop a protocol to fabricate microfluidic devices and then use these devices to isolate viral RNA from serum for the measurement of human immunodeficiency virus (HIV) viral load. Results obtained from this method show significantly reduced error compared with similar nonautomated sample preparation processes. © 2014 Society for Laboratory Automation and Screening.

Rapid and amplification-free detection of fish pathogens by utilizing a molecular beacon-based microfluidic system.

PubMed

Su, Yi-Chih; Wang, Chih-Hung; Chang, Wen-Hsin; Chen, Tzong-Yueh; Lee, Gwo-Bin

2015-01-15

Nervous necrosis virus (NNV) and iridovirus are highly infectious pathogens that can cause lethal diseases in various species of fish. These infectious diseases have no effective treatments and the mortality rate is over 80%, which could cause dramatic economic losses in the aquaculture industry. Conventional diagnostic methods of NNV or iridovirus infected fishes, such as virus culture, enzyme-linked immunosorbent assays and nucleic acid assays usually require time-consuming and complex procedures performed by specialized technicians with delicate laboratory facilities. Rapid, simple, accurate and on-site detection of NNV and iridovirus infections would enable timely preventive measures such as immediate sacrifice of infected fishes, and is therefore critically needed for the aquaculture industry. In this study, a microfluidic-based assay that employ magnetic beads conjugated with viral deoxyribonucleic acid (DNA) capturing probes and fluorescent DNA molecular beacons were developed to rapidly detect NNV and iridovirus. Importantly, this new assay was realized in an integrated microfluidic system with a custom-made control system. With this approach, direct and automated NNV and iridovirus detection from infected fishes can be achieved in less than 30 min. Therefore, this molecular-beacon based microfluidic system presents a potentially promising tool for rapid diagnosis of fish pathogens in the field in the future. Copyright © 2014 Elsevier B.V. All rights reserved.

The agony of choice in dermatophyte diagnostics-performance of different molecular tests and culture in the detection of Trichophyton rubrum and Trichophyton interdigitale.

PubMed

Kupsch, C; Ohst, T; Pankewitz, F; Nenoff, P; Uhrlaß, S; Winter, I; Gräser, Y

2016-08-01

Dermatophytosis caused by dermatophytes of the genera Trichophyton and Microsporum belong to the most frequent mycoses worldwide. Molecular detection methods proved to be highly sensitive and enable rapid and accurate detection of dermatophyte species from clinical specimens. For the first time, we compare the performance of different molecular methods with each other and with conventional diagnostics in the detection of dermatophytoses caused by Trichophyton rubrum and Trichophyton interdigitale in clinical specimens (nail, skin and hair). The compared molecular methods comprise two already published PCR-ELISAs, a published quantitative RT-PCR as well as a newly developed PCR-ELISA targeting the internal transcribed spacer region. We investigated the sensitivity of the assays by analysing 375 clinical samples. In 148 specimens (39.5%) a positive result was gained in at least one of the four molecular tests or by culture, but the number of detected agents differed significantly between some of the assays. The most sensitive assay, a PCR-ELISA targeting a microsatellite region, detected 81 T. rubrum infections followed by an internal transcribed spacer PCR-ELISA (60), quantitative RT-PCR (52) and a topoisomerase II PCR-ELISA (51), whereas cultivation resulted in T. rubrum identification in 37 samples. The pros and cons of all four tests in routine diagnostics are discussed. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Multiplexed Liquid Chromatography-Multiple Reaction Monitoring Mass Spectrometry Quantification of Cancer Signaling Proteins

PubMed Central

Chen, Yi; Fisher, Kate J.; Lloyd, Mark; Wood, Elizabeth R.; Coppola, Domenico; Siegel, Erin; Shibata, David; Chen, Yian A.; Koomen, John M.

2017-01-01

Quantitative evaluation of protein expression across multiple cancer-related signaling pathways (e.g. Wnt/β-catenin, TGF-β, receptor tyrosine kinases (RTK), MAP kinases, NF-κB, and apoptosis) in tumor tissues may enable the development of a molecular profile for each individual tumor that can aid in the selection of appropriate targeted cancer therapies. Here, we describe the development of a broadly applicable protocol to develop and implement quantitative mass spectrometry assays using cell line models and frozen tissue specimens from colon cancer patients. Cell lines are used to develop peptide-based assays for protein quantification, which are incorporated into a method based on SDS-PAGE protein fractionation, in-gel digestion, and liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM/MS). This analytical platform is then applied to frozen tumor tissues. This protocol can be broadly applied to the study of human disease using multiplexed LC-MRM assays. PMID:28808993

Development of 2, 7-Diamino-1, 8-Naphthyridine (DANP) Anchored Hairpin Primers for RT-PCR Detection of Chikungunya Virus Infection

PubMed Central

Chen, Huixin; Parimelalagan, Mariya; Takei, Fumie; Hapuarachchi, Hapuarachchige Chanditha; Koay, Evelyn Siew-Chuan; Ng, Lee Ching; Ho, Phui San; Nakatani, Kazuhiko; Chu, Justin Jang Hann

2016-01-01

A molecular diagnostic platform with DANP-anchored hairpin primer was developed and evaluated for the rapid and cost-effective detection of Chikungunya virus (CHIKV) with high sensitivity and specificity. The molecule 2, 7-diamino-1, 8-naphthyridine (DANP) binds to a cytosine-bulge and emits fluorescence at 450 nm when it is excited by 400 nm light. Thus, by measuring the decline in fluorescence emitted from DANP—primer complexes after PCR reaction, we could monitor the PCR progress. By adapting this property of DANP, we have previously developed the first generation DANP-coupled hairpin RT-PCR assay. In the current study, we improved the assay performance by conjugating the DANP molecule covalently onto the hairpin primer to fix the DANP/primer ratio at 1:1; and adjusting the excitation emission wavelength to 365/430 nm to minimize the background signal and a ‘turn-on’ system is achieved. After optimizing the PCR cycle number to 30, we not only shortened the total assay turnaround time to 60 minutes, but also further reduced the background fluorescence. The detection limit of our assay was 0.001 PFU per reaction. The DANP-anchored hairpin primer, targeting nsP2 gene of CHIKV genome, is highly specific to CHIKV, having no cross-reactivity to a panel of other RNA viruses tested. In conclusion, we report here a molecular diagnostic assay that is sensitive, specific, rapid and cost effective for CHIKV detection and can be performed where no real time PCR instrumentation is required. Our results from patient samples indicated 93.62% sensitivity and 100% specificity of this method, ensuring that it can be a useful tool for rapid detection of CHIKV for outbreaks in many parts of the world. PMID:27571201

Development of 2, 7-Diamino-1, 8-Naphthyridine (DANP) Anchored Hairpin Primers for RT-PCR Detection of Chikungunya Virus Infection.

PubMed

Chen, Huixin; Parimelalagan, Mariya; Takei, Fumie; Hapuarachchi, Hapuarachchige Chanditha; Koay, Evelyn Siew-Chuan; Ng, Lee Ching; Ho, Phui San; Nakatani, Kazuhiko; Chu, Justin Jang Hann

2016-08-01

A molecular diagnostic platform with DANP-anchored hairpin primer was developed and evaluated for the rapid and cost-effective detection of Chikungunya virus (CHIKV) with high sensitivity and specificity. The molecule 2, 7-diamino-1, 8-naphthyridine (DANP) binds to a cytosine-bulge and emits fluorescence at 450 nm when it is excited by 400 nm light. Thus, by measuring the decline in fluorescence emitted from DANP-primer complexes after PCR reaction, we could monitor the PCR progress. By adapting this property of DANP, we have previously developed the first generation DANP-coupled hairpin RT-PCR assay. In the current study, we improved the assay performance by conjugating the DANP molecule covalently onto the hairpin primer to fix the DANP/primer ratio at 1:1; and adjusting the excitation emission wavelength to 365/430 nm to minimize the background signal and a 'turn-on' system is achieved. After optimizing the PCR cycle number to 30, we not only shortened the total assay turnaround time to 60 minutes, but also further reduced the background fluorescence. The detection limit of our assay was 0.001 PFU per reaction. The DANP-anchored hairpin primer, targeting nsP2 gene of CHIKV genome, is highly specific to CHIKV, having no cross-reactivity to a panel of other RNA viruses tested. In conclusion, we report here a molecular diagnostic assay that is sensitive, specific, rapid and cost effective for CHIKV detection and can be performed where no real time PCR instrumentation is required. Our results from patient samples indicated 93.62% sensitivity and 100% specificity of this method, ensuring that it can be a useful tool for rapid detection of CHIKV for outbreaks in many parts of the world.

Different Roles of GRP78 on Cell Proliferation and Apoptosis in Cartilage Development.

PubMed

Xiong, Zhangyuan; Jiang, Rong; Li, Xiangzhu; Liu, Yanna; Guo, Fengjin

2015-09-07

Eukaryotic cells possess several mechanisms to adapt to endoplasmic reticulum (ER) stress and thereby survive. ER stress activates a set of signaling pathways collectively termed as the unfolded protein response (UPR). We previously reported that Bone morphogenetic protein 2 (BMP2) mediates mild ER stress and activates UPR signal molecules in chondrogenesis. The mammalian UPR protects the cell against the stress of misfolded proteins in the endoplasmic reticulum. Failure to adapt to ER stress causes the UPR to trigger apoptosis. Glucose regulated protein 78 (GRP78), as an important molecular chaperone in UPR signaling pathways, is responsible for binding to misfolded or unfolded protein during ER stress. However the influence on GRP78 in BMP2-induced chondrocyte differentiation has not yet been elucidated and the molecular mechanism underlyng these processes remain unexplored. Herein we demonstrate that overexpression of GRP78 enhanced cell proliferation in chondrocyte development with G1 phase advance, S phase increasing and G2-M phase transition. Furthermore, overexpression of GRP78 inhibited ER stress-mediated apoptosis and then reduced apoptosis in chondrogenesis induced by BMP2, as assayed by cleaved caspase3, caspase12, C/EBP homologous protein (CHOP/DDIT3/GADD153), p-JNK (phosphorylated c-Jun N-terminal kinase) expression during the course of chondrocyte differentiation by Western blot. In addition, flow cytometry (FCM) assay, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assay and immune-histochemistry analysis also proved this result in vitro and in vivo. It was demonstrated that GRP78 knockdown via siRNA activated the ER stress-specific caspase cascade in developing chondrocyte tissue. Collectively, these findings reveal a novel critical role of GRP78 in regulating ER stress-mediated apoptosis in cartilage development and the molecular mechanisms involved.

Public data and open source tools for multi-assay genomic investigation of disease.

PubMed

Kannan, Lavanya; Ramos, Marcel; Re, Angela; El-Hachem, Nehme; Safikhani, Zhaleh; Gendoo, Deena M A; Davis, Sean; Gomez-Cabrero, David; Castelo, Robert; Hansen, Kasper D; Carey, Vincent J; Morgan, Martin; Culhane, Aedín C; Haibe-Kains, Benjamin; Waldron, Levi

2016-07-01

Molecular interrogation of a biological sample through DNA sequencing, RNA and microRNA profiling, proteomics and other assays, has the potential to provide a systems level approach to predicting treatment response and disease progression, and to developing precision therapies. Large publicly funded projects have generated extensive and freely available multi-assay data resources; however, bioinformatic and statistical methods for the analysis of such experiments are still nascent. We review multi-assay genomic data resources in the areas of clinical oncology, pharmacogenomics and other perturbation experiments, population genomics and regulatory genomics and other areas, and tools for data acquisition. Finally, we review bioinformatic tools that are explicitly geared toward integrative genomic data visualization and analysis. This review provides starting points for accessing publicly available data and tools to support development of needed integrative methods. © The Author 2015. Published by Oxford University Press.

In vitro assays of molecular motors--impact of motor-surface interactions.

PubMed

Mansson, Alf; Balaz, Martina; Albet-Torres, Nuria; Rosengren, K Johan

2008-05-01

In many types of biophysical studies of both single molecules and ensembles of molecular motors the motors are adsorbed to artificial surfaces. Some of the most important assay systems of this type (in vitro motility assays and related single molecule techniques) will be briefly described together with an account of breakthroughs in the understanding of actomyosin function that have resulted from their use. A poorly characterized, but potentially important, entity in these studies is the mechanism of motor adsorption to surfaces and the effects of motor surface interactions on experimental results. A better understanding of these phenomena is also important for the development of commercially viable nanotechnological applications powered by molecular motors. Here, we will consider several aspects of motor surface interactions with a particular focus on heavy meromyosin (HMM) from skeletal muscle. These aspects will be related to heavy meromyosin structure and relevant parts of the vast literature on protein-surface interactions for non-motor proteins. An overview of methods for studying motor-surface interactions will also be given. The information is used as a basis for further development of a model for HMM-surface interactions and is discussed in relation to experiments where nanopatterning has been employed for in vitro reconstruction of actomyosin order. The challenges and potentials of this approach in biophysical studies, compared to the use of self-assembly of biological components into supramolecular protein aggregates (e.g. myosin filaments) will be considered. Finally, this review will consider the implications for further developments of motor-powered lab-on-a-chip devices.

New Horizons on Molecular Pharmacology Applied to Drug Discovery: When Resonance Overcomes Radioligand Binding.

PubMed

Pernomian, Larissa; Gomes, Mayara Santos; Moreira, Josimar Dornelas; da Silva, Carlos Henrique Tomich de Paula; Rosa, Joaquin Maria Campos; Cardoso, Cristina Ribeiro de Barros

2017-01-01

One of the cornerstones of rational drug development is the measurement of molecular parameters derived from ligand-receptor interaction, which guides therapeutic windows definition. Over the last decades, radioligand binding has provided valuable contributions in this field as key method for such purposes. However, its limitations spurred the development of more exquisite techniques for determining such parameters. For instance, safety risks related to radioactivity waste, expensive and controlled disposal of radioisotopes, radiotracer separation-dependence for affinity analysis, and one-site mathematical models-based fitting of data make radioligand binding a suboptimal approach in providing measures of actual affinity conformations from ligands and G proteincoupled receptors (GPCR). Current advances on high-throughput screening (HTS) assays have markedly extended the options of sparing sensitive ways for monitoring ligand affinity. The advent of the novel bioluminescent donor NanoLuc luciferase (Nluc), engineered from Oplophorus gracilirostris luciferase, allowed fitting bioluminescence resonance energy transfer (BRET) for monitoring ligand binding. Such novel approach named Nluc-based BRET (NanoBRET) binding assay consists of a real-time homogeneous proximity assay that overcomes radioligand binding limitations but ensures the quality in affinity measurements. Here, we cover the main advantages of NanoBRET protocol and the undesirable drawbacks of radioligand binding as molecular methods that span pharmacological toolbox applied to Drug Discovery. Also, we provide a novel perspective for the application of NanoBRET technology in affinity assays for multiple-state binding mechanisms involving oligomerization and/or functional biased selectivity. This new angle was proposed based on specific biophysical criteria required for the real-time homogeneity assigned to the proximity NanoBRET protocol. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Development and Validation of an Ultradeep Next-Generation Sequencing Assay for Testing of Plasma Cell-Free DNA from Patients with Advanced Cancer.

PubMed

Janku, Filip; Zhang, Shile; Waters, Jill; Liu, Li; Huang, Helen J; Subbiah, Vivek; Hong, David S; Karp, Daniel D; Fu, Siqing; Cai, Xuyu; Ramzanali, Nishma M; Madwani, Kiran; Cabrilo, Goran; Andrews, Debra L; Zhao, Yue; Javle, Milind; Kopetz, E Scott; Luthra, Rajyalakshmi; Kim, Hyunsung J; Gnerre, Sante; Satya, Ravi Vijaya; Chuang, Han-Yu; Kruglyak, Kristina M; Toung, Jonathan; Zhao, Chen; Shen, Richard; Heymach, John V; Meric-Bernstam, Funda; Mills, Gordon B; Fan, Jian-Bing; Salathia, Neeraj S

2017-09-15

Purpose: Tumor-derived cell-free DNA (cfDNA) in plasma can be used for molecular testing and provide an attractive alternative to tumor tissue. Commonly used PCR-based technologies can test for limited number of alterations at the time. Therefore, novel ultrasensitive technologies capable of testing for a broad spectrum of molecular alterations are needed to further personalized cancer therapy. Experimental Design: We developed a highly sensitive ultradeep next-generation sequencing (NGS) assay using reagents from TruSeqNano library preparation and NexteraRapid Capture target enrichment kits to generate plasma cfDNA sequencing libraries for mutational analysis in 61 cancer-related genes using common bioinformatics tools. The results were retrospectively compared with molecular testing of archival primary or metastatic tumor tissue obtained at different points of clinical care. Results: In a study of 55 patients with advanced cancer, the ultradeep NGS assay detected 82% (complete detection) to 87% (complete and partial detection) of the aberrations identified in discordantly collected corresponding archival tumor tissue. Patients with a low variant allele frequency (VAF) of mutant cfDNA survived longer than those with a high VAF did ( P = 0.018). In patients undergoing systemic therapy, radiological response was positively associated with changes in cfDNA VAF ( P = 0.02), and compared with unchanged/increased mutant cfDNA VAF, decreased cfDNA VAF was associated with longer time to treatment failure (TTF; P = 0.03). Conclusions: Ultradeep NGS assay has good sensitivity compared with conventional clinical mutation testing of archival specimens. A high VAF in mutant cfDNA corresponded with shorter survival. Changes in VAF of mutated cfDNA were associated with TTF. Clin Cancer Res; 23(18); 5648-56. ©2017 AACR . ©2017 American Association for Cancer Research.

Development of real-time PCR for detection and quantitation of Streptococcus parauberis.

PubMed

Nguyen, T L; Lim, Y J; Kim, D-H; Austin, B

2016-01-01

Streptococcus parauberis is an increasing threat to aquaculture of olive flounder, Paralichthys olivaceus Temminck & Schlegel, in South Korea. We developed a real-time polymerase chain reaction (PCR) method using the TaqMan probe assay to detect and quantify S. parauberis by targeting the gyrB gene sequences, which are effective for molecular analysis of the genus Streptococcus. Our real-time PCR assay is capable of detecting 10 fg of genomic DNA per reaction. The intra- and interassay coefficient of variation (CV) values ranged from 0.42-1.95%, demonstrating that the assay has good reproducibility. There was not any cross-reactivity to Streptococcus iniae or to other streptococcal/lactococcal fish pathogens, such as S. agalactiae and Lactococcus garvieae, indicating that the assay is highly specific to S. parauberis. The results of the real-time PCR assay corresponded well to those of conventional culture assays for S. parauberis from inoculated tissue homogenates (r = 0.957; P < 0.05). Hence, this sensitive and specific real-time PCR is a valuable tool for diagnostic quantitation of S. parauberis in clinical samples. © 2014 John Wiley & Sons Ltd.

Nano-plasmonic exosome diagnostics

PubMed Central

Im, Hyungsoon; Shao, Huilin; Weissleder, Ralph; Castro, Cesar M.; Lee, Hakho

2015-01-01

Exosomes have emerged as a promising biomarker. These vesicles abound in biofluids and harbor molecular constituents from their parent cells, thereby offering a minimally-invasive avenue for molecular analyses. Despite such clinical potential, routine exosomal analysis, particularly the protein assay, remains challenging, due to requirements for large sample volumes and extensive processing. We have been developing miniaturized systems to facilitate clinical exosome studies. These systems can be categorized into two components: microfluidics for sample preparation and analytical tools for protein analyses. In this report, we review a new assay platform, nano-plasmonic exosome (nPLEX), in which sensing is based on surface plasmon resonance to achieve label-free exosome detection. Looking forward, we also discuss some potential challenges and improvements in exosome studies. PMID:25936957

Development of an opioid self-administration assay to study drug seeking in zebrafish.

PubMed

Bossé, Gabriel D; Peterson, Randall T

2017-09-29

The zebrafish (Danio rerio) has become an excellent tool to study mental health disorders, due to its physiological and genetic similarity to humans, ease of genetic manipulation, and feasibility of small molecule screening. Zebrafish have been shown to exhibit characteristics of addiction to drugs of abuse in non-contingent assays, including conditioned place preference, but contingent assays have been limited to a single assay for alcohol consumption. Using inexpensive electronic, mechanical, and optical components, we developed an automated opioid self-administration assay for zebrafish, enabling us to measure drug seeking and gain insight into the underlying biological pathways. Zebrafish trained in the assay for five days exhibited robust self-administration, which was dependent on the function of the μ-opioid receptor. In addition, a progressive ratio protocol was used to test conditioned animals for motivation. Furthermore, conditioned fish continued to seek the drug despite an adverse consequence and showed signs of stress and anxiety upon withdrawal of the drug. Finally, we validated our assay by confirming that self-administration in zebrafish is dependent on several of the same molecular pathways as in other animal models. Given the ease and throughput of this assay, it will enable identification of important biological pathways regulating drug seeking and could lead to the development of new therapeutic molecules to treat addiction. Copyright © 2017 Elsevier B.V. All rights reserved.

Commercial Molecular Tests for Fungal Diagnosis from a Practical Point of View.

PubMed

Lackner, Michaela; Lass-Flörl, Cornelia

2017-01-01

The increasing interest in molecular diagnostics is a result of tremendously improved knowledge on fungal infections in the past 20 years and the rapid development of new methods, in particular polymerase chain reaction. High expectations have been placed on molecular diagnostics, and the number of laboratories now using the relevant technology is rapidly increasing-resulting in an obvious need for standardization and definition of laboratory organization. In the past 10 years, multiple new molecular tools were marketed for the detection of DNA, antibodies, cell wall components, or other antigens. In contrast to classical culture methods, molecular methods do not detect a viable organisms, but only molecules which indicate its presence; this can be nucleic acids, cell components (antigens), or antibodies (Fig. 1). In this chapter, an overview is provided on commercially available detection tools, their strength and how to use them. A main focus is laid on providing tips and tricks that make daily life easier. We try to focus and mention methodical details which are not highlighted in the manufacturer's instructions of these test kits, but are based on our personal experience in the laboratory. Important to keep in mind is that molecular tools cannot replace culture, microscopy, or a critical view on patients' clinical history, signs, and symptoms, but provide a valuable add on tool. Diagnosis should not be based solely on a molecular test, but molecular tools might deliver an important piece of information that helps matching the diagnostic puzzle to a diagnosis, in particular as few tests are in vitro diagnostic tests (IVD) or only part of the whole test carries the IVD certificate (e.g., DNA extraction is often not included). Please be aware that the authors do not claim to provide a complete overview on all commercially available diagnostic assays being currently marketed for fungal detection, as those are subject to constant change. A main focus is put on commonly used panfungal assays and pathogen-specific assays, including Aspergillus-specific, Candida-specific, Cryptococcus specific, Histoplasma-specific, and Pneumocystis-specific assays. Assays are categorized according to their underlying principle in either antigen-detecting or antibody-detecting or DNA-detecting (Fig. 1). Other non-DNA-detecting nucleic acid methods such as FISH and PNA FISH are not summarized in this chapter and an overview on test performance, common false positives, and the clinical evaluation of commercial tests in studies is provided already in a previous book series by Javier Yugueros Marcos and David H. Pincus (Marcos and Pincus, Methods Mol Biol 968:25-54, 2013).

An accelerated assay for the identification of lifespan-extending interventions in Drosophila melanogaster.

PubMed

Bauer, Johannes H; Goupil, Stephan; Garber, Graham B; Helfand, Stephen L

2004-08-31

Recent advances in aging research have uncovered genes and genetic pathways that influence lifespan in such diverse organisms as yeast, nematodes, flies, and mice. The discovery of genes and drugs that affect lifespan has been delayed by the absence of a phenotype other than survivorship, which depends on the measurement of age at death of individuals in a population. The use of survivorship to identify genetic and pharmacological interventions that prolong life is time-consuming and requires a large number of homogeneous animals. Here, we report the development of an assay in Drosophila melanogaster using the expression of molecular biomarkers that accelerates the ability to evaluate potential lifespan-altering interventions. Coupling the expression of an age-dependent molecular biomarker to a lethal toxin reduces the time needed to perform lifespan studies by 80%. The assay recapitulates the effect of the three best known environmental life-span-extending interventions in the fly: ambient temperature, reproductive status, and calorie reduction. Single gene mutations known to extend lifespan in the fly such as Indy and rpd3 also extend lifespan in this assay. We used this assay as a screen to identify drugs that extend lifespan in flies. Lipoic acid and resveratrol were identified as being beneficial in our assay and shown to extend lifespan under normal laboratory conditions. We propose that this assay can be used to screen pharmacological as well as genetic interventions more rapidly for positive effects on lifespan. Copyright 2004 The National Academy of Sciencs of the USA

An accelerated assay for the identification of lifespan-extending interventions in Drosophila melanogaster

PubMed Central

Bauer, Johannes H.; Goupil, Stephan; Garber, Graham B.; Helfand, Stephen L.

2004-01-01

Recent advances in aging research have uncovered genes and genetic pathways that influence lifespan in such diverse organisms as yeast, nematodes, flies, and mice. The discovery of genes and drugs that affect lifespan has been delayed by the absence of a phenotype other than survivorship, which depends on the measurement of age at death of individuals in a population. The use of survivorship to identify genetic and pharmacological interventions that prolong life is time-consuming and requires a large number of homogeneous animals. Here, we report the development of an assay in Drosophila melanogaster using the expression of molecular biomarkers that accelerates the ability to evaluate potential lifespan-altering interventions. Coupling the expression of an age-dependent molecular biomarker to a lethal toxin reduces the time needed to perform lifespan studies by 80%. The assay recapitulates the effect of the three best known environmental life-span-extending interventions in the fly: ambient temperature, reproductive status, and calorie reduction. Single gene mutations known to extend lifespan in the fly such as Indy and rpd3 also extend lifespan in this assay. We used this assay as a screen to identify drugs that extend lifespan in flies. Lipoic acid and resveratrol were identified as being beneficial in our assay and shown to extend lifespan under normal laboratory conditions. We propose that this assay can be used to screen pharmacological as well as genetic interventions more rapidly for positive effects on lifespan. PMID:15328413

Transition-ready technologies and expertise from the Chemical and Biological National Security Program at LLNL

DOE Office of Scientific and Technical Information (OSTI.GOV)

Folta, P A; McBride, M T

2006-02-22

HSARPA has initiated a new Bioinformatics and Assay Development solicitation, BIAD2 (BAA 06-01), to address a number of technology gaps and requirements for biodetection (www.hsarpabaa.com). This solicitation will leverage the vast research and development capabilities of the private sector and academia in order to meet the needs of HSARPA and Homeland Security. In order to meet these requirements, this solicitation will: (1) Develop and validate actionable assays for the public and private sector; (2) Develop and validate new assays and novel assay methodologies to enhance existing detection systems and enable future detection platforms; (3) Develop next generation assays which aremore » robust against novel, emerging and engineered threats; (4) Develop novel assays that detect low levels of ribonucleic acid (RNA)-based viral threats in complex backgrounds; (5) Develop novel assays to characterize the viability, degree of virulence or toxicity, and countermeasure resistance of a biological agent; and (6) Develop new bioinformatics tools to support assay development and assay validation The Lawrence Livermore National Laboratory (LLNL) Bioassays and Signature Program (BSP) develops nationally-validated detection and identification assays to cover the full range of biological threat agents, starting from human, animal, and plant pathogens on the Select Agent list. The assays that have been co-developed by the CDC and the BSP are used internationally and represent the gold standard for molecular detection of select agent pathogens for the public health community. They are also used in the DHS environmental monitoring operations such as BioWatch and DHS National Security Special Events support. These reagents have been used to process and analyze more than 5 million samples and have delivered exceptional performance for the end users, with zero false positives since their deployment. Currently, highly-multiplexed nucleic acid assays that represent the ''next-generation'' in BioWatch assays have been developed at LLNL and are in final evaluation at CDC. LLNL is a full partner to the LRN network, as well as a formal LRN member, sanctioned to perform these diagnostics on field samples. The Program has assay extensive experience developing, testing, validating, deploying, and commercializing biodetection assays and instrumentation. The following pages contain additional descriptions of our Program's capabilities. We have a demonstrated track record of established collaborations and look forward to partnering with offerers to the HSARPA BAA to continue to meet the national needs and requirements.« less

Epidemiological, bacteriological and molecular studies on caseous lymphadenitis in Sirohi goats of Rajasthan, India.

PubMed

Kumar, Jyoti; Singh, Fateh; Tripathi, Bhupendra Nath; Kumar, Rajiv; Dixit, Shivendra Kumar; Sonawane, Ganesh Gangaram

2012-10-01

Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis (CL), a chronic debilitating disease of goats. In the present study, a total of 575 goats of Sirohi breed on an organized farm situated in the semi-arid tropical region of Rajasthan, India were clinically examined. Pus samples from superficial lymph nodes of 27 (4.7%) adult goats presenting clinical lesions suggestive of CL were collected for bacteriological and molecular analyses. Of these goats, 51.9% yielded C. pseudotuberculosis on the basis of morphological, cultural and biochemical characteristics. A polymerase chain reaction (PCR) assay targeting proline iminopeptidase gene specific to C. pseudotuberculosis was developed that confirmed all 14 bacterial isolates. The specificity of the PCR product was confirmed by sequencing of the 551-bp amplicon in both senses, showing 98-100% homology with published sequences. Thus, overall prevalence rate based on clinical, bacterial culture and PCR assay were found to be 4.7%, 2.4% and 2.4%, respectively. The PCR assay developed in this study was found to be specific and rapid, and could be used for confirmation of CL in goats as an alternative method to generally cumbersome, time-consuming and less reliable conventional methods.

Linking ToxCast Signatures with Functional Consequences: Proof-of-Concept Study using Known Inhibitors of Vascular Development

EPA Science Inventory

The USEPA’s ToxCast program is developing a novel approach to chemical toxicity testing using high-throughput screening (HTS) assays to rapidly test thousands of chemicals against hundreds of in vitro molecular targets. This approach is based on the premise that in vitro HTS bioa...

Single-Color Digital PCR Provides High-Performance Detection of Cancer Mutations from Circulating DNA.

PubMed

Wood-Bouwens, Christina; Lau, Billy T; Handy, Christine M; Lee, HoJoon; Ji, Hanlee P

2017-09-01

We describe a single-color digital PCR assay that detects and quantifies cancer mutations directly from circulating DNA collected from the plasma of cancer patients. This approach relies on a double-stranded DNA intercalator dye and paired allele-specific DNA primer sets to determine an absolute count of both the mutation and wild-type-bearing DNA molecules present in the sample. The cell-free DNA assay uses an input of 1 ng of nonamplified DNA, approximately 300 genome equivalents, and has a molecular limit of detection of three mutation DNA genome-equivalent molecules per assay reaction. When using more genome equivalents as input, we demonstrated a sensitivity of 0.10% for detecting the BRAF V600E and KRAS G12D mutations. We developed several mutation assays specific to the cancer driver mutations of patients' tumors and detected these same mutations directly from the nonamplified, circulating cell-free DNA. This rapid and high-performance digital PCR assay can be configured to detect specific cancer mutations unique to an individual cancer, making it a potentially valuable method for patient-specific longitudinal monitoring. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Differentiating Pathway-Specific From Nonspecific Effects in High-Throughput Toxicity Data: A Foundation for Prioritizing Adverse Outcome Pathway Development.

PubMed

Fay, Kellie A; Villeneuve, Daniel L; Swintek, Joe; Edwards, Stephen W; Nelms, Mark D; Blackwell, Brett R; Ankley, Gerald T

2018-06-01

The U.S. Environmental Protection Agency's ToxCast program has screened thousands of chemicals for biological activity, primarily using high-throughput in vitro bioassays. Adverse outcome pathways (AOPs) offer a means to link pathway-specific biological activities with potential apical effects relevant to risk assessors. Thus, efforts are underway to develop AOPs relevant to pathway-specific perturbations detected in ToxCast assays. Previous work identified a "cytotoxic burst" (CTB) phenomenon wherein large numbers of the ToxCast assays begin to respond at or near test chemical concentrations that elicit cytotoxicity, and a statistical approach to defining the bounds of the CTB was developed. To focus AOP development on the molecular targets corresponding to ToxCast assays indicating pathway-specific effects, we conducted a meta-analysis to identify which assays most frequently respond at concentrations below the CTB. A preliminary list of potentially important, target-specific assays was determined by ranking assays by the fraction of chemical hits below the CTB compared with the number of chemicals tested. Additional priority assays were identified using a diagnostic-odds-ratio approach which gives greater ranking to assays with high specificity but low responsivity. Combined, the two prioritization methods identified several novel targets (e.g., peripheral benzodiazepine and progesterone receptors) to prioritize for AOP development, and affirmed the importance of a number of existing AOPs aligned with ToxCast targets (e.g., thyroperoxidase, estrogen receptor, aromatase). The prioritization approaches did not appear to be influenced by inter-assay differences in chemical bioavailability. Furthermore, the outcomes were robust based on a variety of different parameters used to define the CTB.

Translational bypass of nonsense mutations in zebrafish rep1, pax2.1 and lamb1 highlights a viable therapeutic option for untreatable genetic eye disease.

PubMed

Moosajee, Mariya; Gregory-Evans, Kevin; Ellis, Charles D; Seabra, Miguel C; Gregory-Evans, Cheryl Y

2008-12-15

The extensive molecular genetic heterogeneity seen with inherited eye disease is a major barrier to the development of gene-based therapeutics. The underlying molecular pathology in a considerable proportion of these diseases however are nonsense mutations leading to premature termination codons. A therapeutic intervention targeted at this abnormality would therefore potentially be relevant to a wide range of inherited eye diseases. We have taken advantage of the ability of aminoglycoside drugs to suppress such nonsense mutations and partially restore full-length, functional protein in a zebrafish model of choroideraemia (chm(ru848); juvenile chorio-retinal degeneration) and in two models of ocular coloboma (noi(tu29a) and gup(m189); congenital optic fissure closure defects). In vitro cell-based assays showed significant readthrough with two drugs, gentamicin and paromomycin, which was confirmed by western blot and in vitro prenylation assays. The presence of either aminoglycoside during zebrafish development in vivo showed remarkable prevention of mutant ocular phenotypes in each model and a reduction in multisystemic defects leading to a 1.5-1.7-fold increase in survival. We also identified a significant reduction in abnormal cell death shown by TUNEL assay. To test the hypothesis that optic fissure closure was apoptosis-dependent, the anti-apoptotic agents, curcumin and zVAD-fmk, were tested in gup(m189) embryos. Both drugs were found to reduce the size of the coloboma, providing molecular evidence that cell death is required for optic fissure remodelling. These findings draw attention to the value of zebrafish models of eye disease as useful preclinical drug screening tools in studies to identify molecular mechanisms amenable to therapeutic intervention.

Microbial detection with low molecular weight RNA.

PubMed

Kourentzi, K D; Fox, G E; Willson, R C

2001-12-01

The need to monitor microorganisms in the environment has increased interest in assays based on hybridization probes that target nucleic acids (e.g., rRNA). We report the development of liquid-phase assays for specific bacterial 5S rRNA sequences or similarly sized artificial RNAs (aRNAs) using molecular beacon technology. These beacons fluoresce only in the presence of specific target sequences, rendering as much as a 27-fold fluorescence enhancement. The assays can be used with both crude cell lysates and purified total RNA preparations. Minimal sample preparation (e.g., heating to promote leakage from cells) is sufficient to detect many Gram-negative bacteria. Using this approach it was possible to detect an aRNA-labeled Escherichia coli strain in the presence of a large background of an otherwise identical E. coli strain. Finally, by using a longer wavelength carboxytetramethylrhodamine beacon it was possible to reduce the fraction of the signal due to cellular autofluorescence to below 0.5%.

Microbial detection with low molecular weight RNA

NASA Technical Reports Server (NTRS)

Kourentzi, K. D.; Fox, G. E.; Willson, R. C.

2001-01-01

The need to monitor microorganisms in the environment has increased interest in assays based on hybridization probes that target nucleic acids (e.g., rRNA). We report the development of liquid-phase assays for specific bacterial 5S rRNA sequences or similarly sized artificial RNAs (aRNAs) using molecular beacon technology. These beacons fluoresce only in the presence of specific target sequences, rendering as much as a 27-fold fluorescence enhancement. The assays can be used with both crude cell lysates and purified total RNA preparations. Minimal sample preparation (e.g., heating to promote leakage from cells) is sufficient to detect many Gram-negative bacteria. Using this approach it was possible to detect an aRNA-labeled Escherichia coli strain in the presence of a large background of an otherwise identical E. coli strain. Finally, by using a longer wavelength carboxytetramethylrhodamine beacon it was possible to reduce the fraction of the signal due to cellular autofluorescence to below 0.5%.

Molecular approaches to Taenia asiatica.

PubMed

Jeon, Hyeong-Kyu; Eom, Keeseon S

2013-02-01

Taenia solium, T. saginata, and T. asiatica are taeniid tapeworms that cause taeniasis in humans and cysticercosis in intermediate host animals. Taeniases remain an important public health concerns in the world. Molecular diagnostic methods using PCR assays have been developed for rapid and accurate detection of human infecting taeniid tapeworms, including the use of sequence-specific DNA probes, PCR-RFLP, and multiplex PCR. More recently, DNA diagnosis using PCR based on histopathological specimens such as 10% formalin-fixed paraffin-embedded and stained sections mounted on slides has been applied to cestode infections. The mitochondrial gene sequence is believed to be a very useful molecular marker for not only studying evolutionary relationships among distantly related taxa, but also for investigating the phylo-biogeography of closely related species. The complete sequence of the human Taenia tapeworms mitochondrial genomes were determined, and its organization and structure were compared to other human-tropic Taenia tapeworms for which complete mitochondrial sequence data were available. The multiplex PCR assay with the Ta4978F, Ts5058F, Tso7421F, and Rev7915 primers will be useful for differential diagnosis, molecular characterization, and epidemiological surveys of human Taenia tapeworms.

Quantitative molecular diagnostic assays of grain washes for Claviceps purpurea are correlated with visual determinations of ergot contamination.

PubMed

Comte, Alexia; Gräfenhan, Tom; Links, Matthew G; Hemmingsen, Sean M; Dumonceaux, Tim J

2017-01-01

We examined the epiphytic microbiome of cereal grain using the universal barcode chaperonin-60 (cpn60). Microbial community profiling of seed washes containing DNA extracts prepared from field-grown cereal grain detected sequences from a fungus identified only to Class Sordariomycetes. To identify the fungal sequence and to improve the reference database, we determined cpn60 sequences from field-collected and reference strains of the ergot fungus, Claviceps purpurea. These data allowed us to identify this fungal sequence as deriving from C. purpurea, and suggested that C. purpurea DNA is readily detectable on agricultural commodities, including those for which ergot was not identified as a grading factor. To get a sense of the prevalence and level of C. purpurea DNA in cereal grains, we developed a quantitative PCR assay based on the fungal internal transcribed spacer (ITS) and applied it to 137 samples from the 2014 crop year. The amount of Claviceps DNA quantified correlated strongly with the proportion of ergot sclerotia identified in each grain lot, although there was evidence that non-target organisms were responsible for some false positives with the ITS-based assay. We therefore developed a cpn60-targeted loop-mediated isothermal amplification assay and applied it to the same grain wash samples. The time to positive displayed a significant, inverse correlation to ergot levels determined by visual ratings. These results indicate that both laboratory-based and field-adaptable molecular diagnostic assays can be used to detect and quantify pathogen load in bulk commodities using cereal grain washes.

Quantitative molecular diagnostic assays of grain washes for Claviceps purpurea are correlated with visual determinations of ergot contamination

PubMed Central

Comte, Alexia; Gräfenhan, Tom; Links, Matthew G.; Hemmingsen, Sean M.

2017-01-01

We examined the epiphytic microbiome of cereal grain using the universal barcode chaperonin-60 (cpn60). Microbial community profiling of seed washes containing DNA extracts prepared from field-grown cereal grain detected sequences from a fungus identified only to Class Sordariomycetes. To identify the fungal sequence and to improve the reference database, we determined cpn60 sequences from field-collected and reference strains of the ergot fungus, Claviceps purpurea. These data allowed us to identify this fungal sequence as deriving from C. purpurea, and suggested that C. purpurea DNA is readily detectable on agricultural commodities, including those for which ergot was not identified as a grading factor. To get a sense of the prevalence and level of C. purpurea DNA in cereal grains, we developed a quantitative PCR assay based on the fungal internal transcribed spacer (ITS) and applied it to 137 samples from the 2014 crop year. The amount of Claviceps DNA quantified correlated strongly with the proportion of ergot sclerotia identified in each grain lot, although there was evidence that non-target organisms were responsible for some false positives with the ITS-based assay. We therefore developed a cpn60-targeted loop-mediated isothermal amplification assay and applied it to the same grain wash samples. The time to positive displayed a significant, inverse correlation to ergot levels determined by visual ratings. These results indicate that both laboratory-based and field-adaptable molecular diagnostic assays can be used to detect and quantify pathogen load in bulk commodities using cereal grain washes. PMID:28257512

Duplex detection of the Mycobacterium tuberculosis complex and medically important non-tuberculosis mycobacteria by real-time PCR based on the rnpB gene.

PubMed

Abdeldaim, Guma; Svensson, Erik; Blomberg, Jonas; Herrmann, Björn

2016-11-01

A duplex real-time PCR based on the rnpB gene was developed for Mycobacterium spp. The assay was specific for the Mycobacterium tuberculosis complex (MTB) and also detected all 19 tested species of non-tuberculous mycobacteria (NTM). The assay was evaluated on 404 clinical samples: 290 respiratory samples and 114 from tissue and other non-respiratory body sites. M. tuberculosis was detected by culture in 40 samples and in 30 samples by the assay. The MTB assay showed a sensitivity similar to Roche Cobas Amplicor MTB-PCR (Roche Molecular Systems, Pleasanton, CA, USA). There were only nine samples with non-tuberculous mycobacteria detected by culture. Six of them were detected by the PCR assay. © 2016 APMIS. Published by John Wiley & Sons Ltd.

Comparison of the BD MAX MRSA XT to the Cepheid™ Xpert® MRSA assay for the molecular detection of methicillin-resistant Staphylococcus aureus from nasal swabs.

PubMed

Mehta, Sanjay R; Estrada, Jasmine; Ybarra, Juan; Fierer, Joshua

2017-04-01

Variation in MRSA genotypes may affect the sensitivity of molecular assays to detect this organism. We compared 2 commonly used screening assays, the Cepheid™ Xpert® MRSA and the BD MAX™ MRSA XT on consecutively obtained nasal swabs from 479 subjects. Specimens giving discordant results were subjected to additional microbiologic and molecular testing. Six hundred forty-two (97.6%) of the 658 test results were concordant. Of the 16 discordant results from 12 subjects, additional results suggested that 9 (60%) of the 15 MRSA XT assays were likely correct, and 6 (40%) of the 15 Xpert® assays were likely correct. One discordant result could not be resolved. A mecA dropout and novel mec right-extremity junction (MREJ) sites led to false-positive and negative results by Xpert®. While both assays performed well, continued vigilance is needed to monitor for Staphylococcus aureus with novel MREJ sites, mecA dropouts, and mecC, leading to inaccurate results in screening assays. Published by Elsevier Inc.

Competitive Reporter Monitored Amplification (CMA) - Quantification of Molecular Targets by Real Time Monitoring of Competitive Reporter Hybridization

PubMed Central

Ullrich, Thomas; Ermantraut, Eugen; Schulz, Torsten; Steinmetzer, Katrin

2012-01-01

Background State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests. Methodology and Principal Findings The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique. Conclusions and Significance The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution for real time monitoring of binding events of fluorescently labelled ligands to surface immobilized probes. With the model assay for the detection of human immunodeficiency virus type 1 and 2 (HIV 1/2), we have been able to observe the amplification kinetics of five targets simultaneously and accommodate two additional hybridization controls with a simple instrument set-up. The ability to accommodate multiple controls and targets into a single assay and to perform the assay on simple and robust instrumentation is a prerequisite for the development of novel molecular point of care tests. PMID:22539973

Eliminating Size-Associated Diffusion Constraints for Rapid On-Surface Bioassays with Nanoparticle Probes.

PubMed

Li, Junwei; Zrazhevskiy, Pavel; Gao, Xiaohu

2016-02-24

Nanoparticle probes enable implementation of advanced on-surface assay formats, but impose often underappreciated size-associated constraints, in particular on assay kinetics and sensitivity. The present study highlights substantially slower diffusion-limited assay kinetics due to the rapid development of a nanoprobe depletion layer next to the surface, which static incubation and mixing of bulk solution employed in conventional assay setups often fail to disrupt. In contrast, cyclic solution draining and replenishing yields reaction-limited assay kinetics irrespective of the probe size. Using common surface bioassays, enzyme-linked immunosorbent assays and immunofluorescence, this study shows that this conceptually distinct approach effectively "erases" size-dependent diffusion constraints, providing a straightforward route to rapid on-surface bioassays employing bulky probes and procedures involving multiple labeling cycles, such as multicycle single-cell molecular profiling. For proof-of-concept, the study demonstrates that the assay time can be shortened from hours to minutes with the same probe concentration and, at a typical incubation time, comparable target labeling can be achieved with up to eight times lower nanoprobe concentration. The findings are expected to enable realization of novel assay formats and stimulate development of rapid on-surface bioassays with nanoparticle probes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The molecular bacterial load assay replaces solid culture for measuring early bactericidal response to antituberculosis treatment.

PubMed

Honeyborne, Isobella; Mtafya, Bariki; Phillips, Patrick P J; Hoelscher, Michael; Ntinginya, Elias N; Kohlenberg, Anke; Rachow, Andrea; Rojas-Ponce, Gabriel; McHugh, Timothy D; Heinrich, Norbert

2014-08-01

We evaluated the use of the molecular bacterial load (MBL) assay, for measuring viable Mycobacterium tuberculosis in sputum, in comparison with solid agar and liquid culture. The MBL assay provides early information on the rate of decline in bacterial load and has technical advantages over culture in either form. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Recent advances in diagnostic microbiology.

PubMed

Bravo, Lulette Tricia C; Procop, Gary W

2009-07-01

The past decade has seen a surge in the development of a variety of molecular diagnostics designed to rapidly identify or characterize medically important microorganisms. We briefly review important advances in molecular microbiology, and then discuss specific assays that have been implemented in clinical microbiology laboratories throughout the country. We also discuss emerging methods and technologies that will soon be more widely used for the prompt and accurate detection of the agents of infectious diseases.

Single Cell Cytochemistry Illustrated by the Demonstration of Glucose-6-Phosphate Dehydrogenase Deficiency in Erythrocytes.

PubMed

Peters, Anna L; van Noorden, Cornelis J F

2017-01-01

Cytochemistry is the discipline that is applied to visualize specific molecules in individual cells and has become an essential tool in life sciences. Immunocytochemistry was developed in the sixties of last century and is the most frequently used cytochemical application. However, metabolic mapping is the oldest cytochemical approach to localize activity of specific enzymes, but in the last decades of the previous century and the first decade of the present century it almost became obsolete. The popularity of this approach revived in the past few years. Metabolism gained interest as player in chronic and complex diseases such as cancer, diabetes, neurodegenerative diseases, and vascular diseases and both enzyme cytochemistry and metabolic mapping have become important tools in life sciences.In this chapter, we present glucose-6-phosphate dehydrogenase (G6PD) deficiency, the most prevalent enzyme deficiency worldwide, to illustrate recent developments in enzyme cytochemistry or metabolic mapping. The first assays which were developed quantified enzyme activity but were unreliable for single cell evaluation. The field has expanded with the development of cytochemical single cell assays and DNA testing. Still, all assays-from the earliest developed tests up to the most recently developed tests-have their place in investigations on G6PD activity. Recently, nanoscopy has become available for light and fluorescence microscopy at the nanoscale. For nanoscopy, cytochemistry is an essential tool to visualize intracellular molecular processes. The ultimate goal in the coming years will be nanoscopy of living cells so that the molecular dynamics can be studied. Cytochemistry will undoubtedly play a critical role in these developments.

Molecular diagnosis of lyssaviruses and sequence comparison of Australian bat lyssavirus samples.

PubMed

Foord, A J; Heine, H G; Pritchard, L I; Lunt, R A; Newberry, K M; Rootes, C L; Boyle, D B

2006-07-01

To evaluate and implement molecular diagnostic tests for the detection of lyssaviruses in Australia. A published hemi-nested reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of all lyssavirus genotypes was modified to a fully nested RT-PCR format and compared with the original assay. TaqMan assays for the detection of Australian bat lyssavirus (ABLV) were compared with both the nested and hemi-nested RT-PCR assays. The sequences of RT-PCR products were determined to assess sequence variations of the target region (nucleocapsid gene) in samples of ABLV originating from different regions. The nested RT-PCR assay was highly analytically specific, and at least as analytically sensitive as the hemi-nested assay. The TaqMan assays were highly analytically specific and more analytically sensitive than either RT-PCR assay, with a detection level of approximately 10 genome equivalents per microl. Sequence of the first 544 nucleotides of the nucleocapsid protein coding sequence was obtained from all samples of ABLV received at Australian Animal Health Laboratory during the study period. The nested RT-PCR provided a means for molecular diagnosis of all tested genotypes of lyssavirus including classical rabies virus and Australian bat lyssavirus. The published TaqMan assay proved to be superior to the RT-PCR assays for the detection of ABLV in terms of analytical sensitivity. The TaqMan assay would also be faster and cross contamination is less likely. Nucleotide sequence analyses of samples of ABLV from a wide geographical range in Australia demonstrated the conserved nature of this region of the genome and therefore the suitability of this region for molecular diagnosis.

Development of a Vaccine for Bacterial Kidney Disease in Salmon, 1984 Annual Report.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaattari, Stephen L.

1985-06-01

The data presented here demonstrate that there is some variability to the antigenic structure of KDB. Although gel filtration of all antigenic preparations revealed a wide range of sizes for antigens, resolution on a denaturing gel revealed relatively few protein bands and immunological assays revealed the same (3) low number of antigens. It is of particular interest that there seems to be a protein of 60 kd in all preparations, but that there are not larger individual molecular species. This, in turn indicates that the larger molecular weight species detected in gel filtration are most likely aggregates or membrane fragmentsmore » composed of a lower molecular weight subunit. Use of ultrafiltration of KDM-2 medium appears to be successful in eliminating contamination of high molecular weight material found in KDM-2. There appears to be no alteration in the number of soluble antigens produced by growth in either medium, nor in the number of proteins, as detected by SDS-PAGE. However, soluble antigens isolated from UF-KDM-2 does appear to have greater heterogeneity in their isoelectric focusing (IEF) patterns than those from UF-KDM-2. Also, although there does appear to be an extended lag period in KDB growth on UF-KDM-2, there is no alteration in final O.D. or wet weight of cells. Thus, it appears that UF-KDM-2 may be an alternate medium for those wishing to isolate purified bacterial proteins or antigens. ELISA assays have been developed for the detection of soluble KDB antigens. This system is currently being developed as a sensitive measure of the presence of soluble antigen in serum and tissues of fish. Such a sensitive assay may also allow for the detection of KD+ spawners by the testing of ovarian fluid or serum. ELISA assays have also been developed to detect antibodies to soluble and cellular antigens of KDB. These systems have been proven successful in the detection of rabbit and murine monoclonal antibodies against KDB antigens. Future work will develop the use of anti-fish immunoglobulin (Ig) reagents to detect the presence of fish antibodies to KDB. This would be an extremely useful tool to be used in monitoring the immune response of salmon to the various test vaccines. The various antigens characterized in this study, along with whole KDB cells are currently being conjugated to various immunopotentiating agents. Testing of these prototype vaccines is currently under study.« less

Demonstrating the feasibility of large-scale development of standardized assays to quantify human proteins

PubMed Central

Kennedy, Jacob J.; Abbatiello, Susan E.; Kim, Kyunggon; Yan, Ping; Whiteaker, Jeffrey R.; Lin, Chenwei; Kim, Jun Seok; Zhang, Yuzheng; Wang, Xianlong; Ivey, Richard G.; Zhao, Lei; Min, Hophil; Lee, Youngju; Yu, Myeong-Hee; Yang, Eun Gyeong; Lee, Cheolju; Wang, Pei; Rodriguez, Henry; Kim, Youngsoo; Carr, Steven A.; Paulovich, Amanda G.

2014-01-01

The successful application of MRM in biological specimens raises the exciting possibility that assays can be configured to measure all human proteins, resulting in an assay resource that would promote advances in biomedical research. We report the results of a pilot study designed to test the feasibility of a large-scale, international effort in MRM assay generation. We have configured, validated across three laboratories, and made publicly available as a resource to the community 645 novel MRM assays representing 319 proteins expressed in human breast cancer. Assays were multiplexed in groups of >150 peptides and deployed to quantify endogenous analyte in a panel of breast cancer-related cell lines. Median assay precision was 5.4%, with high inter-laboratory correlation (R2 >0.96). Peptide measurements in breast cancer cell lines were able to discriminate amongst molecular subtypes and identify genome-driven changes in the cancer proteome. These results establish the feasibility of a scaled, international effort. PMID:24317253

A novel multiplex PCR assay for simultaneous detection of nine clinically significant bacterial pathogens associated with bovine mastitis.

PubMed

Ashraf, Aqeela; Imran, Muhammad; Yaqub, Tahir; Tayyab, Muhammad; Shehzad, Wasim; Thomson, Peter C

2017-06-01

For rapid and simultaneous detection of nine bovine mastitic pathogens, a sensitive and specific multiplex PCR assay was developed. The assay was standardized using reference strains and validated on mastitic milk cultures which were identified to species level based on 16S rRNA sequencing. Multiplex PCR assay also efficiently detected the target bacterial strains directly from milk. The detection limit of the assay was up to 50 pg for DNA isolated from pure cultures and 10 4  CFU/ml for spiked milk samples. As estimated by latent class analysis, the assay was sensitive up to 88% and specific up to 98% for targeted mastitic pathogens, compared with the bacterial culture method and the 16S rRNA sequence analysis. This novel molecular assay could be useful for monitoring and maintaining the bovine udder health, ensuring the bacteriological safety of milk, and conducting epidemiological studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

Timing fungicide application intervals based on airborne Erysiphe necator concentrations

USDA-ARS?s Scientific Manuscript database

Management of grape powdery mildew (Erysiphe necator) and other polycyclic diseases relies on numerous fungicide applications that follow a calendar or model-based application intervals, both of which assume that inoculum is always present. Quantitative molecular assays have been previously develope...

Disruption of Embryonic Vascular Development in Predictive Toxicology

EPA Science Inventory

Toxicity testing in the 21st century is moving toward using high-throughput screening assays to rapidly test thousands of chemicals against hundreds of molecular targets and biological pathways, and to provide mechanistic information on chemical effects in human cells and small m...

Using pathway modules as targets for assay development in xenobiotic screening

EPA Science Inventory

Toxicology and pharmaceutical research is increasingly making use of high throughout-screening (HTS) methods to assess the effects of chemicals on molecular pathways, cells and tissues. Whole-genome microarray analysis provides broad information on the response of biological syst...

Founder lines for improved citrus biotechnology

USDA-ARS?s Scientific Manuscript database

This article discusses the research needed to develop the RMCE strategy and molecular assays for site-specific recombinases as tools for genome manipulation. Explanation of genetic engineering used to generate transgenic citrus plants to exhibit a novel phenotype, but not to contain the recombinase...

Construction Strategy for an Internal Amplification Control for Real-Time Diagnostic Assays Using Nucleic Acid Sequence-Based Amplification: Development and Clinical Application

PubMed Central

Rodríguez-Lázaro, David; D'Agostino, Martin; Pla, Maria; Cook, Nigel

2004-01-01

An important analytical control in molecular amplification-based methods is an internal amplification control (IAC), which should be included in each reaction mixture. An IAC is a nontarget nucleic acid sequence which is coamplified simultaneously with the target sequence. With negative results for the target nucleic acid, the absence of an IAC signal indicates that amplification has failed. A general strategy for the construction of an IAC for inclusion in molecular beacon-based real-time nucleic acid sequence-based amplification (NASBA) assays is presented. Construction proceeds in two phases. In the first phase, a double-stranded DNA molecule that contains nontarget sequences flanked by target sequences complementary to the NASBA primers is produced. At the 5′ end of this DNA molecule is a T7 RNA polymerase binding sequence. In the second phase of construction, RNA transcripts are produced from the DNA by T7 RNA polymerase. This RNA is the IAC; it is amplified by the target NASBA primers and is detected by a molecular beacon probe complementary to the internal nontarget sequences. As a practical example, an IAC for use in an assay for the detection of Mycobacterium avium subsp. paratuberculosis is described, its incorporation and optimization within the assay are detailed, and its application to spiked and natural clinical samples is shown to illustrate the correct interpretation of the diagnostic results. PMID:15583319

Breakpoint structure of the Anopheles gambiae 2Rb chromosomal inversion.

PubMed

Lobo, Neil F; Sangaré, Djibril M; Regier, Allison A; Reidenbach, Kyanne R; Bretz, David A; Sharakhova, Maria V; Emrich, Scott J; Traore, Sekou F; Costantini, Carlo; Besansky, Nora J; Collins, Frank H

2010-10-25

Alternative arrangements of chromosome 2 inversions in Anopheles gambiae are important sources of population structure, and are associated with adaptation to environmental heterogeneity. The forces responsible for their origin and maintenance are incompletely understood. Molecular characterization of inversion breakpoints provides insight into how they arose, and provides the basis for development of molecular karyotyping methods useful in future studies. Sequence comparison of regions near the cytological breakpoints of 2Rb allowed the molecular delineation of breakpoint boundaries. Comparisons were made between the standard 2R+b arrangement in the An. gambiae PEST reference genome and the inverted 2Rb arrangements in the An. gambiae M and S genome assemblies. Sequence differences between alternative 2Rb arrangements were exploited in the design of a PCR diagnostic assay, which was evaluated against the known chromosomal banding pattern of laboratory colonies and field-collected samples from Mali and Cameroon. The breakpoints of the 7.55 Mb 2Rb inversion are flanked by extensive runs of the same short (72 bp) tandemly organized sequence, which was likely responsible for chromosomal breakage and rearrangement. Application of the molecular diagnostic assay suggested that 2Rb has a single common origin in An. gambiae and its sibling species, Anopheles arabiensis, and also that the standard arrangement (2R+b) may have arisen twice through breakpoint reuse. The molecular diagnostic was reliable when applied to laboratory colonies, but its accuracy was lower in natural populations. The complex repetitive sequence flanking the 2Rb breakpoint region may be prone to structural and sequence-level instability. The 2Rb molecular diagnostic has immediate application in studies based on laboratory colonies, but its usefulness in natural populations awaits development of complementary molecular tools.

Long noncoding RNA GAPLINC promotes gastric cancer cell proliferation by acting as a molecular sponge of miR-378 to modulate MAPK1 expression.

PubMed

Diao, Lingyun; Wang, Shengying; Sun, Zhiguang

2018-01-01

Dysregulated long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) play key roles in the development of human cancers. LncRNA GAPLINC has been reported to be increased in gastric cancer (GC) tissues. Real-time PCR assays were used to measure expressions of GAPLINC, miR-378, and MAPK1 mRNA. Western blot assays were employed to examine MAPK1 protein expression. Cell proliferation and cell cycle were measured by CCK-8 and propidium iodide-detection assays, respectively. The interaction between GAPLINC and miR-378 was confirmed by site-directed mutagenesis and luciferase assays. Luciferase assays were also used to study whether GAPLINC was able to act as a molecular sponge of miR-378 to modulate MAPK1 expression. The lncRNA GAPLINC expression was upregulated and positively correlated with MAPK1 expression in gastric cancer tissues and cells. Additionally, lncRNA GAPLINC promoted the expression of MAPK1 and the enhancement of GC cell proliferation and cell cycle progression by LncRNA GAPLINC was dependent on MAPK1 in vitro and in vivo. Consequently, we found that miR-378 expression was inversely correlated with GAPLINC expression in GC tissues and cells. miR-378 could directly bind to GAPLINC and decreased GAPLINC expression, thus reducing MAPK1 expression. Furthermore, overexpression of miR-378 inhibited MAPK1 expression, cell proliferation, and cell cycle progression of gastric cancer cells, while these effects were abrogated by upregulating lncRNA GAPLINC expression. Taken together, lncRNA GAPLINC promotes gastric cancer cell proliferation by acting as a molecular sponge of miR-378 to modulate MAPK1 expression.

COLD-PCR: improving the sensitivity of molecular diagnostics assays

PubMed Central

Milbury, Coren A; Li, Jin; Liu, Pingfang; Makrigiorgos, G Mike

2011-01-01

The detection of low-abundance DNA variants or mutations is of particular interest to medical diagnostics, individualized patient treatment and cancer prognosis; however, detection sensitivity for low-abundance variants is a pronounced limitation of most currently available molecular assays. We have recently developed coamplification at lower denaturation temperature-PCR (COLD-PCR) to resolve this limitation. This novel form of PCR selectively amplifies low-abundance DNA variants from mixtures of wild-type and mutant-containing (or variant-containing) sequences, irrespective of the mutation type or position on the amplicon, by using a critical denaturation temperature. The use of a lower denaturation temperature in COLD-PCR results in selective denaturation of amplicons with mutation-containing molecules within wild-type mutant heteroduplexes or with a lower melting temperature. COLD-PCR can be used in lieu of conventional PCR in several molecular applications, thus enriching the mutant fraction and improving the sensitivity of downstream mutation detection by up to 100-fold. PMID:21405967

Comparative study of Factor Xa fluorogenic substrates and their influence on the quantification of LMWHs.

PubMed

Castro-López, Vanessa; Harris, Leanne F; O'Donnell, James S; Killard, Anthony J

2011-01-01

Low molecular weight heparins (LMWHs) are recognised as the preferred anticoagulants in the prevention and treatment of venous thromboembolism. Anti-Factor Xa (anti-FXa) levels are used to monitor the anticoagulant effect of LMWHs and such assays are routinely employed in hospital diagnostic laboratories. In this study, a fluorogenic anti-FXa assay was developed using a commercially available fluorogenic substrate with an attached 6-amino-1-naphthalene-sulfonamide (ANSN) fluorophore and was used for the determination of two LMWHs, enoxaparin and tinzaparin and the heparinoid, danaparoid. The assay was based on the complexation of heparinised plasma with 100 nM exogenous FXa and 25 μM of the fluorogenic substrate Mes-D-LGR-ANSN (C(2)H(5))(2) (SN-7). The assay was tested with pooled plasma samples spiked with anticoagulant concentrations in the range 0-1.6 U mL(-1). The statistically sensitive assay range was 0-0.4 U mL(-1) for enoxaparin and tinzaparin and 0-0.2 U mL(-1) for danaparoid, with assay variation typically below 10.5%. This assay was then compared with a previously published fluorogenic anti-FXa assay developed with the peptide substrate, methylsulfonyl-D: -cyclohexylalanyl-glycyl-arginine-7-amino-4-methylcoumarin acetate (Pefafluor FXa). Both assays were compared in terms of fluorescence intensity, lag times and sensitivity to anticoagulants.

Sensitivity and specificity: twin goals of proteomics assays. Can they be combined?

PubMed

Wilson, Robert

2013-04-01

A major ambition of proteomics is the provision of assays that can diagnose disease and monitor therapies. These assays are required to be sensitive and specific for individual proteins, and in most cases to quantify more than one protein in the same sample. The two main technologies currently used for proteomics assays are based on mass spectrometry and panels of affinity molecules such as antibodies. In the first part of this review the most sensitive existing assays based on these technologies are described and compared with the gold standard of ELISA. Analytical sensitivity is defined and related to the limit of detection, and analytical specificity is defined and shown to depend on molecular proofreading steps, similar to those applied in living systems whenever there is a need for high fidelity. It is shown that at present neither mass spectrometry nor panels of affinity molecules offer the necessary combination of sensitivity and specificity required for multiplexed assays. In the second part of this review the growing numbers of assays that use additional proofreading steps to combine sensitivity with specificity are described. These include assays based on proximity ligation and slow off-rate modified aptamers. Finally the review considers what improvements might be possible in the near future, and concludes that further development of proteomics assays incorporating advanced proofreading steps are most likely to provide the necessary combination of sensitivity and specificity, without incurring high development costs.

Development of a diagnostic real-time polymerase chain reaction assay for the detection of invasive Haemophilus influenzae in clinical samples.

PubMed

Meyler, Kenneth L; Meehan, Mary; Bennett, Desiree; Cunney, Robert; Cafferkey, Mary

2012-12-01

Since the introduction of the Haemophilus influenzae serotype b vaccine, invasive H. influenzae disease has become dominated by nontypeable (NT) strains. Several widely used molecular diagnostic methods have been shown to lack sensitivity or specificity in the detection of some of these strains. Novel real-time assays targeting the fucK, licA, and ompP2 genes were developed and evaluated. The fucK assay detected all strains of H. influenzae tested (n = 116) and had an analytical sensitivity of 10 genome copies/polymerase chain reaction (PCR). This assay detected both serotype b and NT H. influenzae in 12 previously positive specimens (culture and/or bexA PCR) and also detected H. influenzae in a further 5 of 883 culture-negative blood and cerebrospinal fluid (CSF) samples. The fucK assay has excellent potential as a diagnostic test for detection of typeable and nontypeable strains of invasive H. influenzae in clinical samples of blood and CSF. Copyright © 2012 Elsevier Inc. All rights reserved.

Development of a rapid assay to detect the jellyfish Cyanea nozakii using a loop-mediated isothermal amplification method.

PubMed

Liu, Zhongyuan; Dong, Zhijun; Liu, Dongyan

2016-07-01

Blooms of the harmful jellyfish Cyanea nozakii, which are a severe nuisance to fisheries and tourisms, frequently occur in the northern East China Sea, Yellow Sea, and Bohai Sea. To provide early warning of this species, a simple and effective molecular method for identifying C. nozakii was developed using the loop-mediated isothermal amplification method (LAMP). The LAMP assay is highly specific and uses a set of four primers that target six different regions on the mitochondrial cytochrome c oxidase subunit I (COI) gene of C. nozakii. The amplification conditions, including the dNTP and betaine concentrations, the inner primer to outer primer concentration ratio, reaction time and temperature, were optimized. The LAMP assay amplified DNA extracted from tissue samples of C. nozakii but did not amplify DNA from other common scyphozoans and hydrozoans collected in the same region. In addition, the LAMP assay was more sensitive than conventional PCR. Therefore, the established LAMP assay is a sensitive, specific, fast, and easily performed method for detection of C. nozakii at different stages in their life cycle.

Molecular Detection of Breast Cancer

DTIC Science & Technology

1998-02-01

treatment-resistant cancer cells. Clearly new approaches are needed to treat these diseases. This project is designed to develop novel approaches to...detect breast cancer cells that contaminate peripheral blood and bone marrow, and to remove such contaminating cells. An RT-PCR assay has been developed ...to detect breast cancer cells, and a novel gene therapy vector has been developed to kill contaminating cancer cells. Blood and bone marrow samples

Exploiting Bacterial Whole-Genome Sequencing Data for Evaluation of Diagnostic Assays: Campylobacter Species Identification as a Case Study.

PubMed

Jansen van Rensburg, Melissa J; Swift, Craig; Cody, Alison J; Jenkins, Claire; Maiden, Martin C J

2016-12-01

The application of whole-genome sequencing (WGS) to problems in clinical microbiology has had a major impact on the field. Clinical laboratories are now using WGS for pathogen identification, antimicrobial susceptibility testing, and epidemiological typing. WGS data also represent a valuable resource for the development and evaluation of molecular diagnostic assays, which continue to play an important role in clinical microbiology. To demonstrate this application of WGS, this study used publicly available genomic data to evaluate a duplex real-time PCR (RT-PCR) assay that targets mapA and ceuE for the detection of Campylobacter jejuni and Campylobacter coli, leading global causes of bacterial gastroenteritis. In silico analyses of mapA and ceuE primer and probe sequences from 1,713 genetically diverse C. jejuni and C. coli genomes, supported by RT-PCR testing, indicated that the assay was robust, with 1,707 (99.7%) isolates correctly identified. The high specificity of the mapA-ceuE assay was the result of interspecies diversity and intraspecies conservation of the target genes in C. jejuni and C. coli Rare instances of a lack of specificity among C. coli isolates were due to introgression in mapA or sequence diversity in ceuE The results of this study illustrate how WGS can be exploited to evaluate molecular diagnostic assays by using publicly available data, online databases, and open-source software. Copyright © 2016 Jansen van Rensburg et al.

Exploiting Bacterial Whole-Genome Sequencing Data for Evaluation of Diagnostic Assays: Campylobacter Species Identification as a Case Study

PubMed Central

Jansen van Rensburg, Melissa J.; Swift, Craig; Cody, Alison J.; Jenkins, Claire

2016-01-01

The application of whole-genome sequencing (WGS) to problems in clinical microbiology has had a major impact on the field. Clinical laboratories are now using WGS for pathogen identification, antimicrobial susceptibility testing, and epidemiological typing. WGS data also represent a valuable resource for the development and evaluation of molecular diagnostic assays, which continue to play an important role in clinical microbiology. To demonstrate this application of WGS, this study used publicly available genomic data to evaluate a duplex real-time PCR (RT-PCR) assay that targets mapA and ceuE for the detection of Campylobacter jejuni and Campylobacter coli, leading global causes of bacterial gastroenteritis. In silico analyses of mapA and ceuE primer and probe sequences from 1,713 genetically diverse C. jejuni and C. coli genomes, supported by RT-PCR testing, indicated that the assay was robust, with 1,707 (99.7%) isolates correctly identified. The high specificity of the mapA-ceuE assay was the result of interspecies diversity and intraspecies conservation of the target genes in C. jejuni and C. coli. Rare instances of a lack of specificity among C. coli isolates were due to introgression in mapA or sequence diversity in ceuE. The results of this study illustrate how WGS can be exploited to evaluate molecular diagnostic assays by using publicly available data, online databases, and open-source software. PMID:27733632

High-sensitivity immunochromatographic assay for fumonisin B1 based on indirect antibody labeling.

PubMed

Urusov, Alexandr E; Petrakova, Alina V; Gubaydullina, Milyausha K; Zherdev, Anatoly V; Eremin, Sergei A; Kong, Dezhao; Liu, Liqiang; Xu, Chuanlai; Dzantiev, Boris B

2017-05-01

To develop a high-sensitivity immunochromatographic test for fumonisin B1 in plant extracts. Unlike conventional immunochromatographic tests, this assay is performed in two stages: competitive reaction with free specific antibodies and identifying immune complexes by their interaction with the anti-species antibody-conjugated gold nanoparticles. The use of a new geometry for the test strip membranes and a novel reagent application method ensures the proper order of these stages without additional manipulations. The contact of the ready-to-use test strip with the liquid sample suffices in initiating all stages of the assay and obtaining test results. The developed test was used on corn extracts; its instrumental limit of fumonisin B1 detection was 0.6 ng ml -1 at 15 min of assay duration. The proposed approach is flexible and can be used for a wide range of low molecular compounds. The use of anti-species antibody-conjugated gold nanoparticles in immunochromatography significantly facilitates the development of test systems by eliminating the need to synthesize and characterize the conjugates with specific antibodies for each new compound to be detected.

Development and Implementation of a High Throughput Screen for the Human Sperm-Specific Isoform of Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDHS)

PubMed Central

Sexton, Jonathan Z; Danshina, Polina V; Lamson, David R; Hughes, Mark; House, Alan J; Yeh, Li-An; O’Brien, Deborah A; Williams, Kevin P

2011-01-01

Glycolytic isozymes that are restricted to the male germline are potential targets for the development of reversible, non-hormonal male contraceptives. GAPDHS, the sperm-specific isoform of glyceraldehyde-3-phosphate dehydrogenase, is an essential enzyme for glycolysis making it an attractive target for rational drug design. Toward this goal, we have optimized and validated a high-throughput spectrophotometric assay for GAPDHS in 384-well format. The assay was stable over time and tolerant to DMSO. Whole plate validation experiments yielded Z’ values >0.8 indicating a robust assay for HTS. Two compounds were identified and confirmed from a test screen of the Prestwick collection. This assay was used to screen a diverse chemical library and identified fourteen small molecules that modulated the activity of recombinant purified GAPDHS with confirmed IC50 values ranging from 1.8 to 42 µM. These compounds may provide useful scaffolds as molecular tools to probe the role of GAPDHS in sperm motility and long term to develop potent and selective GAPDHS inhibitors leading to novel contraceptive agents. PMID:21760877

Armored DNA in recombinant Baculoviruses as controls in molecular genetic assays.

PubMed

Freystetter, Andrea; Paar, Christian; Stekel, Herbert; Berg, Jörg

2017-10-01

The widespread use of molecular PCR-based assays in analytical and clinical laboratories brings about the need for test-specific, stable, and reliable external controls (EC) as well as standards and internal amplification controls (IC), in order to arrive at consistent test results. In addition, there is also a growing need to produce and provide stable, well-characterized molecular controls for quality assurance programs. In this study, we describe a novel approach to generate armored double-stranded DNA controls, which are encapsulated in baculovirus (BV) particles of the species Autographa californica multiple nucleopolyhedrovirus. We used the well-known BacPAK™ Baculovirus Expression System (Takara-Clontech), removed the polyhedrin promoter used for protein expression, and generated recombinant BV-armored DNAs. The obtained BV-armored DNAs were readily extracted by standard clinical DNA extraction methods, showed favorable linearity and performance in our clinical PCR assays, were resistant to DNase I digestion, and exhibited marked stability in human plasma and serum. BV-armored DNA ought to be used as ECs, quantification standards, and ICs in molecular assays, with the latter application allowing for the entire monitoring of clinical molecular assays for sample adequacy. BV-armored DNA may also be used to produce double-stranded DNA reference materials for, e.g., quality assurance programs. The ease to produce BV-armored DNA should make this approach feasible for a broad spectrum of molecular applications. Finally, as BV-armored DNAs are non-infectious to mammals, they may be even more conveniently shipped than clinical specimen.

Collaborative study for the calibration of replacement batches for the heparin low-molecular-mass for assay biological reference preparation.

PubMed

Terao, E; Daas, A

2016-01-01

The European Pharmacopoeia (Ph. Eur.) prescribes the control of the activity of low molecular mass heparins by assays for anti-Xa and anti-IIa activities (monograph 0828), using a reference standard calibrated in International Units (IU). An international collaborative study coded BSP133 was launched in the framework of the Biological Standardisation Programme (BSP) run under the aegis of the Council of Europe and the European Commission to calibrate replacement batches for the dwindling stocks of the Heparin low-molecular-mass for assay Biological Reference Preparation (BRP) batch 8. Thirteen official medicines control and manufacturers laboratories from European and non-European countries took part in this study to calibrate two freeze-dried candidate batches against the 3rd International Standard (IS) for heparin, low molecular weight (11/176; 3rd IS). The Heparin low-molecular-mass for assay BRP (batch 8) was also included in the test panel to check the continuity between subsequent BRP batches. Taking into account the stability data, the results of this collaborative study and on the basis of the central statistical analysis performed at the European Directorate for the Quality of Medicines & HealthCare (EDQM), the 2 candidate batches were officially adopted by the Commission of the European Pharmacopoeia as Heparin low-molecular-mass for assay BRP batches 9 and 10 with assigned anti-Xa activities of 102 and 100 IU/vial and anti-IIa activities of 34 and 33 IU/vial respectively.

ICBP90 Regulation of DNA Methylation, Histone Ubiquitination, and Tumor Suppressor Gene Expression in Breast Cancer Cells

DTIC Science & Technology

2013-09-01

accomplishments include creation of relevant plant lines, development of in vitro assays, and profiling of mRNA expression in null mutants. 15. SUBJECT TERMS...DNA methylation, UHRF1, VIM1, ubiquitination, epigenetics, chromatin 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF...Molecular Basis of Human Disease ,” which covered several weeks’ worth of material specifically related to the molecular and epigenetic basis of cancer

Long noncoding RNA MINCR regulates cellular proliferation, migration, and invasion in hepatocellular carcinoma.

PubMed

Cao, Jinyu; Zhang, Deyuan; Zeng, Liangtao; Liu, Fanrong

2018-06-01

Accumulating evidence indicates that long noncoding RNAs (lncRNAs) are aberrantly expressed in many cancer types, including hepatocellular carcinoma (HCC). lncRNA MYC-induced long non-coding RNA (MINCR) were revealed to be markedly up-regulated in gallbladder cancer and Burkitt lymphoma cells. However, the biological role and function of MINCR in HCC progression are still unknown. The expression of MINCR in HCC tissues and cell lines was determined using quantitative real-time polymerase chain reaction assays. The effects of MINCR in HCC cell proliferation, migration, and invasion were determined using cell-counting kit 8 (CCK8) assay, wound healing assay, and Transwell assays in vitro. MINCR expression was up-regulated in HCC tissues and cell lines as compared with that in the negative control. The decreased expression of MINCR in vitro markedly inhibited HCC cell proliferation, migration, and invasion. Our results showed that MINCR is important in HCC development and may act as a therapeutic target that regulates HCC cellular proliferation, migration, and invasion, which are involved in HCC tumorigenesis. To the best of our know ledge, MINCR in HCC has not been studied. Our findings showed that this study is the first to reveal that MINCR may act as a therapeutic target in HCC. The in-depth exploration of the molecular mechanism is required to illuminate the molecular mechanisms of MINCR in HCC development. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Development Of Antibody-Based Fiber-Optic Sensors

NASA Astrophysics Data System (ADS)

Tromberg, Bruce J.; Sepaniak, Michael J.; Vo-Dinh, Tuan

1988-06-01

The speed and specificity characteristic of immunochemical complex formation has encouraged the development of numerous antibody-based analytical techniques. The scope and versatility of these established methods can be enhanced by combining the principles of conventional immunoassay with laser-based fiber-optic fluorimetry. This merger of spectroscopy and immunochemistry provides the framework for the construction of highly sensitive and selective fiber-optic devices (fluoroimmuno-sensors) capable of in-situ detection of drugs, toxins, and naturally occurring biochemicals. Fluoroimmuno-sensors (FIS) employ an immobilized reagent phase at the sampling terminus of a single quartz optical fiber. Laser excitation of antibody-bound analyte produces a fluorescence signal which is either directly proportional (as in the case of natural fluorophor and "antibody sandwich" assays) or inversely proportional (as in the case of competitive-binding assays) to analyte concentration. Factors which influence analysis time, precision, linearity, and detection limits include the nature (solid or liquid) and amount of the reagent phase, the method of analyte delivery (passive diffusion, convection, etc.), and whether equilibrium or non-equilibrium assays are performed. Data will be presented for optical fibers whose sensing termini utilize: (1) covalently-bound solid antibody reagent phases, and (2) membrane-entrapped liquid antibody reagents. Assays for large-molecular weight proteins (antigens) and small-molecular weight, carcinogenic, polynuclear aromatics (haptens) will be considered. In this manner, the influence of a system's chemical characteristics and measurement requirements on sensor design, and the consequence of various sensor designs on analytical performance will be illustrated.

A multiplex real-time PCR assay for identification of Pneumocystis jirovecii, Histoplasma capsulatum, and Cryptococcus neoformans/Cryptococcus gattii in samples from AIDS patients with opportunistic pneumonia.

PubMed

Gago, Sara; Esteban, Cristina; Valero, Clara; Zaragoza, Oscar; Puig de la Bellacasa, Jorge; Buitrago, María José

2014-04-01

A molecular diagnostic technique based on real-time PCR was developed for the simultaneous detection of three of the most frequent causative agents of fungal opportunistic pneumonia in AIDS patients: Pneumocystis jirovecii, Histoplasma capsulatum, and Cryptococcus neoformans/Cryptococcus gattii. This technique was tested in cultured strains and in clinical samples from HIV-positive patients. The methodology used involved species-specific molecular beacon probes targeted to the internal transcribed spacer regions of the rDNA. An internal control was also included in each assay. The multiplex real-time PCR assay was tested in 24 clinical strains and 43 clinical samples from AIDS patients with proven fungal infection. The technique developed showed high reproducibility (r(2) of >0.98) and specificity (100%). For H. capsulatum and Cryptococcus spp., the detection limits of the method were 20 and 2 fg of genomic DNA/20 μl reaction mixture, respectively, while for P. jirovecii the detection limit was 2.92 log10 copies/20 μl reaction mixture. The sensitivity in vitro was 100% for clinical strains and 90.7% for clinical samples. The assay was positive for 92.5% of the patients. For one of the patients with proven histoplasmosis, P. jirovecii was also detected in a bronchoalveolar lavage sample. No PCR inhibition was detected. This multiplex real-time PCR technique is fast, sensitive, and specific and may have clinical applications.

Molecular Force Spectroscopy on Cells

NASA Astrophysics Data System (ADS)

Liu, Baoyu; Chen, Wei; Zhu, Cheng

2015-04-01

Molecular force spectroscopy has become a powerful tool to study how mechanics regulates biology, especially the mechanical regulation of molecular interactions and its impact on cellular functions. This force-driven methodology has uncovered a wealth of new information of the physical chemistry of molecular bonds for various biological systems. The new concepts, qualitative and quantitative measures describing bond behavior under force, and structural bases underlying these phenomena have substantially advanced our fundamental understanding of the inner workings of biological systems from the nanoscale (molecule) to the microscale (cell), elucidated basic molecular mechanisms of a wide range of important biological processes, and provided opportunities for engineering applications. Here, we review major force spectroscopic assays, conceptual developments of mechanically regulated kinetics of molecular interactions, and their biological relevance. We also present current challenges and highlight future directions.

Recent patents on self-quenching DNA probes.

PubMed

Knemeyer, Jens-Peter; Marmé, Nicole

2007-01-01

In this review, we report on patents concerning self-quenching DNA probes for assaying DNA during or after amplification as well as for direct assaying DNA or RNA, for example in living cells. Usually the probes consist of fluorescently labeled oligonucleotides whose fluorescence is quenched in the absence of the matching target DNA. Thereby the fluorescence quenching is based on fluorescence resonance energy transfer (FRET), photoinduced electron transfer (PET), or electronically interactions between dye and quencher. However, upon hybridization to the target or after the degradation during a PCR, the fluorescence of the dye is restored. Although the presented probes were originally developed for use in homogeneous assay formats, most of them are also appropriate to improve surface-based assay methods. In particular we describe patents for self-quenching primers, self-quenching probes for TaqMan assays, probes based on G-quartets, Molecular Beacons, Smart Probes, and Pleiades Probes.

Clinical challenges in the molecular characterization of circulating tumour cells in breast cancer.

PubMed

Lianidou, E S; Mavroudis, D; Georgoulias, V

2013-06-25

Blood testing for circulating tumour cells (CTC) has emerged as one of the hottest fields in cancer research. CTC detection and enumeration can serve as a 'liquid biopsy' and an early marker of response to systemic therapy, whereas their molecular characterisation has a strong potential to be translated to individualised targeted treatments and spare breast cancer (BC) patients unnecessary and ineffective therapies. Different analytical systems for CTC detection and isolation have been developed and new areas of research are directed towards developing novel assays for CTC molecular characterisation. Molecular characterisation of single CTC holds considerable promise for predictive biomarker assessment and to explore CTC heterogeneity. The application of extremely powerful next-generation sequencing technologies in the area of CTC molecular characterisation in combination with reliable single CTC isolation opens new frontiers for the management of patients in the near future. This review is mainly focused on the clinical potential of the molecular characterisation of CTC in BC.

Clinical challenges in the molecular characterization of circulating tumour cells in breast cancer

PubMed Central

Lianidou, E S; Mavroudis, D; Georgoulias, V

2013-01-01

Blood testing for circulating tumour cells (CTC) has emerged as one of the hottest fields in cancer research. CTC detection and enumeration can serve as a ‘liquid biopsy' and an early marker of response to systemic therapy, whereas their molecular characterisation has a strong potential to be translated to individualised targeted treatments and spare breast cancer (BC) patients unnecessary and ineffective therapies. Different analytical systems for CTC detection and isolation have been developed and new areas of research are directed towards developing novel assays for CTC molecular characterisation. Molecular characterisation of single CTC holds considerable promise for predictive biomarker assessment and to explore CTC heterogeneity. The application of extremely powerful next-generation sequencing technologies in the area of CTC molecular characterisation in combination with reliable single CTC isolation opens new frontiers for the management of patients in the near future. This review is mainly focused on the clinical potential of the molecular characterisation of CTC in BC. PMID:23756869

Ten years of R&D and full automation in molecular diagnosis.

PubMed

Greub, Gilbert; Sahli, Roland; Brouillet, René; Jaton, Katia

2016-01-01

A 10-year experience of our automated molecular diagnostic platform that carries out 91 different real-time PCR is described. Progresses and future perspectives in molecular diagnostic microbiology are reviewed: why automation is important; how our platform was implemented; how homemade PCRs were developed; the advantages/disadvantages of homemade PCRs, including the critical aspects of troubleshooting and the need to further reduce the turnaround time for specific samples, at least for defined clinical settings such as emergencies. The future of molecular diagnosis depends on automation, and in a novel perspective, it is time now to fully acknowledge the true contribution of molecular diagnostic and to reconsider the indication for PCR, by also using these tests as first-line assays.

Recombinant to modified factor VIII and factor IX - chromogenic and one-stage assays issues.

PubMed

Kitchen, S; Kershaw, G; Tiefenbacher, S

2016-07-01

The recent development of modified recombinant factor VIII (FVIII) and factor IX (FIX) therapeutic products with extended half-lives will create challenges for the haemostasis laboratory in obtaining recovery estimates of these products in clinical samples using existing assays. The new long-acting therapeutic concentrates contain molecular modifications of Fc fusion, site-specific of polyethylene glycol or albumin fusion. The optimum methods for monitoring each new product will need to be assessed individually and laboratories should select an assay which gives similar results to the assay used to assign potency to the product in question. For some extended half-life FVIII and FIX products some one stage assays are entirely unsuitable for monitoring purposes. For most products and assay reagents studied so far, and reviewed in this manuscript, chromogenic FVIII or FIX assays can be safely used with conventional plasma standards. If one stage assays are used then they should be performed using carefully selected reagents/methods which have been shown to recover activity close to the labelled potency for the specific product being monitored. © 2016 John Wiley & Sons Ltd.

A rapid enzymatic assay for high-throughput screening of adenosine-producing strains

PubMed Central

Dong, Huina; Zu, Xin; Zheng, Ping; Zhang, Dawei

2015-01-01

Adenosine is a major local regulator of tissue function and industrially useful as precursor for the production of medicinal nucleoside substances. High-throughput screening of adenosine overproducers is important for industrial microorganism breeding. An enzymatic assay of adenosine was developed by combined adenosine deaminase (ADA) with indophenol method. The ADA catalyzes the cleavage of adenosine to inosine and NH3, the latter can be accurately determined by indophenol method. The assay system was optimized to deliver a good performance and could tolerate the addition of inorganic salts and many nutrition components to the assay mixtures. Adenosine could be accurately determined by this assay using 96-well microplates. Spike and recovery tests showed that this assay can accurately and reproducibly determine increases in adenosine in fermentation broth without any pretreatment to remove proteins and potentially interfering low-molecular-weight molecules. This assay was also applied to high-throughput screening for high adenosine-producing strains. The high selectivity and accuracy of the ADA assay provides rapid and high-throughput analysis of adenosine in large numbers of samples. PMID:25580842

Quantitative PCR assay to determine prevalence and intensity of MSX (Haplosporidium nelsoni) in North Carolina and Rhode Island oysters Crassostrea virginica.

PubMed

Wilbur, Ami E; Ford, Susan E; Gauthier, Julie D; Gomez-Chiarri, Marta

2012-12-27

The continuing challenges to the management of both wild and cultured eastern oyster Crassostrea virginica populations resulting from protozoan parasites has stimulated interest in the development of molecular assays for their detection and quantification. For Haplosporidium nelsoni, the causative agent of multinucleated sphere unknown (MSX) disease, diagnostic evaluations depend extensively on traditional but laborious histological approaches and more recently on rapid and sensitive (but not quantitative) end-point polymerase chain reaction (PCR) assays. Here, we describe the development and application of a quantitative PCR (qPCR) assay for H. nelsoni using an Applied Biosystems TaqMan® assay designed with minor groove binder (MGB) probes. The assay was highly sensitive, detecting as few as 20 copies of cloned target DNA. Histologically evaluated parasite density was significantly correlated with the quantification cycle (Cq), regardless of whether quantification was categorical (r2 = 0.696, p < 0.0001) or quantitative (r2 = 0.797, p < 0.0001). Application in field studies conducted in North Carolina, USA (7 locations), revealed widespread occurrence of the parasite with moderate to high intensities noted in some locations. In Rhode Island, USA, application of the assay on oysters from 2 locations resulted in no positives.

Molecular method for detection of total coliforms in drinking water samples.

PubMed

Maheux, Andrée F; Boudreau, Dominique K; Bisson, Marc-Antoine; Dion-Dupont, Vanessa; Bouchard, Sébastien; Nkuranga, Martine; Bergeron, Michel G; Rodriguez, Manuel J

2014-07-01

This work demonstrates the ability of a bacterial concentration and recovery procedure combined with three different PCR assays targeting the lacZ, wecG, and 16S rRNA genes, respectively, to detect the presence of total coliforms in 100-ml samples of potable water (presence/absence test). PCR assays were first compared to the culture-based Colilert and MI agar methods to determine their ability to detect 147 coliform strains representing 76 species of Enterobacteriaceae encountered in fecal and environmental settings. Results showed that 86 (58.5%) and 109 (74.1%) strains yielded a positive signal with Colilert and MI agar methods, respectively, whereas the lacZ, wecG, and 16S rRNA PCR assays detected 133 (90.5%), 111 (75.5%), and 146 (99.3%) of the 147 total coliform strains tested. These assays were then assessed by testing 122 well water samples collected in the Québec City region of Canada. Results showed that 97 (79.5%) of the samples tested by culture-based methods and 95 (77.9%), 82 (67.2%), and 98 (80.3%) of samples tested using PCR-based methods contained total coliforms, respectively. Consequently, despite the high genetic variability of the total coliform group, this study demonstrated that it is possible to use molecular assays to detect total coliforms in potable water: the 16S rRNA molecular assay was shown to be as efficient as recommended culture-based methods. This assay might be used in combination with an Escherichia coli molecular assay to assess drinking water quality. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Molecular Method for Detection of Total Coliforms in Drinking Water Samples

PubMed Central

Boudreau, Dominique K.; Bisson, Marc-Antoine; Dion-Dupont, Vanessa; Bouchard, Sébastien; Nkuranga, Martine; Bergeron, Michel G.; Rodriguez, Manuel J.

2014-01-01

This work demonstrates the ability of a bacterial concentration and recovery procedure combined with three different PCR assays targeting the lacZ, wecG, and 16S rRNA genes, respectively, to detect the presence of total coliforms in 100-ml samples of potable water (presence/absence test). PCR assays were first compared to the culture-based Colilert and MI agar methods to determine their ability to detect 147 coliform strains representing 76 species of Enterobacteriaceae encountered in fecal and environmental settings. Results showed that 86 (58.5%) and 109 (74.1%) strains yielded a positive signal with Colilert and MI agar methods, respectively, whereas the lacZ, wecG, and 16S rRNA PCR assays detected 133 (90.5%), 111 (75.5%), and 146 (99.3%) of the 147 total coliform strains tested. These assays were then assessed by testing 122 well water samples collected in the Québec City region of Canada. Results showed that 97 (79.5%) of the samples tested by culture-based methods and 95 (77.9%), 82 (67.2%), and 98 (80.3%) of samples tested using PCR-based methods contained total coliforms, respectively. Consequently, despite the high genetic variability of the total coliform group, this study demonstrated that it is possible to use molecular assays to detect total coliforms in potable water: the 16S rRNA molecular assay was shown to be as efficient as recommended culture-based methods. This assay might be used in combination with an Escherichia coli molecular assay to assess drinking water quality. PMID:24771030

Development of potent ALK inhibitor and its molecular inhibitory mechanism against NSCLC harboring EML4-ALK proteins.

PubMed

Kang, Chung Hyo; Yun, Jeong In; Lee, Kwangho; Lee, Chong Ock; Lee, Heung Kyoung; Yun, Chang-Soo; Hwang, Jong Yeon; Cho, Sung Yun; Jung, Heejung; Kim, Pilho; Ha, Jae Du; Jeon, Jeong Hee; Choi, Sang Un; Jeong, Hye Gwang; Kim, Hyoung Rae; Park, Chi Hoon

2015-08-28

Here, we show the newly synthesized and potent ALK inhibitor having similar scaffold to KRCA-0008, which was reported previously, and its molecular mechanism against cancer cells harboring EML4-ALK fusion protein. Through ALK wild type enzyme assay, we selected two compounds, KRCA-0080 and KRCA-0087, which have trifluoromethyl instead of chloride in R2 position. We characterized these newly synthesized compounds by in vitro and in vivo assays. Enzyme assay shows that KRCA-0080 is more potent against various ALK mutants, including L1196M, G1202R, T1151_L1152insT, and C1156Y, which are seen in crizotinib-resistant patients, than KRCA-0008 is. Cell based assays demonstrate our compounds downregulate the cellular signaling, such as Akt and Erk, by suppressing ALK activity to inhibit the proliferation of the cells harboring EML4-ALK. Interestingly, our compounds induced strong G1/S arrest in H3122 cells leading to the apoptosis, which is proved by PARP-1 cleavage. In vivo H3122 xenograft assay, we found that KRCA-0080 shows significant reduction in tumor size compared to crizotinib and KRCA-0008 by 15-20%. Conclusively, we report a potent ALK inhibitor which shows significant in vivo efficacy as well as excellent inhibitory activity against various ALK mutants. Copyright © 2015 Elsevier Inc. All rights reserved.

Comparison of Five Assays for Detection of Clostridium difficile Toxin

PubMed Central

Chapin, Kimberle C.; Dickenson, Roberta A.; Wu, Fongman; Andrea, Sarah B.

2011-01-01

Performance characteristics of five assays for detection of Clostridium difficile toxin were compared using fresh stool samples from patients with C. difficile infection (CDI). Assays were performed simultaneously and according to the manufacturers' instructions. Patients were included in the study if they exhibited clinical symptoms consistent with CDI. Nonmolecular assays included glutamate dehydrogenase antigen tests, with positive findings followed by the Premier Toxin A and B Enzyme Immunoassay (GDH/EIA), and the C. Diff Quik Chek Complete test. Molecular assays (PCR) included the BD GeneOhm Cdiff Assay, the Xpert C. difficile test, and the ProGastro Cd assay. Specimens were considered true positive if results were positive in two or more assays. For each method, the Youden index was calculated and cost-effectiveness was analyzed. Of 81 patients evaluated, 26 (32.1%) were positive for CDI. Sensitivity of the BD GeneOhm Cdiff assay, the Xpert C. difficile test, the ProGastro Cd assay, C. Diff Quik Chek Complete test, and two-step GDH/EIA was 96.2%, 96.2%, 88.5%, 61.5%, and 42.3%, respectively. Specificity of the Xpert C. difficile test was 96.4%, and for the other four assays was 100%. Compared with nonmolecular methods, molecular methods detected 34.7% more positive specimens. Assessment of performance characteristics and cost-effectiveness demonstrated that the BD GeneOhm Cdiff assay yielded the best results. While costly, the Xpert C. difficile test required limited processing and yielded rapid results. Because of discordant results, specimen processing, and extraction equipment requirements, the ProGastro Cd assay was the least favored molecular assay. The GDH/EIA method lacked sufficient sensitivity to be recommended. PMID:21704273

BioAssay Research Database (BARD): chemical biology and probe-development enabled by structured metadata and result types

PubMed Central

Howe, E.A.; de Souza, A.; Lahr, D.L.; Chatwin, S.; Montgomery, P.; Alexander, B.R.; Nguyen, D.-T.; Cruz, Y.; Stonich, D.A.; Walzer, G.; Rose, J.T.; Picard, S.C.; Liu, Z.; Rose, J.N.; Xiang, X.; Asiedu, J.; Durkin, D.; Levine, J.; Yang, J.J.; Schürer, S.C.; Braisted, J.C.; Southall, N.; Southern, M.R.; Chung, T.D.Y.; Brudz, S.; Tanega, C.; Schreiber, S.L.; Bittker, J.A.; Guha, R.; Clemons, P.A.

2015-01-01

BARD, the BioAssay Research Database (https://bard.nih.gov/) is a public database and suite of tools developed to provide access to bioassay data produced by the NIH Molecular Libraries Program (MLP). Data from 631 MLP projects were migrated to a new structured vocabulary designed to capture bioassay data in a formalized manner, with particular emphasis placed on the description of assay protocols. New data can be submitted to BARD with a user-friendly set of tools that assist in the creation of appropriately formatted datasets and assay definitions. Data published through the BARD application program interface (API) can be accessed by researchers using web-based query tools or a desktop client. Third-party developers wishing to create new tools can use the API to produce stand-alone tools or new plug-ins that can be integrated into BARD. The entire BARD suite of tools therefore supports three classes of researcher: those who wish to publish data, those who wish to mine data for testable hypotheses, and those in the developer community who wish to build tools that leverage this carefully curated chemical biology resource. PMID:25477388

Reverse Transcription Recombinase Polymerase Amplification Assay for the Detection of Middle East Respiratory Syndrome Coronavirus

PubMed Central

Abd El Wahed, Ahmed; Patel, Pranav; Heidenreich, Doris; Hufert, Frank T.; Weidmann, Manfred

2013-01-01

The emergence of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in the eastern Mediterranean and imported cases to Europe has alerted public health authorities. Currently, detection of MERS-CoV in patient samples is done by real-time RT-PCR. Samples collected from suspected cases are sent to highly-equipped centralized laboratories for screening. A rapid point-of-care test is needed to allow more widespread mobile detection of the virus directly from patient material. In this study, we describe the development of a reverse transcription isothermal Recombinase Polymerase Amplification (RT-RPA) assay for the identification of MERS-CoV. A partial nucleocapsid gene RNA molecular standard of MERS-coronavirus was used to determine the assay sensitivity. The isothermal (42°C) MERS-CoV RT-RPA was as sensitive as real-time RT-PCR (10 RNA molecules), rapid (3-7 minutes) and mobile (using tubescanner weighing 1kg). The MERS-CoV RT-RPA showed cross-detection neither of any of the RNAs of several coronaviruses and respiratory viruses affecting humans nor of the human genome. The developed isothermal real-time RT-RPA is ideal for rapid mobile molecular MERS-CoV monitoring in acute patients and may also facilitate the search for the animal reservoir of MERS-CoV. PMID:24459611

Molecular identification of python species: development and validation of a novel assay for forensic investigations.

PubMed

Ciavaglia, Sherryn A; Tobe, Shanan S; Donnellan, Stephen C; Henry, Julianne M; Linacre, Adrian M T

2015-05-01

Python snake species are often encountered in illegal activities and the question of species identity can be pertinent to such criminal investigations. Morphological identification of species of pythons can be confounded by many issues and molecular examination by DNA analysis can provide an alternative and objective means of identification. Our paper reports on the development and validation of a PCR primer pair that amplifies a segment of the mitochondrial cytochrome b gene that has been suggested previously as a good candidate locus for differentiating python species. We used this DNA region to perform species identification of pythons, even when the template DNA was of poor quality, as might be the case with forensic evidentiary items. Validation tests are presented to demonstrate the characteristics of the assay. Tests involved the cross-species amplification of this marker in non-target species, minimum amount of DNA template required, effects of degradation on product amplification and a blind trial to simulate a casework scenario that provided 100% correct identity. Our results demonstrate that this assay performs reliably and robustly on pythons and can be applied directly to forensic investigations where the presence of a species of python is in question. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection.

PubMed

Buh Gasparic, Meti; Tengs, Torstein; La Paz, Jose Luis; Holst-Jensen, Arne; Pla, Maria; Esteve, Teresa; Zel, Jana; Gruden, Kristina

2010-03-01

Several techniques have been developed for detection and quantification of genetically modified organisms, but quantitative real-time PCR is by far the most popular approach. Among the most commonly used real-time PCR chemistries are TaqMan probes and SYBR green, but many other detection chemistries have also been developed. Because their performance has never been compared systematically, here we present an extensive evaluation of some promising chemistries: sequence-unspecific DNA labeling dyes (SYBR green), primer-based technologies (AmpliFluor, Plexor, Lux primers), and techniques involving double-labeled probes, comprising hybridization (molecular beacon) and hydrolysis (TaqMan, CPT, LNA, and MGB) probes, based on recently published experimental data. For each of the detection chemistries assays were included targeting selected loci. Real-time PCR chemistries were subsequently compared for their efficiency in PCR amplification and limits of detection and quantification. The overall applicability of the chemistries was evaluated, adding practicability and cost issues to the performance characteristics. None of the chemistries seemed to be significantly better than any other, but certain features favor LNA and MGB technology as good alternatives to TaqMan in quantification assays. SYBR green and molecular beacon assays can perform equally well but may need more optimization prior to use.

Potential biological targets for bioassay development in drug discovery of Sturge-Weber syndrome.

PubMed

Mohammadipanah, Fatemeh; Salimi, Fatemeh

2017-04-29

Sturge-Weber Syndrome (SWS) is among the neurocutaneous diseases, which has several clinical manifestations of ocular (glaucoma), cutaneous (port-wine stain), neurological (seizures) and vascular problems. Molecular mechanisms of SWS pathogenesis are initiated by the somatic mutation in GNAQ. Therefore, no definite treatments exist for the SWS and treatment options only mitigate the intensity of its clinical manifestations. Biological assay design for drug discovery against this syndrome demands comprehensive knowledge on mechanisms which are involved in its pathogenesis. By analysis of the interrelated molecular targets of SWS, some in vitro bioassay systems can be allotted for drug screening against this syndrome. Development of such platforms of bioassay can bring along the implementation of high throughput screening of natural or synthetic compounds in drug discovery programs. Regarding the fact that study of biological targets and their integration in biological assay design can facilitate the process of effective drug discovery; some potential biological targets and their respective biological assay for SWS drug discovery are propounded in this review. For this purpose, some biological targets for SWS drug discovery such as acetylcholine esterase, alkaline phosphatase, gamma-aminobutyricacidergic, Hypoxia-Inducible Factor (HIF) -1α and 2α are suggested. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Biological monitoring of 2,4,5-trichlorophenol (I): preparation of antibodies and development of an immunoassay using theoretical models.

PubMed

Nichkova, Mikaela; Galve, Roger; Marco, M-Pilar

2002-11-01

Antibodies against 2,4,5-trichlorophenol have been prepared after theoretical and molecular modeling chemical studies of three potential immunizing haptens with the aim to find out the one mimicking best the target analyte. Competitive direct and indirect ELISAs have been developed after screening a battery of haptenized enzyme tracers and coating antigens, respectively. The relation between the degree of heterology of the competitor and the resulting immunoassay detectability has been investigated according to the electronic similarities of the competitor haptens with the target analyte taking in consideration their pK(a) values. These studies have been performed using theoretical and molecular modeling tools to find out their electronic distribution at their minimum energetic levels. The results suggest that the competitors should have a high homology to produced assays with good detectability values. On the other hand detectability improves when lowering the hapten density of the competitors. An indirect competitive ELISA has been finally selected for further investigation. The immunoassay has an IC(50) value of 0.6 microg L(-)(1) and a limit of detection of 0.084 microg L(-)(1). The selectivity of the assay is high in relation to other chlorophenols frequently present in real samples. In contrast, the brominated analogues may also be recognized with this assay.

Standardized Methods to Generate Mock (Spiked) Clinical Specimens by Spiking Blood or Plasma with Cultured Pathogens

PubMed Central

Dong, Ming; Fisher, Carolyn; Añez, Germán; Rios, Maria; Nakhasi, Hira L.; Hobson, J. Peyton; Beanan, Maureen; Hockman, Donna; Grigorenko, Elena; Duncan, Robert

2016-01-01

Aims To demonstrate standardized methods for spiking pathogens into human matrices for evaluation and comparison among diagnostic platforms. Methods and Results This study presents detailed methods for spiking bacteria or protozoan parasites into whole blood and virus into plasma. Proper methods must start with a documented, reproducible pathogen source followed by steps that include standardized culture, preparation of cryopreserved aliquots, quantification of the aliquots by molecular methods, production of sufficient numbers of individual specimens and testing of the platform with multiple mock specimens. Results are presented following the described procedures that showed acceptable reproducibility comparing in-house real-time PCR assays to a commercially available multiplex molecular assay. Conclusions A step by step procedure has been described that can be followed by assay developers who are targeting low prevalence pathogens. Significance and Impact of Study The development of diagnostic platforms for detection of low prevalence pathogens such as biothreat or emerging agents is challenged by the lack of clinical specimens for performance evaluation. This deficit can be overcome using mock clinical specimens made by spiking cultured pathogens into human matrices. To facilitate evaluation and comparison among platforms, standardized methods must be followed in the preparation and application of spiked specimens. PMID:26835651

Development and characterization of monoclonal antibody to the lymphocystis disease virus of Japanese flounder Paralichthys olivaceus isolated from China.

PubMed

Cheng, Shunfeng; Zhan, Wenbin; Xing, Jing; Sheng, Xiuzhen

2006-08-01

Lymphocystis disease virus (LCDV) can infect, both naturally and experimentally, about 100 different teleost fish species. In this study, LCDV was purified using differential and gradient centrifugation from skin tumours of Japanese flounder Paralichthys olivaceus. A panel of five monoclonal antibodies (Mabs) against LCDV were produced by immunization of Balb/c mice with purified virus preparations. Analysed by the indirect enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), Western blot and immunogold electron microscopy (IEM), they showed specificity for LCDV. Immunofluorescent studies showed that the specific fluorescence signals appeared at the peripheral zone of hypertrophied cells cytoplasm where was the cytoplasmic inclusion bodies location and many of them formed ribbon-shaped. Western blot analysis demonstrated that two Mabs 1D7 and 2B6 reacted specifically to a single protein with an approximately molecular weight of 116kDa, Mab 3G3 reacted with two LCDV proteins at molecular mass of approximately 116 and 90kDa. Immunogold transmission electron-microscopy provided visualized evidence that the epitopes recognized by these Mabs were located on the outer surface of virions. The Mabs characterized should prove useful for developing LCDV diagnostic assays and for studying the biology of infection and pathogenesis of disease.

A human ether-á-go-go-related (hERG) ion channel atomistic model generated by long supercomputer molecular dynamics simulations and its use in predicting drug cardiotoxicity.

PubMed

Anwar-Mohamed, Anwar; Barakat, Khaled H; Bhat, Rakesh; Noskov, Sergei Y; Tyrrell, D Lorne; Tuszynski, Jack A; Houghton, Michael

2014-11-04

Acquired cardiac long QT syndrome (LQTS) is a frequent drug-induced toxic event that is often caused through blocking of the human ether-á-go-go-related (hERG) K(+) ion channel. This has led to the removal of several major drugs post-approval and is a frequent cause of termination of clinical trials. We report here a computational atomistic model derived using long molecular dynamics that allows sensitive prediction of hERG blockage. It identified drug-mediated hERG blocking activity of a test panel of 18 compounds with high sensitivity and specificity and was experimentally validated using hERG binding assays and patch clamp electrophysiological assays. The model discriminates between potent, weak, and non-hERG blockers and is superior to previous computational methods. This computational model serves as a powerful new tool to predict hERG blocking thus rendering drug development safer and more efficient. As an example, we show that a drug that was halted recently in clinical development because of severe cardiotoxicity is a potent inhibitor of hERG in two different biological assays which could have been predicted using our new computational model. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic laboratory setting

USGS Publications Warehouse

Purcell, Maureen K.; Getchell, Rodman G.; McClure, Carol A.; Weber, S.E.; Garver, Kyle A.

2011-01-01

Real-time, or quantitative, polymerase chain reaction (qPCR) is quickly supplanting other molecular methods for detecting the nucleic acids of human and other animal pathogens owing to the speed and robustness of the technology. As the aquatic animal health community moves toward implementing national diagnostic testing schemes, it will need to evaluate how qPCR technology should be employed. This review outlines the basic principles of qPCR technology, considerations for assay development, standards and controls, assay performance, diagnostic validation, implementation in the diagnostic laboratory, and quality assurance and control measures. These factors are fundamental for ensuring the validity of qPCR assay results obtained in the diagnostic laboratory setting.

Charge–transfer reaction of 2,3-dichloro-1,4-naphthoquinone with crizotinib: Spectrophotometric study, computational molecular modeling and use in development of microwell assay for crizotinib

PubMed Central

Alzoman, Nourah Z.; Alshehri, Jamilah M.; Darwish, Ibrahim A.; Khalil, Nasr Y.; Abdel-Rahman, Hamdy M.

2014-01-01

The reaction of 2,3-dichloro-1,4-naphthoquinone (DCNQ) with crizotinib (CZT; a novel drug used for treatment of non-small cell lung cancer) was investigated in different solvents of varying dielectric constants and polarity indexes. The reaction produced a red-colored product. Spectrophotometric investigations confirmed that the reaction proceeded through charge–transfer (CT) complex formation. The molar absorptivity of the complex was found to be linearly correlated with the dielectric constant and polarity index of the solvent; the correlation coefficients were 0.9567 and 0.9069, respectively. The stoichiometric ratio of DCNQ:CZT was found to be 2:1 and the association constant of the complex was found to be 1.07 × 102 l/mol. The kinetics of the reaction was studied; the order of the reaction, rate and rate constant were determined. Computational molecular modeling for the complex between DCNQ and CZT was conducted, the sites of interaction on CZT molecule were determined, and the mechanism of the reaction was postulated. The reaction was employed as a basis in the development of a novel 96-microwell assay for CZT in a linear range of 4–500 μg/ml. The assay limits of detection and quantitation were 2.06 and 6.23 μg/ml, respectively. The assay was validated as per the guidelines of the International Conference on Harmonization (ICH) and successfully applied to the analysis of CZT in its bulk and capsules with good accuracy and precision. The assay has high throughput and consumes a minimum volume of organic solvents thus it reduces the exposures of the analysts to the toxic effects of organic solvents, and significantly reduces the analysis cost. PMID:25685046

New Molecular Assay for the Proliferation Signature in Mantle Cell Lymphoma Applicable to Formalin-Fixed Paraffin-Embedded Biopsies

PubMed Central

Abrisqueta, Pau; Wright, George W.; Slack, Graham W.; Mottok, Anja; Villa, Diego; Jares, Pedro; Rauert-Wunderlich, Hilka; Royo, Cristina; Clot, Guillem; Pinyol, Magda; Boyle, Merrill; Chan, Fong Chun; Braziel, Rita M.; Chan, Wing C.; Weisenburger, Dennis D.; Cook, James R.; Greiner, Timothy C.; Fu, Kai; Ott, German; Delabie, Jan; Smeland, Erlend B.; Holte, Harald; Jaffe, Elaine S.; Steidl, Christian; Connors, Joseph M.; Gascoyne, Randy D.; Rosenwald, Andreas; Staudt, Louis M.; Campo, Elias; Rimsza, Lisa M.

2017-01-01

Purpose Mantle cell lymphoma is an aggressive B-cell neoplasm that displays heterogeneous outcomes after treatment. In 2003, the Lymphoma/Leukemia Molecular Profiling Project described a powerful biomarker—the proliferation signature—using gene expression in fresh frozen material. Herein, we describe the training and validation of a new assay that measures the proliferation signature in RNA derived from routinely available formalin-fixed paraffin-embedded (FFPE) biopsies. Methods Forty-seven FFPE biopsies were used to train an assay on the NanoString platform, using microarray gene expression data of matched fresh frozen biopsies as a gold standard. The locked assay was applied to pretreatment FFPE lymph node biopsies from an independent cohort of 110 patients uniformly treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone. Seventeen biopsies were tested across three laboratories to assess assay reproducibility. Results The MCL35 assay, which contained a 17-gene proliferation signature, yielded gene expression of sufficient quality to assign an assay score and risk group in 108 (98%) of 110 archival FFPE biopsies. The MCL35 assay assigned patients to high-risk (26%), standard-risk (29%), and low-risk (45%) groups, with different lengths of overall survival (OS): a median of 1.1, 2.6, and 8.6 years, respectively (log-rank for trend, P < .001). In multivariable analysis, these risk groups and the Mantle Cell Lymphoma International Prognostic Index were independently associated with OS (P < .001 for both variables). Concordance of risk assignment across the three independent laboratories was 100%. Conclusion The newly developed and validated MCL35 assay for FFPE biopsies uses the proliferation signature to define groups of patients with significantly different OS independent of the Mantle Cell Lymphoma International Prognostic Index. Importantly, the analytic and clinical validity of this assay defines it as a reliable biomarker to support risk-adapted clinical trials. PMID:28291392

Molecular typing of Salmonella enterica serovar typhi isolates from various countries in Asia by a multiplex PCR assay on variable-number tandem repeats.

PubMed

Liu, Yichun; Lee, May-Ann; Ooi, Eng-Eong; Mavis, Yeo; Tan, Ai-Ling; Quek, Hung-Hiang

2003-09-01

A multiplex PCR method incorporating primers flanking three variable-number tandem repeat (VNTR) loci (arbitrarily labeled TR1, TR2, and TR3) in the CT18 strain of Salmonella enterica serovar Typhi has been developed for molecular typing of S. enterica serovar Typhi clinical isolates from several Asian countries, including Singapore, Indonesia, India, Bangladesh, Malaysia, and Nepal. We have demonstrated that the multiplex PCR could be performed on crude cell lysates and that the VNTR banding profiles produced could be easily analyzed by visual inspection after conventional agarose gel electrophoresis. The assay was highly discriminative in identifying 49 distinct VNTR profiles among 59 individual isolates. A high level of VNTR profile heterogeneity was observed in isolates from within the same country and among countries. These VNTR profiles remained stable after the strains were passaged extensively under routine laboratory culture conditions. In contrast to the S. enterica serovar Typhi isolates, an absence of TR3 amplicons and a lack of length polymorphisms in TR1 and TR2 amplicons were observed for other S. enterica serovars, such as Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Paratyphi A, B, and C. DNA sequencing of the amplified VNTR regions substantiated these results, suggesting the high stability of the multiplex PCR assay. The multiplex-PCR-based VNTR profiling developed in this study provides a simple, rapid, reproducible, and high-resolution molecular tool for the epidemiological analysis of S. enterica serovar Typhi strains.

C/EBPβ Promotes STAT3 Expression and Affects Cell Apoptosis and Proliferation in Porcine Ovarian Granulosa Cells.

PubMed

Yuan, Xiaolong; Zhou, Xiaofeng; He, Yingting; Zhong, Yuyi; Zhang, Ailing; Zhang, Zhe; Zhang, Hao; Li, Jiaqi

2018-06-13

Previous studies suggest that signal transducer and activator of transcription 3 (STAT3) and CCAAT/enhancer binding protein beta (C/EBPβ) play an essential role in ovarian granulosa cells (GCs) for mammalian follicular development. Several C/EBPβ putative binding sites were previously predicted on the STAT3 promoter in mammals. However, the molecular regulation of C/EBPβ on STAT3 and their effects on cell proliferation and apoptosis remain virtually unexplored in GCs. Using porcine GCs as a model, the 5′-deletion, luciferase report assay, mutation, chromatin immunoprecipitation, Annexin-V/PI staining and EdU assays were applied to investigate the molecular mechanism for C/EBPβ regulating the expression of STAT3 and their effects on the cell proliferation and apoptosis ability. We found that over and interfering with the expression of C/EBPβ significantly increased and decreased the messenger RNA (mRNA) and protein levels of STAT3 , respectively. The dual luciferase reporter assay showed that C/EBPβ directly bound at −1397/−1387 of STAT3 to positively regulate the mRNA and protein expressions of STAT3 . Both C/EBPβ and STAT3 were observed to inhibit cell apoptosis and promote cell proliferation. Furthermore, C/EBPβ might enhance the antiapoptotic and pro-proliferative effects of STAT3 . These results would be of great insight in further exploring the molecular mechanism of C/EBPβ and STAT3 on the function of GCs and the development of ovarian follicles in mammals.

POLYCLONAL ANTISERA AGAINST ESTUARINE CRUSTACEAN VITELLINS: A MOLECULAR APPROACH TO REPRODUCTIVE ENDOCRINOLOGY AND TOXICOLOGY

EPA Science Inventory

To fully elucidate the action of crustacean hormones, or their agonists, on reproduction and vitellogenesis it has become increasingly important to develop sensitive assays that indicate a stimulatory or inhibitory effect on easily measured endpoints. Because of the relative abun...

POLYCLONAL ANTISERA AGAINST ESTUARINE CRUSTACEAN VITELLINS: A MOLECULAR APPROACH TO REPRODUCTIVE ENDOCRINOLOGY AND TOXICOLOGY.

EPA Science Inventory

To fully elucidate the action of crustacean hormones, or their agonists, on vitellogenesis and reproduction, it has become increasingly important to develop sensitive assays that indicate a stimulatory or inhibitory effect on easily measured endpoints. Because of the relative ab...

A REACTIVITY PATTERN OF DISCRIMINATION OF ER AGONISM AND ANTAGONISM BASED ON 3-D MOLECULAR ATTRIBUTES

EPA Science Inventory

Various models have been developed to predict the relative binding affinity (RBA) of chemicals to estrogen receptors (ER). These models are important for prioritizing chemicals for screening in biological assays assessing the potential for endocrine disruption. One shortcoming of...

LAMPhimerus: A novel LAMP assay for detecting Amphimerus sp. DNA in human stool samples

PubMed Central

Calvopiña, Manuel; Fontecha-Cuenca, Cristina; Sugiyama, Hiromu; Sato, Megumi; López Abán, Julio; Vicente, Belén; Muro, Antonio

2017-01-01

Background Amphimeriasis is a fish-borne disease caused by the liver fluke Amphimerus spp. that has recently been reported as endemic in the tropical Pacific side of Ecuador with a high prevalence in humans and domestic animals. The diagnosis is based on the stool examination to identify parasite eggs, but it lacks sensitivity. Additionally, the morphology of the eggs may be confounded with other liver and intestinal flukes. No immunological or molecular methods have been developed to date. New diagnostic techniques for specific and sensitive detection of Amphimerus spp. DNA in clinical samples are needed. Methodology/Principal findings A LAMP targeting a sequence of the Amphimerus sp. internal transcribed spacer 2 region was designed. Amphimerus sp. DNA was obtained from adult worms recovered from animals and used to optimize the molecular assays. Conventional PCR was performed using outer primers F3-B3 to verify the proper amplification of the Amphimerus sp. DNA target sequence. LAMP was optimized using different reaction mixtures and temperatures, and it was finally set up as LAMPhimerus. The specificity and sensitivity of both PCR and LAMP were evaluated. The detection limit was 1 pg of genomic DNA. Field testing was done using 44 human stool samples collected from localities where fluke is endemic. Twenty-five samples were microscopy positive for Amphimerus sp. eggs detection. In molecular testing, PCR F3-B3 was ineffective when DNA from fecal samples was used. When testing all human stool samples included in our study, the diagnostic parameters for the sensitivity and specificity were calculated for our LAMPhimerus assay, which were 76.67% and 80.77%, respectively. Conclusions/Significance We have developed and evaluated, for the first time, a specific and sensitive LAMP assay for detecting Amphimerus sp. in human stool samples. The procedure has been named LAMPhimerus method and has the potential to be adapted for field diagnosis and disease surveillance in amphimeriasis-endemic areas. Future large-scale studies will assess the applicability of this novel LAMP assay. PMID:28628614

Optical Traps to Study Properties of Molecular Motors

PubMed Central

Spudich, James A.; Rice, Sarah E.; Rock, Ronald S.; Purcell, Thomas J.; Warrick, Hans M.

2016-01-01

In vitro motility assays enabled the analysis of coupling between ATP hydrolysis and movement of myosin along actin filaments or kinesin along microtubules. Single-molecule assays using laser trapping have been used to obtain more detailed information about kinesins, myosins, and processive DNA enzymes. The combination of in vitro motility assays with laser-trap measurements has revealed detailed dynamic structural changes associated with the ATPase cycle. This article describes the use of optical traps to study processive and nonprocessive molecular motor proteins, focusing on the design of the instrument and the assays to characterize motility. PMID:22046048

Using yeast to determine the functional consequences of mutations in the human p53 tumor suppressor gene: An introductory course-based undergraduate research experience in molecular and cell biology.

PubMed

Hekmat-Scafe, Daria S; Brownell, Sara E; Seawell, Patricia Chandler; Malladi, Shyamala; Imam, Jamie F Conklin; Singla, Veena; Bradon, Nicole; Cyert, Martha S; Stearns, Tim

2017-03-04

The opportunity to engage in scientific research is an important, but often neglected, component of undergraduate training in biology. We describe the curriculum for an innovative, course-based undergraduate research experience (CURE) appropriate for a large, introductory cell and molecular biology laboratory class that leverages students' high level of interest in cancer. The course is highly collaborative and emphasizes the analysis and interpretation of original scientific data. During the course, students work in teams to characterize a collection of mutations in the human p53 tumor suppressor gene via expression and analysis in yeast. Initially, student pairs use both qualitative and quantitative assays to assess the ability of their p53 mutant to activate expression of reporter genes, and they localize their mutation within the p53 structure. Through facilitated discussion, students suggest possible molecular explanations for the transactivation defects displayed by their p53 mutants and propose experiments to test these hypotheses that they execute during the second part of the course. They use a western blot to determine whether mutant p53 levels are reduced, a DNA-binding assay to test whether recognition of any of three p53 target sequences is compromised, and fluorescence microscopy to assay nuclear localization. Students studying the same p53 mutant periodically convene to discuss and interpret their combined data. The course culminates in a poster session during which students present their findings to peers, instructors, and the greater biosciences community. Based on our experience, we provide recommendations for the development of similar large introductory lab courses. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(2):161-178, 2017. © 2016 The International Union of Biochemistry and Molecular Biology.

Measurement issues associated with quantitative molecular biology analysis of complex food matrices for the detection of food fraud.

PubMed

Burns, Malcolm; Wiseman, Gordon; Knight, Angus; Bramley, Peter; Foster, Lucy; Rollinson, Sophie; Damant, Andrew; Primrose, Sandy

2016-01-07

Following a report on a significant amount of horse DNA being detected in a beef burger product on sale to the public at a UK supermarket in early 2013, the Elliott report was published in 2014 and contained a list of recommendations for helping ensure food integrity. One of the recommendations included improving laboratory testing capacity and capability to ensure a harmonised approach for testing for food authenticity. Molecular biologists have developed exquisitely sensitive methods based on the polymerase chain reaction (PCR) or mass spectrometry for detecting the presence of particular nucleic acid or peptide/protein sequences. These methods have been shown to be specific and sensitive in terms of lower limits of applicability, but they are largely qualitative in nature. Historically, the conversion of these qualitative techniques into reliable quantitative methods has been beset with problems even when used on relatively simple sample matrices. When the methods are applied to complex sample matrices, as found in many foods, the problems are magnified resulting in a high measurement uncertainty associated with the result which may mean that the assay is not fit for purpose. However, recent advances in the technology and the understanding of molecular biology approaches have further given rise to the re-assessment of these methods for their quantitative potential. This review focuses on important issues for consideration when validating a molecular biology assay and the various factors that can impact on the measurement uncertainty of a result associated with molecular biology approaches used in detection of food fraud, with a particular focus on quantitative PCR-based and proteomics assays.

Development and application of a universal Hemoplasma screening assay based on the SYBR green PCR principle.

PubMed

Willi, Barbara; Meli, Marina L; Lüthy, Ruedi; Honegger, Hanspeter; Wengi, Nicole; Hoelzle, Ludwig E; Reusch, Claudia E; Lutz, Hans; Hofmann-Lehmann, Regina

2009-12-01

Hemotropic mycoplasmas (hemoplasmas) are the causative agents of infectious anemia in several mammalian species. Their zoonotic potential has recently been substantiated by the identification of a feline hemoplasma isolate in an immunocompromised human patient. Although species-specific diagnostic molecular methods have been developed, their application as screening tools is limited due to the species diversity of hemoplasmas. The goals of this study were to develop a universal hemoplasma screening assay with broad specificity based on the SYBR green PCR principle, to compare the assay with hemoplasma-specific TaqMan PCR, and to analyze potential tick vectors and human blood samples to address the zoonotic potential. The newly developed PCR assay based on the 16S rRNA gene amplified feline, canine, bovine, porcine, camelid, and murine hemoplasmas, as well as Mycoplasma penetrans and Mycoplasma pneumoniae. The lower detection limit for feline and canine hemoplasmas was 1 to 10 copies/PCR. The assay exhibited 98.2% diagnostic sensitivity and 92.1% diagnostic specificity for feline hemoplasmas. All 1,950 Ixodes ticks were PCR negative, suggesting that Ixodes ticks are not relevant vectors for the above-mentioned hemoplasma species in Switzerland. None of the 414 blood samples derived from anemic or immunocompromised human patients revealed a clear positive result. The SYBR green PCR assay described here is a suitable tool to screen for known and so-far-undiscovered hemoplasma species. Positive results should be confirmed by specific TaqMan PCR or sequencing.

Slow Off-Rate Modified Aptamer (SOMAmer) as a Novel Reagent in Immunoassay Development for Accurate Soluble Glypican-3 Quantification in Clinical Samples.

PubMed

Duo, Jia; Chiriac, Camelia; Huang, Richard Y-C; Mehl, John; Chen, Guodong; Tymiak, Adrienne; Sabbatini, Peter; Pillutla, Renuka; Zhang, Yan

2018-04-17

Accurate quantification of soluble glypican-3 in clinical samples using immunoassays is challenging, because of the lack of appropriate antibody reagents to provide a full spectrum measurement of all potential soluble glypican-3 fragments in vivo. Glypican-3 SOMAmer (slow off-rate modified aptamer) is a novel reagent that binds, with high affinity, to a far distinct epitope of glypican-3, when compared to all available antibody reagents generated in-house. This paper describes an integrated analytical approach to rational selection of key reagents based on molecular characterization by epitope mapping, with the focus on our work using a SOMAmer as a new reagent to address development challenges with traditional antibody reagents for the soluble glypican-3 immunoassay. A qualified SOMAmer-based assay was developed and used for soluble glypican-3 quantification in hepatocellular carcinoma (HCC) patient samples. The assay demonstrated good sensitivity, accuracy, and precision. Data correlated with those obtained using the traditional antibody-based assay were used to confirm the clinically relevant soluble glypican-3 forms in vivo. This result was reinforced by a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay quantifying signature peptides generated from trypsin digestion. The work presented here offers an integrated strategy for qualifying aptamers as an alternative affinity platform for immunoassay reagents that can enable speedy assay development, especially when traditional antibody reagents cannot meet assay requirements.

Molecular Approaches to Taenia asiatica

PubMed Central

Jeon, Hyeong-Kyu

2013-01-01

Taenia solium, T. saginata, and T. asiatica are taeniid tapeworms that cause taeniasis in humans and cysticercosis in intermediate host animals. Taeniases remain an important public health concerns in the world. Molecular diagnostic methods using PCR assays have been developed for rapid and accurate detection of human infecting taeniid tapeworms, including the use of sequence-specific DNA probes, PCR-RFLP, and multiplex PCR. More recently, DNA diagnosis using PCR based on histopathological specimens such as 10% formalin-fixed paraffin-embedded and stained sections mounted on slides has been applied to cestode infections. The mitochondrial gene sequence is believed to be a very useful molecular marker for not only studying evolutionary relationships among distantly related taxa, but also for investigating the phylo-biogeography of closely related species. The complete sequence of the human Taenia tapeworms mitochondrial genomes were determined, and its organization and structure were compared to other human-tropic Taenia tapeworms for which complete mitochondrial sequence data were available. The multiplex PCR assay with the Ta4978F, Ts5058F, Tso7421F, and Rev7915 primers will be useful for differential diagnosis, molecular characterization, and epidemiological surveys of human Taenia tapeworms. PMID:23467738

Molecular Diversity of Bacteroidales in Fecal and Environmental Samples and Swine-Associated Subpopulations

EPA Science Inventory

Several swine-specific microbial source tracking methods are based on PCR assays targeting Bacteroidales 16S rRNA gene sequences. The limited application of these assays can be explained by the poor understanding of their molecular diversity in fecal sources and environmental wat...

Development of real-time recombinase polymerase amplification assay for rapid and sensitive detection of canine parvovirus 2.

PubMed

Geng, Yunyun; Wang, Jianchang; Liu, Libing; Lu, Yan; Tan, Ke; Chang, Yan-Zhong

2017-11-06

Canine parvovirus 2, a linear single-stranded DNA virus belonging to the genus Parvovirus within the family Parvoviridae, is a highly contagious pathogen of domestic dogs and several wild canidae species. Early detection of canine parvovirus (CPV-2) is crucial to initiating appropriate outbreak control strategies. Recombinase polymerase amplification (RPA), a novel isothermal gene amplification technique, has been developed for the molecular detection of diverse pathogens. In this study, a real-time RPA assay was developed for the detection of CPV-2 using primers and an exo probe targeting the CPV-2 nucleocapsid protein gene. The real-time RPA assay was performed successfully at 38 °C, and the results were obtained within 4-12 min for 10 5 -10 1 molecules of template DNA. The assay only detected CPV-2, and did not show cross-detection of other viral pathogens, demonstrating a high level of specificity. The analytical sensitivity of the real-time RPA was 10 1 copies/reaction of a standard DNA template, which was 10 times more sensitive than the common RPA method. The clinical sensitivity of the real-time RPA assay matched 100% (n = 91) to the real-time PCR results. The real-time RPA assay is a simple, rapid, reliable and affordable method that can potentially be applied for the detection of CPV-2 in the research laboratory and point-of-care diagnosis.

Analytical applications of MIPs in diagnostic assays: future perspectives.

PubMed

Bedwell, Thomas S; Whitcombe, Michael J

2016-03-01

Many efforts have been made to produce artificial materials with biomimetic properties for applications in binding assays. Among these efforts, the technique of molecular imprinting has received much attention because of the high selectivity obtainable for molecules of interest, robustness of the produced polymers, simple and short synthesis, and excellent cost efficiency. In this review, progress in the field of molecularly imprinted sorbent assays is discussed-with a focus on work conducted from 2005 to date.

Development and validation of sensitive LC-MS/MS assays for quantification of HP-β-CD in human plasma and CSF.

PubMed

Jiang, Hui; Sidhu, Rohini; Fujiwara, Hideji; De Meulder, Marc; de Vries, Ronald; Gong, Yong; Kao, Mark; Porter, Forbes D; Yanjanin, Nicole M; Carillo-Carasco, Nuria; Xu, Xin; Ottinger, Elizabeth; Woolery, Myra; Ory, Daniel S; Jiang, Xuntian

2014-07-01

2-Hydroxypropyl-β-cyclodextrin (HP-β-CD), a widely used excipient for drug formulation, has emerged as an investigational new drug for the treatment of Niemann-Pick type C1 (NPC1) disease, a neurodegenerative cholesterol storage disorder. Development of a sensitive quantitative LC-MS/MS assay to monitor the pharmacokinetics (PKs) of HP-β-CD required for clinical trials has been challenging owing to the dispersity of the HP-β-CD. To support a phase 1 clinical trial for ICV delivery of HP-β-CD in NPC1 patients, novel methods for quantification of HP-β-CD in human plasma and cerebrospinal fluid (CSF) using LC-MS/MS were developed and validated: a 2D-LC-in-source fragmentation-MS/MS (2D-LC-IF-MS/MS) assay and a reversed phase ultra performance LC-MS/MS (RP-UPLC-MS/MS) assay. In both assays, protein precipitation and "dilute and shoot" procedures were used to process plasma and CSF, respectively. The assays were fully validated and in close agreement, and allowed determination of PK parameters for HP-β-CD. The LC-MS/MS methods are ∼100-fold more sensitive than the current HPLC assay, and were successfully employed to analyze HP-β-CD in human plasma and CSF samples to support the phase 1 clinical trial of HP-β-CD in NPC1 patients. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

Loop-mediated isothermal amplification (LAMP) assays for the species-specific detection of Eimeria that infect chickens

PubMed Central

2011-01-01

Background Eimeria parasites can cause the disease coccidiosis in poultry and even subclinical infection can incur economic loss. Diagnosis of infection predominantly relies on traditional techniques including lesion scoring and faecal microscopy despite the availability of sensitive molecular assays, largely due to cost and the requirement for specialist equipment. Despite longstanding proven efficacy these traditional techniques demand time and expertise, can be highly subjective and may under-diagnose subclinical disease. Recognition of the tight economic margins prevailing in modern poultry production and the impact of avian coccidiosis on poverty in many parts of the world has highlighted a requirement for a panel of straightforward and sensitive, but cost-effective, Eimeria species-specific diagnostic assays. Results Loop-mediated isothermal amplification (LAMP) is an uncomplicated, quick and relatively inexpensive diagnostic tool. In this study we have developed a panel of species-specific LAMP assays targeting the seven Eimeria species that infect the chicken. Each assay has been shown to be genuinely species-specific with the capacity to detect between one and ten eimerian genomes, equivalent to less than a single mature schizont. Development of a simple protocol for template DNA preparation from tissue collected post mortem with no requirement for specialist laboratory equipment supports the use of these assays in routine diagnosis of eimerian infection. Preliminary field testing supports this hypothesis. Conclusions Development of a panel of sensitive species-specific LAMP assays introduces a valuable new cost-effective tool for use in poultry husbandry. PMID:22053893

Characterization of vitellogenin gene expression in round goby (Neogobius melanostomus) using a quantitative polymerase chain reaction assay.

PubMed

Bowley, Lucas A; Alam, Farhana; Marentette, Julie R; Balshine, Sigal; Wilson, Joanna Y

2010-12-01

A growing concern over endocrine disruption in aquatic species has prompted the development of molecular assays to monitor environmental impacts. This study describes the development of quantitative polymerase chain reaction (qPCR) assays to characterize the expression of two vitellogenin (Vtg) genes in the invasive round goby (Neogobius melanostomus). Fragments from the 18SrRNA (housekeeping gene), Vtg II, and Vtg III genes were cloned and sequenced. The qPCR assays were developed to detect hepatic Vtg expression in goby. The assays detected induction of both Vtg genes in nonreproductive males following a two-week laboratory exposure to 17β-estradiol (≥1 mg/kg i.p. injection). The assays were applied to goby from Hamilton Harbour, Lake Ontario (Canada), including those from sites where feminization and intersex of goby has been documented. Both Vtg genes had significantly higher expression in females compared to males. Male reproductive goby adopt either parental or sneaker tactics; Vtg II expression was higher in sneaker than in parental males but parental and nonreproductive males did not differ from each other. The Vtg III expression was significantly higher in sneaker males followed by parental males and nonreproductive males, respectively. The Vtg II and III expression in nonreproductive males was elevated in the contaminated site with documented intersex. This assay provides an important tool for the use of an invasive species in monitoring endocrine disruption in the Great Lakes region. Copyright © 2010 SETAC.

A comprehensive pharmacokinetic/pharmacodynamics analysis of the novel IGF1R/INSR inhibitor BI 893923 applying in vitro, in vivo and in silico modeling techniques.

PubMed

Titze, Melanie I; Schaaf, Otmar; Hofmann, Marco H; Sanderson, Michael P; Zahn, Stephan K; Quant, Jens; Lehr, Thorsten

2016-06-01

BI 893923 is a novel IGF1R/INSR tyrosine kinase inhibitor demonstrating anti-tumor efficacy and good tolerability. We aimed to characterize the relationship between BI 893923 plasma concentration, tumor biomarker modulation, tumor growth and hyperglycemia in mice using in silico modeling analyses. In vitro molecular and cellular assays were used to demonstrate the potency and selectivity of BI 893923. Diverse in vitro DMPK assays were used to characterize the compound's drug-like properties. Mice xenografted with human GEO tumors were treated with different doses of BI 893923 to demonstrate the compound's efficacy, biomarker modulation and tolerability. PK/PD analyses were performed using nonlinear mixed-effects modeling. BI 893923 demonstrated potent and selective molecular inhibition of the IGF1R and INSR and demonstrated attractive drug-like properties (permeability, bioavailability). BI 893923 dose-dependently reduced GEO tumor growth and demonstrated good tolerability, characterized by transient hyperglycemia and normal body weight gain. A population PK/PD model was developed, which established relationships between BI 893923 pharmacokinetics, hyperglycemia, pIGF1R reduction and tumor growth. BI 893923 demonstrates molecular properties consistent with a highly attractive inhibitor of the IGF1R/INSR. A generic PK/PD model was developed to support preclinical drug development and dose finding in mice.

Crizotinib for the Treatment of ALK-Rearranged Non-Small Cell Lung Cancer: A Success Story to Usher in the Second Decade of Molecular Targeted Therapy in Oncology

PubMed Central

Bartlett, Cynthia Huang; Mino-Kenudson, Mari; Cui, Jean; Iafrate, A. John

2012-01-01

Crizotinib, an ALK/MET/ROS1 inhibitor, was approved by the U.S. Food and Drug Administration for the treatment of anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC) in August 2011, merely 4 years after the first publication of ALK-rearranged NSCLC. The crizotinib approval was accompanied by the simultaneous approval of an ALK companion diagnostic fluorescent in situ hybridization assay for the detection of ALK-rearranged NSCLC. Crizotinib continued to be developed as an ALK and MET inhibitor in other tumor types driven by alteration in ALK and MET. Crizotinib has recently been shown to be an effective ROS1 inhibitor in ROS1-rearranged NSCLC, with potential future clinical applications in ROS1-rearranged tumors. Here we summarize the heterogeneity within the ALK- and ROS1-rearranged molecular subtypes of NSCLC. We review the past and future clinical development of crizotinib for ALK-rearranged NSCLC and the diagnostic assays to detect ALK-rearranged NSCLC. We highlight how the success of crizotinib has changed the paradigm of future drug development for targeted therapies by targeting a molecular-defined subtype of NSCLC despite its rarity and affected the practice of personalized medicine in oncology, emphasizing close collaboration between clinical oncologists, pathologists, and translational scientists. PMID:22989574

Method: a single nucleotide polymorphism genotyping method for Wheat streak mosaic virus.

PubMed

Rogers, Stephanie M; Payton, Mark; Allen, Robert W; Melcher, Ulrich; Carver, Jesse; Fletcher, Jacqueline

2012-05-17

The September 11, 2001 attacks on the World Trade Center and the Pentagon increased the concern about the potential for terrorist attacks on many vulnerable sectors of the US, including agriculture. The concentrated nature of crops, easily obtainable biological agents, and highly detrimental impacts make agroterrorism a potential threat. Although procedures for an effective criminal investigation and attribution following such an attack are available, important enhancements are still needed, one of which is the capability for fine discrimination among pathogen strains. The purpose of this study was to develop a molecular typing assay for use in a forensic investigation, using Wheat streak mosaic virus (WSMV) as a model plant virus. This genotyping technique utilizes single base primer extension to generate a genetic fingerprint. Fifteen single nucleotide polymorphisms (SNPs) within the coat protein and helper component-protease genes were selected as the genetic markers for this assay. Assay optimization and sensitivity testing was conducted using synthetic targets. WSMV strains and field isolates were collected from regions around the world and used to evaluate the assay for discrimination. The assay specificity was tested against a panel of near-neighbors consisting of genetic and environmental near-neighbors. Each WSMV strain or field isolate tested produced a unique SNP fingerprint, with the exception of three isolates collected within the same geographic location that produced indistinguishable fingerprints. The results were consistent among replicates, demonstrating the reproducibility of the assay. No SNP fingerprints were generated from organisms included in the near-neighbor panel, suggesting the assay is specific for WSMV. Using synthetic targets, a complete profile could be generated from as low as 7.15 fmoles of cDNA. The molecular typing method presented is one tool that could be incorporated into the forensic science tool box after a thorough validation study. This method incorporates molecular biology techniques that are already well established in research and diagnostic laboratories, allowing for an easy introduction of this method into existing laboratories. single nucleotide polymorphisms, genotyping, plant pathology, viruses, microbial forensics, Single base primer extension, SNaPshot Multiplex Kit.

Method: a single nucleotide polymorphism genotyping method for Wheat streak mosaic virus

PubMed Central

2012-01-01

Background The September 11, 2001 attacks on the World Trade Center and the Pentagon increased the concern about the potential for terrorist attacks on many vulnerable sectors of the US, including agriculture. The concentrated nature of crops, easily obtainable biological agents, and highly detrimental impacts make agroterrorism a potential threat. Although procedures for an effective criminal investigation and attribution following such an attack are available, important enhancements are still needed, one of which is the capability for fine discrimination among pathogen strains. The purpose of this study was to develop a molecular typing assay for use in a forensic investigation, using Wheat streak mosaic virus (WSMV) as a model plant virus. Method This genotyping technique utilizes single base primer extension to generate a genetic fingerprint. Fifteen single nucleotide polymorphisms (SNPs) within the coat protein and helper component-protease genes were selected as the genetic markers for this assay. Assay optimization and sensitivity testing was conducted using synthetic targets. WSMV strains and field isolates were collected from regions around the world and used to evaluate the assay for discrimination. The assay specificity was tested against a panel of near-neighbors consisting of genetic and environmental near-neighbors. Result Each WSMV strain or field isolate tested produced a unique SNP fingerprint, with the exception of three isolates collected within the same geographic location that produced indistinguishable fingerprints. The results were consistent among replicates, demonstrating the reproducibility of the assay. No SNP fingerprints were generated from organisms included in the near-neighbor panel, suggesting the assay is specific for WSMV. Using synthetic targets, a complete profile could be generated from as low as 7.15 fmoles of cDNA. Conclusion The molecular typing method presented is one tool that could be incorporated into the forensic science tool box after a thorough validation study. This method incorporates molecular biology techniques that are already well established in research and diagnostic laboratories, allowing for an easy introduction of this method into existing laboratories. Keywords: single nucleotide polymorphisms, genotyping, plant pathology, viruses, microbial forensics, Single base primer extension, SNaPshot Multiplex Kit PMID:22594601

Molecularly imprinted polymer nanoparticle-based assay (MINA): application for fumonisin B1 determination.

PubMed

Munawar, Hasim; Smolinska-Kempisty, Katarzyna; Cruz, Alvaro Garcia; Canfarotta, Francesco; Piletska, Elena; Karim, Khalku; Piletsky, Sergey A

2018-06-20

The enzyme-linked immunosorbent assay (ELISA) has been used as a standard tool for monitoring food and animal feed contamination from the carcinogenic fumonisin B1 (FB1). Unfortunately, ELISA is not always efficient due to the instability of the antibody and enzyme components in the immunoassay, the presence of natural enzyme inhibitors in the samples and the high levels of non-specific protein binding. Additionally, the production of antibodies for ELISA can be time-consuming and costly, due to the involvement of animals in the manufacturing process. To overcome these limiting factors, a molecularly imprinted nanoparticle based assay (MINA) has been developed, where the molecularly imprinted nanoparticles (nanoMIPs) replace the primary antibody used in a competitive ELISA. Herein, computational modelling was used to design the nanoMIPs by selecting monomers that specifically interact with FB1. The affinity of the monomers to FB1 was verified by measuring their binding in affinity chromatography experiments. The nanoMIPs were produced by solid phase synthesis and the results showed that nanoMIPs had a hydrodynamic diameter of around 249 ± 29 nm. The assay tested in model samples is highly selective and does not show cross-reactivity with other mycotoxins such as fumonisin B2 (FB2), aflatoxin B1 (AFB1), citrinin (CTT), zearalenone (ZEA), and deoxynivalenol (DON). The MINA allows the detection of FB1 in the concentration range of 10 pM-10 nM with a detection limit of 1.9 pM and a recovery of 108.13-113.76%.

Lung Adenocarcinoma with Anaplastic Lymphoma Kinase (ALK) Rearrangement Presenting as Carcinoma of Unknown Primary Site: Recognition and Treatment Implications.

PubMed

Hainsworth, John D; Anthony Greco, F

Molecular cancer classifier assays are being used with increasing frequency to predict tissue of origin and direct site-specific therapy for patients with carcinoma of unknown primary site (CUP). We postulated some CUP patients predicted to have non-small-cell lung cancer (NSCLC) by molecular cancer classifier assay may have anaplastic lymphoma kinase (ALK) rearranged tumors, and benefit from treatment with ALK inhibitors. We retrospectively reviewed CUP patients who had the 92-gene molecular cancer classifier assay (CancerTYPE ID; bioTheranostics, Inc.) performed on tumor biopsies to identify patients predicted to have NSCLC. Beginning in 2011, we have tested these patients for ALK rearrangements and epidermal growth factor receptor (EGFR) activating mutations, based on the proven therapeutic value of these targets in NSCLC. We identified CUP patients with predicted NSCLC who were subsequently found to have ALK rearrangements. NSCLC was predicted by the molecular cancer classifier assay in 37 of 310 CUP patients. Twenty-one of these patients were tested for ALK rearrangements, and four had an EML4-ALK fusion gene detected. The diagnosis of lung cancer was strongly suggested in only one patient prior to molecular testing. One patient received ALK inhibitor treatment and has had prolonged benefit. We report on patients with lung adenocarcinoma and ALK rearrangements originally diagnosed as CUP who were identified using a molecular cancer classifier assay. Although ALK inhibitors treatment experience is limited, this newly identifiable group of lung cancer patients should be considered for therapy according to guidelines for stage IV ALK-positive NSCLC.

Lung Adenocarcinoma with Anaplastic Lymphoma Kinase (ALK) Rearrangement Presenting as Carcinoma of Unknown Primary Site: Recognition and Treatment Implications.

PubMed

Hainsworth, John D; Anthony Greco, F

2016-03-01

Molecular cancer classifier assays are being used with increasing frequency to predict tissue of origin and direct site-specific therapy for patients with carcinoma of unknown primary site (CUP). We postulated some CUP patients predicted to have non-small-cell lung cancer (NSCLC) by molecular cancer classifier assay may have anaplastic lymphoma kinase (ALK) rearranged tumors, and benefit from treatment with ALK inhibitors. We retrospectively reviewed CUP patients who had the 92-gene molecular cancer classifier assay (CancerTYPE ID; bioTheranostics, Inc.) performed on tumor biopsies to identify patients predicted to have NSCLC. Beginning in 2011, we have tested these patients for ALK rearrangements and epidermal growth factor receptor (EGFR) activating mutations, based on the proven therapeutic value of these targets in NSCLC. We identified CUP patients with predicted NSCLC who were subsequently found to have ALK rearrangements. NSCLC was predicted by the molecular cancer classifier assay in 37 of 310 CUP patients. Twenty-one of these patients were tested for ALK rearrangements, and four had an EML4-ALK fusion gene detected. The diagnosis of lung cancer was strongly suggested in only one patient prior to molecular testing. One patient received ALK inhibitor treatment and has had prolonged benefit. We report on patients with lung adenocarcinoma and ALK rearrangements originally diagnosed as CUP who were identified using a molecular cancer classifier assay. Although ALK inhibitors treatment experience is limited, this newly identifiable group of lung cancer patients should be considered for therapy according to guidelines for stage IV ALK-positive NSCLC.

Electrochemical detection of microRNAs via gap hybridization assay.

PubMed

Pöhlmann, Christopher; Sprinzl, Mathias

2010-06-01

MicroRNAs have recently been associated with cancer development by acting as tumor suppressors or oncogenes and could therefore be applied as molecular markers for early diagnosis of cancer. In this work, we established a rapid, selective, and sensitive gap hybridization assay for detection of mature microRNAs based on four components DNA/RNA hybridization and electrochemical detection using esterase 2-oligodeoxynucleotide conjugates. Complementary binding of microRNA to a gap built of capture and detector oligodeoxynucleotide, the reporter enzyme is brought to the vicinity of the electrode and produces enzymatically an electrochemical signal. In the absence of microRNA, the gap between capture and detector oligodeoxynucleotide is not filled, and missing base stacking energy destabilizes the hybridization complex. The gap hybridization assay demonstrates selective detection of miR-16 within a mixture of other miRNAs, including the feasibility of single mismatch discrimination. Applying the biosensor assay, a detection limit of 2 pM or 2 amol of miR-16 was obtained. Using isolated total RNA from human breast adenocarcinoma MCF-7 cells, the assay detected specifically miR-21 and miR-16 in parallel, and higher expression of oncogene miR-21 compared to miR-16 was demonstrated. Including RNA isolation, the gap hybridization assay was developed with a total assay time of 60 min and without the need for reverse transcription PCR amplification of the sample. The characteristics of the assay developed in this work could satisfy the need for rapid and easy methods for early cancer marker detection in clinical diagnostics.

Multiplex real-time RT-PCR assay for bovine viral diarrhea virus type 1, type 2 and HoBi-like pestivirus.

PubMed

Mari, Viviana; Losurdo, Michele; Lucente, Maria Stella; Lorusso, Eleonora; Elia, Gabriella; Martella, Vito; Patruno, Giovanni; Buonavoglia, Domenico; Decaro, Nicola

2016-03-01

HoBi-like pestiviruses are emerging pestiviruses that infect cattle causing clinical forms overlapping to those induced by bovine viral diarrhea virus (BVDV) 1 and 2. As a consequence of their widespread distribution reported in recent years, molecular tools for rapid discrimination among pestiviruses infecting cattle are needed. The aim of the present study was to develop a multiplex real-time RT-PCR assay, based on the TaqMan technology, for the rapid and unambiguous characterisation of all bovine pestiviruses, including the emerging HoBi-like strains. The assay was found to be sensitive, specific and repeatable, ensuring detection of as few as 10(0)-10(1) viral RNA copies. No cross-reactions between different pestiviral species were observed even in samples artificially contaminated with more than one pestivirus. Analysis of field samples tested positive for BVDV-1, BVDV-2 or HoBi-like virus by a nested PCR protocol revealed that the developed TaqMan assay had equal or higher sensitivity and was able to discriminate correctly the viral species in all tested samples, whereas a real-time RT-PCR assay previously developed for HoBi-like pestivirus detection showed cross-reactivity with few high-titre BVDV-2 samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Giant magnetoresistive biosensors for molecular diagnosis: surface chemistry and assay development

NASA Astrophysics Data System (ADS)

Yu, Heng; Osterfeld, Sebastian J.; Xu, Liang; White, Robert L.; Pourmand, Nader; Wang, Shan X.

2008-08-01

Giant magnetoresistive (GMR) biochips using magnetic nanoparticle as labels were developed for molecular diagnosis. The sensor arrays consist of GMR sensing strips of 1.5 μm or 0.75 μm in width. GMR sensors are exquisitely sensitive yet very delicate, requiring ultrathin corrosion-resistive passivation and efficient surface chemistry for oligonucleotide probe immobilization. A mild and stable surface chemistry was first developed that is especially suitable for modifying delicate electronic device surfaces, and a practical application of our GMR biosensors was then demonstrated for detecting four most common human papillomavirus (HPV) subtypes in plasmids. We also showed that the DNA hybridization time could potentially be reduced from overnight to about ten minutes using microfluidics.

Analytical validation of viral CNS Flow Chip kit for detection of acute meningitis and encephalitis.

PubMed

Pérez-Ruiz, Mercedes; Pedrosa-Corral, Irene; Sanbonmatsu-Gámez, Sara; Gómez-Camarasa, Cristina; Navarro-Marí, José María

2018-06-12

A new molecular assay (Viral CNS Flow Chip kit, Master Diagnóstica, Spain) has been developed for the detection of eight viruses causing acute meningitis and encephalitis, i.e. herpes simplex viruses 1-2, varicella zoster virus, human enterovirus, human parechovirus, Toscana virus, human cytomegalovirus and Epstein Barr virus. The new assay is a multiplex one-step RT-PCR followed by automatic flow-through hybridization, colorimetric detection and image analysis. The limit of detection was 50 copies/reaction, and 10 copies/reaction for human enterovirus and the other seven viruses, respectively. The analytical validation was performed with nucleic acids extracted from 268 cerebrospinal fluid samples and the results were compared with routine molecular assays. An excellent coefficient of agreement was observed between V-CNS and routine assays [kappa index: 0.948 (95%CI: 0.928-0.968)]. The overall sensitivity and specificity was 95.9% (95%CI: 91.2-98.3%) and 99.9% (95%CI: 99.6-100%), respectively. Viral CNS Flow Chip kit is an efficient multiplex platform for the detection of the main viruses involved in acute meningitis and encephalitis. The inclusion of a TOSV genome target may improve the laboratory diagnosis of viral neurological infections in endemic areas. Copyright © 2018 Elsevier B.V. All rights reserved.

Three RNases in Senescent and Nonsenescent Wheat Leaves 1

PubMed Central

Blank, A.; McKeon, Thomas A.

1991-01-01

We have described three RNases in wheat leaves (Triticum aestivum L. cv Chinese Spring) and developed assays for measuring each RNase individually in crude leaf extracts. We initially used activity staining in sodium dodecyl sulfate-polyacrylamide gels to characterize RNases in extracts of primary and flag leaves. We thus identified acid RNase (EC 3.1.27.1, here designated RNase WLA), and two apparently novel enzymes, designated RNases WLB and WLC. RNase WLB activity displays a distinctive isozyme pattern, a molecular mass of 26 kilodaltons (major species), a broad pH range with an optimum near neutrality, insensitivity to EDTA, and stimulation by moderate concentrations of KCl and by MgCl2. RNase WLC activity exhibits a molecular mass of 27 kilodaltons, a neutral pH optimum, insensitivity to EDTA, and inhibition by KCl, MgCl2, and tri-(hydroxymethyl)aminomethane. Based on distinctive catalytic properties established in gels, we designed conventional solution assays for selective quantitation of each RNase activity. We used the assays to monitor the individual RNases after gel filtration chromatography and native gel electrophoresis of extracts. In accompanying work, we used the assays to monitor RNases WLA, WLB, and WLC, which are present in senescent and nonsenescent leaves, during the course of leaf senescence. ImagesFigure 1Figure 3Figure 4 PMID:16668563

Split-luciferase complementary assay: applications, recent developments, and future perspectives.

PubMed

Azad, Taha; Tashakor, Amin; Hosseinkhani, Saman

2014-09-01

Bioluminescent systems are considered as potent reporter systems for bioanalysis since they have specific characteristics, such as relatively high quantum yields and photon emission over a wide range of colors from green to red. Biochemical events are mostly accomplished through large protein machines. These molecular complexes are built from a few to many proteins organized through their interactions. These protein-protein interactions are vital to facilitate the biological activity of cells. The split-luciferase complementation assay makes the study of two or more interacting proteins possible. In this technique, each of the two domains of luciferase is attached to each partner of two interacting proteins. On interaction of those proteins, luciferase fragments are placed close to each other and form a complemented luciferase, which produces a luminescent signal. Split luciferase is an effective tool for assaying biochemical metabolites, where a domain or an intact protein is inserted into an internally fragmented luciferase, resulting in ligand binding, which causes a change in the emitted signals. We review the various applications of this novel luminescent biosensor in studying protein-protein interactions and assaying metabolites involved in analytical biochemistry, cell communication and cell signaling, molecular biology, and the fate of the whole cell, and show that luciferase-based biosensors are powerful tools that can be applied for diagnostic and therapeutic purposes.

Kim Magrini | NREL

Science.gov Websites

Developing char-based soil amendments Soil carbon and char analysis via molecular beam mass spectrometry depth profiling, Fourier transform infrared [FTIR]) Rapid soil carbon analysis using analytical DRIFTS, 13C NMR, and py-MBMS to Characterize the Effects of Soil Science Oxidation Assays on Soil Organic

Overview of the ToxCast Research Program: Applications to Predictive Toxicology and Chemical Prioritization

EPA Science Inventory

EPA’s ToxCast program, the NTP’s HTS initiative, and the NCGC’s Molecular Libraries Initiative into a collaborative research program focused on identifying toxicity pathways and developing in vitro assays to characterize the ability of chemicals to perturb those pathways. The go...

Considerations in Use of the EPA’s ToxCast Data for Environmental Toxicology (SETAC)

EPA Science Inventory

The US EPA has developed the ToxCast program to prioritize chemicals for selective toxicity testing. ToxCast relies on extensive bioactivity profiling using a panel of biochemical and cellular assays that measure chemicals effects on potential molecular initiating events and key ...

Simulating Limb Formation in the U.S. EPA Virtual Embryo - Risk Assessment Project

EPA Science Inventory

The U.S. EPA’s Virtual Embryo project (v-Embryo™) is a computer model simulation of morphogenesis that integrates cell and molecular level data from mechanistic and in vitro assays with knowledge about normal development processes to assess in silico the effects of chemicals on d...

Development of LAMP assays for the molecular detection of taeniid infection in canine in Tibetan rural area.

PubMed

Feng, Kai; Li, Wei; Guo, Zhihong; Duo, Hong; Fu, Yong; Shen, Xiuying; Tie, Cheng; E, Rijie; Xiao, Changqin; Luo, Yanhong; Qi, Guo; Ni, Ma; Ma, Qingmei; Yamazaki, Wataru; Yoshida, Ayako; Horii, Yoichiro; Yagi, Kinpei; Nonaka, Nariaki

2017-12-22

For field-identification of taeniid cestodes in canine animals in Tibetan area, loop-mediated isothermal amplification (LAMP) assays for Echinococcus multilocularis, E. shiquicus, Taenia hydatigena, T. multiceps, T. pisiformis and T. crassiceps were developed and evaluated along with the reported assay for E. granulosus. The LAMP assays showed specific reaction with their corresponding target species DNA with the detection limit of 1 to 10 pg. Moreover, the assays for E. granulosus, E. multilocularis, T. hydatigena and T. multiceps could detect DNA extracted from 3 or more eggs of their corresponding target species. Then, the LAMP assays were applied on samples containing 3 to 35 taeniid eggs obtained from 61 field-collected canine feces in Qinghai, and the result was compared with a reported multiplex PCR and sequence analysis. The LAMP assays and the PCR detected single species DNA of E. granulosus, E. shiquicus, T. hydatigena and T. multiceps in 5, 2, 44 and 2 samples, respectively. In the rest 8 samples, DNA of both E. granulosus and T. hydatigena were detected by the PCR but the LAMP assays detected those DNAs in 2 samples and only T. hydatigena DNA in 6 samples. It was assumed that less than 3 E. granulosus eggs were mixed in the samples although the samples contained 21 to 27 eggs in total. In conclusion, the LAMP assays were less sensitive than the multiplex PCR, but would have adequate sensitivity for field use in Tibetan area.

Specific and straightforward molecular investigation of β-thalassemia mutations in the Malaysian Malays and Chinese using direct TaqMan genotyping assays.

PubMed

Kho, S L; Chua, K H; George, E; Tan, J A M A

2013-07-15

Beta-thalassemia is a life-threatening inherited blood disorder. Rapid characterization of β-globin gene mutations is necessary because of the high frequency of Malaysian β-thalassemia carriers. A combination real-time polymerase chain reaction genotyping assay using TaqMan probes was developed to confirm β-globin gene mutations. In this study, primers and probes were designed to specifically identify 8 common β-thalassemia mutations in the Malaysian Malay and Chinese ethnic groups using the Primer Express software. "Blind tests" using DNA samples from healthy individuals and β-thalassemia patients with different genotypes were performed to determine the specificity and sensitivity of this newly designed assay. Our results showed 100% sensitivity and specificity for this novel assay. In conclusion, the TaqMan genotyping assay is a straightforward assay that allows detection of β-globin gene mutations in less than 40 min. The simplicity and reproducibility of the TaqMan genotyping assay permit its use in laboratories as a rapid and cost-effective diagnostic tool for confirmation of common β-thalassemia mutations in Malaysia.

A molecular identification system for grasses: a novel technology for forensic botany.

PubMed

Ward, J; Peakall, R; Gilmore, S R; Robertson, J

2005-09-10

Our present inability to rapidly, accurately and cost-effectively identify trace botanical evidence remains the major impediment to the routine application of forensic botany. Grasses are amongst the most likely plant species encountered as forensic trace evidence and have the potential to provide links between crime scenes and individuals or other vital crime scene information. We are designing a molecular DNA-based identification system for grasses consisting of several PCR assays that, like a traditional morphological taxonomic key, provide criteria that progressively identify an unknown grass sample to a given taxonomic rank. In a prior study of DNA sequences across 20 phylogenetically representative grass species, we identified a series of potentially informative indels in the grass mitochondrial genome. In this study we designed and tested five PCR assays spanning these indels and assessed the feasibility of these assays to aid identification of unknown grass samples. We confirmed that for our control set of 20 samples, on which the design of the PCR assays was based, the five primer combinations produced the expected results. Using these PCR assays in a 'blind test', we were able to identify 25 unknown grass samples with some restrictions. Species belonging to genera represented in our control set were all correctly identified to genus with one exception. Similarly, genera belonging to tribes in the control set were correctly identified to the tribal level. Finally, for those samples for which neither the tribal or genus specific PCR assays were designed, we could confidently exclude these samples from belonging to certain tribes and genera. The results confirmed the utility of the PCR assays and the feasibility of developing a robust full-scale usable grass identification system for forensic purposes.

Protein Science by DNA Sequencing: How Advances in Molecular Biology Are Accelerating Biochemistry.

PubMed

Higgins, Sean A; Savage, David F

2018-01-09

A fundamental goal of protein biochemistry is to determine the sequence-function relationship, but the vastness of sequence space makes comprehensive evaluation of this landscape difficult. However, advances in DNA synthesis and sequencing now allow researchers to assess the functional impact of every single mutation in many proteins, but challenges remain in library construction and the development of general assays applicable to a diverse range of protein functions. This Perspective briefly outlines the technical innovations in DNA manipulation that allow massively parallel protein biochemistry and then summarizes the methods currently available for library construction and the functional assays of protein variants. Areas in need of future innovation are highlighted with a particular focus on assay development and the use of computational analysis with machine learning to effectively traverse the sequence-function landscape. Finally, applications in the fundamentals of protein biochemistry, disease prediction, and protein engineering are presented.

Development and validation of panoptic Meso scale discovery assay to quantify total systemic interleukin-6

PubMed Central

Chaturvedi, Shalini; Siegel, Derick; Wagner, Carrie L; Park, Jaehong; van de Velde, Helgi; Vermeulen, Jessica; Fung, Man-Cheong; Reddy, Manjula; Hall, Brett; Sasser, Kate

2015-01-01

Aim Interleukin-6 (IL-6), a multifunctional cytokine, exists in several forms ranging from a low molecular weight (MW 20–30 kDa) non-complexed form to high MW (200–450 kDa), complexes. Accurate baseline IL-6 assessment is pivotal to understand clinical responses to IL-6-targeted treatments. Existing assays measure only the low MW, non-complexed IL-6 form. The present work aimed to develop a validated assay to measure accurately total IL-6 (complexed and non-complexed) in serum or plasma as matrix in a high throughput and easily standardized format for clinical testing. Methods Commercial capture and detection antibodies were screened against humanized IL-6 and evaluated in an enzyme-linked immunosorbent assay format. The best antibody combinations were screened to identify an antibody pair that gave minimum background and maximum recovery of IL-6 in the presence of 100% serum matrix. A plate-based total IL-6 assay was developed and transferred to the Meso Scale Discovery (MSD) platform for large scale clinical testing. Results The top-performing antibody pair from 36 capture and four detection candidates was validated on the MSD platform. The lower limit of quantification in human serum samples (n = 6) was 9.77 pg l–1, recovery ranged from 93.13–113.27%, the overall pooled coefficients of variation were 20.12% (inter-assay) and 8.67% (intra-assay). High MW forms of IL-6, in size fractionated serum samples from myelodysplastic syndrome and rheumatoid arthritis patients, were detected by the assay but not by a commercial kit. Conclusion This novel panoptic (sees all forms) IL-6 MSD assay that measures both high and low MW forms may have clinical utility. PMID:25847183

Design and optimization of a novel reverse transcription linear-after-the-exponential PCR for the detection of foot-and-mouth disease virus.

PubMed

Pierce, K E; Mistry, R; Reid, S M; Bharya, S; Dukes, J P; Hartshorn, C; King, D P; Wangh, L J

2010-07-01

A novel molecular assay for the detection of foot-and-mouth disease virus (FMDV) was developed using linear-after-the-exponential polymerase chain reaction (LATE-PCR). Pilot experiments using synthetic DNA targets demonstrated the ability of LATE-PCR to quantify initial target concentration through endpoint detection. A two-step protocol involving reverse transcription (RT) followed by LATE-PCR was then used to confirm the ability of the assay to detect FMDV RNA. Finally, RT and LATE-PCR were combined in a one-step duplex assay for co-amplification of an FMDV RNA segment and an internal control comprised of an Armored RNA. In that form, each of the excess primers in the reaction mixture hybridize to their respective RNA targets during a short pre-incubation, then generate cDNA strands during a 3-min RT step at 60°C, and the resulting cDNA is amplified by LATE-PCR without intervening sample processing. The RT-LATE-PCR assay generates fluorescent signals at endpoint that are proportional to the starting number of RNA targets and can detect a range of sequence variants using a single mismatch-tolerant probe. In addition to offering improvements over current laboratory-based molecular diagnostic assays for FMDV, this new assay is compatible with a novel portable ('point-of-care') device, the BioSeeq II, designed for the rapid diagnosis of FMD in the field. © 2009 The Authors. Journal compilation © 2009 The Society for Applied Microbiology.

Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation

PubMed Central

Loibner, Martina; Oberauner-Wappis, Lisa; Viertler, Christian; Groelz, Daniel; Zatloukal, Kurt

2017-01-01

Morphologic assessment of formalin-fixed, paraffin-embedded (FFPE) tissue samples has been the gold standard for cancer diagnostics for decades due to its excellent preservation of morphology. Personalized medicine increasingly provides individually adapted and targeted therapies for characterized individual diseases enabled by combined morphological and molecular analytical technologies and diagnostics. Performance of morphologic and molecular assays from the same FFPE specimen is challenging because of the negative impact of formalin due to chemical modification and cross-linking of nucleic acids and proteins. A non-cross-linking, formalin-free tissue fixative has been recently developed to fulfil both requirements, i.e., to preserve morphology like FFPE and biomolecules like cryo-preservation. Since FISH is often required in combination with histopathology and molecular diagnostics, we tested the applicability of FISH protocols on tissues treated with this new fixative. We found that formalin post-fixation of histological sections of non-cross-linking, formalin-free and paraffin-embedded (NCFPE) breast cancer tissue generated equivalent results to those with FFPE tissue in human epidermal growth factor receptor 2 (HER2) FISH analysis. This protocol describes how a FISH assay originally developed and validated for FFPE tissue can be used for NCFPE tissues by a simple post-fixation step of histological sections. PMID:29364207

Multiplex detection of microRNAs by combining molecular beacon probes with T7 exonuclease-assisted cyclic amplification reaction.

PubMed

Liu, Yacui; Zhang, Jiangyan; Tian, Jingxiao; Fan, Xiaofei; Geng, Hao; Cheng, Yongqiang

2017-01-01

A simple, highly sensitive, and specific assay was developed for the homogeneous and multiplex detection of microRNAs (miRNAs) by combining molecular beacon (MB) probes and T7 exonuclease-assisted cyclic amplification. An MB probe with five base pairs in the stem region without special modification can effectively prevent the digestion by T7 exonuclease. Only in the presence of target miRNA is the MB probe hybridized with the target miRNA, and then digested by T7 exonuclease in the 5' to 3' direction. At the same time, the target miRNA is released and subsequently initiates the nuclease-assisted cyclic digestion process, generating enhanced fluorescence signal significantly. The results show that the combination of T7 exonuclease-assisted cyclic amplification reaction and MB probe possesses higher sensitivity for miRNA detection. Moreover, multiplex detection of miRNAs was successfully achieved by designing two MB probes labeled with FAM and Cy3, respectively. As a result, the method opens a new pathway for the sensitive and multiplex detection of miRNAs as well as clinical diagnosis. Graphical Abstract A simple, highly sensitive, and specific assay was developed for the detection of microRNAs by combining molecular beacon probes with T7 exonuclease-assisted cyclic amplification reaction.

Prostate Cancer: Serum and Tissue Markers

PubMed Central

Miller, Gary J; Brawer, Michael K; Sakr, Wael A; Thrasher, J Brantley; Townsend, Ronald

2001-01-01

The detection of prostate cancer, its clinical staging, and the prediction of its prognosis remain topics of paramount importance in clinical management. The digital rectal exam, although once the “gold standard,” has been largely supplanted by a variety of techniques including serum and tissue-based assays. This article reviews recent progress in the development of prostate-specific antigen assays with greater specificity; molecular markers for prostate cancer (DNA ploidy, nuclear morphometry, markers of proliferation, and cell adhesion molecules); the link between vitamin D deficiency and the clinical emergence of prostate cancer; the possible correlation of serum insulin-like growth factor levels with the risk for developing prostate cancer; and the latest advances in radiologic staging. PMID:16985995

Support for HIV-1 Intervention Therapy

DTIC Science & Technology

1993-10-01

I. Kiselev, and E. S. Severin. 1990. Amplification of DNA 46 sequences of Epstein - Barr and human immunodeficiency viruses using DNA-polymerase from... develop and validate assays that predict or demonstrate disease progression for use in interventional trials with an emphasis on molecular biologic...to stay on the leading edge of technology development . A potential problem in obtaining quality sequence information is the occurrence of template

Immunocapture loop-mediated isothermal amplification assays for the detection of canine parvovirus.

PubMed

Sun, Yu-Ling; Yen, Chon-Ho; Tu, Ching-Fu

2017-11-01

A loop-mediated isothermal amplification (LAMP) assay was used for rapid canine parvovirus (CPV) diagnosis. To reduce the time required and increase the sensitivity of the assay, an immunocapture (IC) technique was developed in this study to exclude the DNA extraction step in molecular diagnostic procedures for CPV. A polyclonal rabbit anti-CPV serum was produced against VP2-EpC that was cloned via DNA recombination. The polyclonal anti-VP2-EpC serum was used for virus capture to prepare microtubes. IC-LAMP was performed to amplify a specific CPV target gene sequence from the CPV viral particles that were captured on the microtubes, and the amplicons were analyzed using agarose electrophoresis or enzyme-linked immunosorbent assay (IC-LAMP-ELISA) and lateral-flow dipstick (IC-LAMP-LFD). The detection sensitivities of IC-LAMP, IC-LAMP-ELISA, and IC-LAMP-LFD were 10 -1 , 10 -1 , and 10 -1 TCID 50 /mL, respectively. Using the IC-LAMP-ELISA and IC-LAMP-LFD assays, the complete CPV diagnostic process can be achieved within 1.5h. Both of the developed IC-LAMP-based assays are simple, direct visual and efficient techniques that are applicable to the detection of CPV. Copyright © 2017 Elsevier B.V. All rights reserved.

Rapid and sensitive detection of mink circovirus by recombinase polymerase amplification.

PubMed

Ge, Junwei; Shi, Yunjia; Cui, Xingyang; Gu, Shanshan; Zhao, Lili; Chen, Hongyan

2018-06-01

To date, the pathogenic role of mink circovirus (MiCV) remains unclear, and its prevalence and economic importance are unknown. Therefore, a rapid and sensitive molecular diagnosis is necessary for disease management and epidemiological surveillance. However, only PCR methods can identify MiCV infection at present. In this study, we developed a nested PCR and established a novel recombinase polymerase amplification (RPA) assay for MiCV detection. Sensitivity analysis showed that the detection limit of nested PCR and RPA assay was 10 1 copies/reaction, and these methods were more sensitive than conventional PCR, which has a detection limit of 10 5 copies/reaction. The RPA assay had no cross-reactivity with other related viral pathogens, and amplification was completed in less than 20 min with a simple device. Further assessment of clinical samples showed that the two assays were accurate in identifying positive and negative conventional PCR samples. The detection rate of MiCV by the RPA assay in clinical samples was 38.09%, which was 97% consistent with that by the nested PCR. The developed nested PCR is a highly sensitive tool for practical use, and the RPA assay is a simple, sensitive, and potential alternative method for rapid and accurate MiCV diagnosis. Copyright © 2018 Elsevier B.V. All rights reserved.

Stationary microfluidics: molecular diagnostic assays by moving magnetic beads through non-moving liquids

NASA Astrophysics Data System (ADS)

Becker, Holger; Carstens, Cornelia; Kuhlmeier, Dirk; Sandetskaya, Natalia; Schröter, Nicole; Zilch, Christian; Gärtner, Claudia

2013-03-01

Commonly, microfluidic devices are based on the movement of fluids. For molecular diagnostics assays which often include steps like PCR, this practically always involves a more or less complicated set of external pumps, valves and liquid controls. In the presented paper, we follow a different approach in which the fluid after sample introduction remains stationary and the main bioactive sample molecules are moved through a chain of reaction compartments which contain the different reagents necessary for the assay. The big advantage of this concept is the lack of any external fluid actuation/control. Results on sample carry-over experiments and complete assays will be given.

Molecular techniques and genetic alterations in head and neck cancer

PubMed Central

Ha, Patrick K; Chang, Steven S; Glazer, Chad A; Califano, Joseph A; Sidransky, David

2009-01-01

It is well known that cellular DNA alterations can lead to the formation of cancer, and there has been much discovery in the pathways involved in the development of head and neck squamous cell carcinoma (HNSCC). With novel genome-wide molecular assays, our ability to detect these abnormalities has increased. We now have a better understanding of the molecular complexity of HNSCC, but there is still much research to be done. In this review, we discuss the well described genetic alterations and touch on the newer findings, as well as some of the future directions of head and neck cancer research. PMID:18674960

Bio-Mimetic Sensors Based on Molecularly Imprinted Membranes

PubMed Central

Algieri, Catia; Drioli, Enrico; Guzzo, Laura; Donato, Laura

2014-01-01

An important challenge for scientific research is the production of artificial systems able to mimic the recognition mechanisms occurring at the molecular level in living systems. A valid contribution in this direction resulted from the development of molecular imprinting. By means of this technology, selective molecular recognition sites are introduced in a polymer, thus conferring it bio-mimetic properties. The potential applications of these systems include affinity separations, medical diagnostics, drug delivery, catalysis, etc. Recently, bio-sensing systems using molecularly imprinted membranes, a special form of imprinted polymers, have received the attention of scientists in various fields. In these systems imprinted membranes are used as bio-mimetic recognition elements which are integrated with a transducer component. The direct and rapid determination of an interaction between the recognition element and the target analyte (template) was an encouraging factor for the development of such systems as alternatives to traditional bio-assay methods. Due to their high stability, sensitivity and specificity, bio-mimetic sensors-based membranes are used for environmental, food, and clinical uses. This review deals with the development of molecularly imprinted polymers and their different preparation methods. Referring to the last decades, the application of these membranes as bio-mimetic sensor devices will be also reported. PMID:25196110

A Novel Application for 222Rn Emanation Standards

PubMed Central

Laureano-Perez, L.; Collé, R.; Jacobson, D.R.; Fitzgerald, R.; Khan, N.S.; Dmochowski, I.J.

2013-01-01

In collaboration with the University of Pennsylvania, a 222Rn emanation source was used for the determination of the binding affinity of radon to a cryptophane molecular host. This source was similar to a 222Rn emanation standard that was developed and disseminated by the National Institute of Standards and Technology (NIST). The novel experimental design involved performing the reactions at femtomole levels, developing exacting gravimetric sampling methods and making precise 222Rn assays by liquid scintillation counting. A cryptophane-radon association constant was determined, KA = (49,000 ± 12,000) L· mol−1 at 293 K, which was the first measurement of radon binding to a molecular host. PMID:22455833

Development of polymerase chain reaction-based diagnostic tests for detection of Malsoor virus & adenovirus isolated from Rousettus species of bats in Maharashtra, India.

PubMed

Shete, Anita M; Yadav, Pragya; Kumar, Vimal; Nikam, Tushar; Mehershahi, Kurosh; Kokate, Prasad; Patil, Deepak; Mourya, Devendra T

2017-01-01

Bats are recognized as important reservoirs for emerging infectious disease and some unknown viral diseases. Two novel viruses, Malsoor virus (family Bunyaviridae, genus, Phlebovirus) and a novel adenovirus (AdV) (family, Adenoviridae genus, Mastadenovirus), were identified from Rousettus bats in the Maharashtra State of India. This study was done to develop and optimize real time reverse transcription - polymerase chain reaction (RT-PCR) assays for Malsoor virus and real time and nested PCR for adenovirus from Rousettus bats. For rapid and accurate screening of Malsoor virus and adenovirus a nested polymerase chain reaction and TaqMan-based real-time PCR were developed. Highly conserved region of nucleoprotein gene of phleboviruses and polymerase gene sequence from the Indian bat AdV isolate polyprotein gene were selected respectively for diagnostic assay development of Malsoor virus and AdV. Sensitivity and specificity of assays were calculated and optimized assays were used to screen bat samples. Molecular diagnostic assays were developed for screening of Malsoor virus and AdV and those were found to be specific. Based on the experiments performed with different parameters, nested PCR was found to be more sensitive than real-time PCR; however, for rapid screening, real-time PCR can be used and further nested PCR can be used for final confirmation or in those laboratories where real-time facility/expertise is not existing. This study reports the development and optimization of nested RT-PCR and a TaqMan-based real-time PCR for Malsoor virus and AdV. The diagnostic assays can be used for rapid detection of these novel viruses to understand their prevalence among bat population.

Development of a quantitative loop-mediated isothermal amplification assay for the field detection of Erysiphe necator.

PubMed

Thiessen, Lindsey D; Neill, Tara M; Mahaffee, Walter F

2018-01-01

Plant pathogen detection systems have been useful tools to monitor inoculum presence and initiate management schedules. More recently, a loop-mediated isothermal amplification (LAMP) assay was successfully designed for field use in the grape powdery mildew pathosystem; however, false negatives or false positives were prevalent in grower-conducted assays due to the difficulty in perceiving the magnesium pyrophosphate precipitate at low DNA concentrations. A quantitative LAMP (qLAMP) assay using a fluorescence resonance energy transfer-based probe was assessed by grape growers in the Willamette Valley of Oregon. Custom impaction spore samplers were placed at a research vineyard and six commercial vineyard locations, and were tested bi-weekly by the lab and by growers. Grower-conducted qLAMP assays used a beta-version of the Smart-DART handheld LAMP reaction devices (Diagenetix, Inc., Honolulu, HI, USA), connected to Android 4.4 enabled, Bluetooth-capable Nexus 7 tablets for output. Quantification by a quantitative PCR assay was assumed correct to compare the lab and grower qLAMP assay quantification. Growers were able to conduct and interpret qLAMP results; however, the Erysiphe necator inoculum quantification was unreliable using the beta-Smart-DART devices. The qLAMP assay developed was sensitive to one spore in early testing of the assay, but decreased to >20 spores by the end of the trial. The qLAMP assay is not likely a suitable management tool for grape powdery mildew due to losses in sensitivity and decreasing costs and portability for other, more reliable molecular tools.

The Intercostal NMJ Assay: a new alternative to the conventional LD50 assay for the determination of the therapeutic potency of botulinum toxin preparations.

PubMed

Huber, Alexander; France, Richard M; Riccalton-Banks, Lisa; McLaren, Jane; Cox, Helen; Quirk, Robin A; Shakesheff, Kevin M; Thompson, David; Panjwani, Naveed; Shipley, Sarah; Pickett, Andy

2008-05-01

Therapeutic botulinum neurotoxin type A preparations have found an increasing number of clinical uses for a large variety of neuromuscular disorders and dermatological conditions. The accurate determination of potency in the clinical application of botulinum toxins is critical to ensuring clinical efficacy and safety, and is currently achieved by using a lethal dose (LD50) assay in mice. Ethical concerns and operational constraints associated with this assay have prompted the development of alternative assay systems that could potentially lead to its replacement. As one such alternative, we describe the development and evaluation of a novel ex vivo assay (the Intercostal Neuromuscular Junction [NMJ] Assay), which uses substantially fewer animals and addresses ethical concerns associated with the LD50 assay. The assay records the decay of force from electrically-stimulated muscle tissue sections in response to the toxin, and thus combines the important mechanisms of receptor binding, translocation, and the enzymatic action of the toxin molecule. Toxin application leads to a time-related and dose-related reduction in contractile force. A regression model describing the relationship between the applied dose and force decay was determined statistically, and was successfully tested as able to correctly predict the potency of an unknown sample. The tissue sections used were found to be highly reproducible, as determined through the innervation pattern and the localisation of NMJs in situ. Furthermore, the efficacy of the assay protocol to successfully deliver the test sample to the cellular target sites, was critically assessed by using molecular tracer molecules.

A paper-based resonance energy transfer nucleic acid hybridization assay using upconversion nanoparticles as donors and quantum dots as acceptors.

PubMed

Doughan, Samer; Uddayasankar, Uvaraj; Krull, Ulrich J

2015-06-09

Monodisperse aqueous upconverting nanoparticles (UCNPs) were covalently immobilized on aldehyde modified cellulose paper via reduction amination to develop a luminescence resonance energy transfer (LRET)-based nucleic acid hybridization assay. This first account of covalent immobilization of UCNPs on paper for a bioassay reports an optically responsive method that is sensitive, reproducible and robust. The immobilized UCNPs were decorated with oligonucleotide probes to capture HPRT1 housekeeping gene fragments, which in turn brought reporter conjugated quantum dots (QDs) in close proximity to the UCNPs for LRET. This sandwich assay could detect unlabeled oligonucleotide target, and had a limit of detection of 13 fmol and a dynamic range spanning nearly 3 orders of magnitude. The use of QDs, which are excellent LRET acceptors, demonstrated improved sensitivity, limit of detection, dynamic range and selectivity compared to similar assays that have used molecular fluorophores as acceptors. The selectivity of the assay was attributed to the decoration of the QDs with polyethylene glycol to eliminate non-specific adsorption. The kinetics of hybridization were determined to be diffusion limited and full signal development occurred within 3 min. Copyright © 2015 Elsevier B.V. All rights reserved.

Development of a real-time loop-mediated isothermal amplification assay for detection of Burkholderia mallei.

PubMed

Pal, V; Saxena, A; Singh, S; Goel, A K; Kumar, J S; Parida, M M; Rai, G P

2018-02-01

Burkholderia mallei is the aetiological agent of glanders, a highly contagious and re-emerging zoonotic disease. Early diagnosis of glanders is critically important to ensure timely treatment with appropriate antibiotics in humans, and to prevent spread of infection in animals. Molecular detection of B. mallei has always been troublesome because of its genetic similarity with Burkholderia pseudomallei, the causative agent of melioidosis. In present investigation, a set of six B. mallei-specific primers were designed and a simple, rapid, specific and sensitive real-time loop-mediated isothermal amplification (LAMP) assay was developed for detection of B. mallei. The LAMP assay could detect as low as 1 pg of B. mallei genomic DNA and 5.5 × 10 3  CFU/ml of B. mallei in spiked human blood. The assay was highly specific for B. mallei as it did not cross-react with other bacterial strains used in the study. The established LAMP assay is field adaptable and can be a better and viable alternative to PCR-based techniques for detection of B. mallei in glanders endemic areas with resource-limited settings. © 2017 Blackwell Verlag GmbH.

Lyssavirus Detection and Typing Using Pyrosequencing▿#‖

PubMed Central

De Benedictis, Paola; De Battisti, Cristian; Dacheux, Laurent; Marciano, Sabrina; Ormelli, Silvia; Salomoni, Angela; Caenazzo, Silvia Tiozzo; Lepelletier, Anthony; Bourhy, Hervé; Capua, Ilaria; Cattoli, Giovanni

2011-01-01

Rabies is a fatal zoonosis caused by a nonsegmented negative-strand RNA virus, namely, rabies virus (RABV). Apart from RABV, at least 10 additional species are known as rabies-related lyssaviruses (RRVs), and some of them are responsible for occasional spillovers into humans. More lyssaviruses have also been detected recently in different bat ecosystems, thanks to the application of molecular diagnostic methods. Due to the variety of the members of the genus Lyssavirus, there is the necessity to develop a reliable molecular assay for rabies diagnosis able to detect and differentiate among the existing rabies and rabies-related viruses. In the present study, a pyrosequencing protocol targeting the 3′ terminus of the nucleoprotein (N) gene was applied for the rapid characterization of lyssaviruses. Correct identification of species was achieved for each sample tested. Results from the pyrosequencing assay were also confirmed by those obtained using the Sanger sequencing method. A pan-lyssavirus one-step reverse transcription (RT)-PCR was developed within the framework of the pyrosequencing procedure. The sensitivity (Se) of the one-step RT-PCR assay was determined by using in vitro-transcribed RNA and serial dilutions of titrated viruses. The assay demonstrated high analytical and relative specificity (Sp) (98.94%) and sensitivity (99.71%). To date, this is the first case in which pyrosequencing has been applied for lyssavirus identification using a cheaper diagnostic approach than the one for all the other protocols for rapid typing that we are acquainted with. Results from this study indicate that this procedure is suitable for lyssavirus detection in samples of both human and animal origin. PMID:21389152

Molecular Identification of an Invasive Wood-Boring Insect Lyctus brunneus (Coleoptera: Bostrichidae: Lyctinae) Using Frass by Loop-Mediated Isothermal Amplification and Nested PCR Assays.

PubMed

Ide, Tatsuya; Kanzaki, Natsumi; Ohmura, Wakako; Okabe, Kimiko

2016-03-27

Lyctus brunneus(Stephens) is one of the most destructive and worldwide invasive pests of seasoned woods for wooden products. This and other pestLyctusspecies have had their distribution expanded by international and domestic human transportation of infested wood and wood products. Rapid detection and accurate identification ofLyctusspecies are effective tools for helping to eradicate them in new introduction sites. The accurate species-level identification of adults requires expert knowledge about their morphology. However, it takes much time and effort to recover suitable adult specimens because they are borers inside wood. Frass ofLyctusspecies can easily be detected and recovered in and around infested wood. Thus, frass was tested to see if it was a suitable sample to allow development of a rapid and technically easy molecular detection and identification method forL.brunneus.Species-specific primers were designed from the cytochrome c oxidase subunit I region ofL.brunneusand used in development and testing of methods for successfully identifying them from their frass using the loop-mediated isothermal amplification (LAMP) or species-specific nested polymerase chain reaction (PCR) assays. The LAMP assay was faster and more sensitive for detecting the presence of DNA derived fromL.brunneusin their frass than the nested PCR assay. These methodologies will be applicable for the rapid detection and identification of other wood-boring invasive pests in regulatory applications. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Anaplasma platys-like strains in ruminants from Tunisia.

PubMed

Ben Said, Mourad; Belkahia, Hanène; El Mabrouk, Narjesse; Saidani, Mariem; Alberti, Alberto; Zobba, Rosanna; Cherif, Amal; Mahjoub, Tarek; Bouattour, Ali; Messadi, Lilia

2017-04-01

Molecular diagnosis of Anaplasma platys and related strains (A. platys-like) in carnivores and ruminants is challenging due to co-infections with cross-reacting strains, and require post-amplification sequencing of the hemi-nested PCR products traditionally generated by targeting the groEL gene. In this study, a Restriction Enzyme Fragment Length Polymorphism (RFLP) assay coupled to hemi-nested groEL PCR was developed to discriminate among A. platys and genetically related strains. This novel approach was used for investigating A. platys-like infection in 963 domesticated ruminants (241 goats, 355 sheep, and 367 cattle) from 22 delegations located in North Tunisia. Overall prevalence rates of A. platys-like were 22.8, 11, and 3.5% in goats, sheep, and cattle, respectively. Alignment, identity comparison, and phylogenetic analysis of the groEL sequence variants obtained in this study confirmed RFLP data suggesting that Tunisian ruminants are infected by novel unclassified Anaplasma strains genetically related to A. platys. Compared to sequencing, RFLP assay allows fast detection of A. platys and A. platys-like pathogens in the same sample and has a potential value especially when screening ticks, cats and ruminants, which can be a common host for these two bacteria. This newly developed molecular technique would provide valuable molecular tool for epidemiological studies related to A. platys as well as remove concern over specificity of serological and molecular methods routinely used to identify diverse Anaplasma strains and species in wild and domestic ruminants. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of a Rickettsia bellii-Specific TaqMan Assay Targeting the Citrate Synthase Gene.

PubMed

Hecht, Joy A; Allerdice, Michelle E J; Krawczak, Felipe S; Labruna, Marcelo B; Paddock, Christopher D; Karpathy, Sandor E

2016-11-01

Rickettsia bellii is a rickettsial species of unknown pathogenicity that infects argasid and ixodid ticks throughout the Americas. Many molecular assays used to detect spotted fever group (SFG) Rickettsia species do not detect R. bellii, so that infection with this bacterium may be concealed in tick populations when assays are used that screen specifically for SFG rickettsiae. We describe the development and validation of a R. bellii-specific, quantitative, real-time PCR TaqMan assay that targets a segment of the citrate synthase (gltA) gene. The specificity of this assay was validated against a panel of DNA samples that included 26 species of Rickettsia, Orientia, Ehrlichia, Anaplasma, and Bartonella, five samples of tick and human DNA, and DNA from 20 isolates of R. bellii, including 11 from North America and nine from South America. A R. bellii control plasmid was constructed, and serial dilutions of the plasmid were used to determine the limit of detection of the assay to be one copy per 4 µl of template DNA. This assay can be used to better determine the role of R. bellii in the epidemiology of tick-borne rickettsioses in the Western Hemisphere. Published by Oxford University Press on behalf of Entomological Society of America 2016. This work is written by US Government employees and is in the public domain in the US.

A novel cell-based assay to measure activity of Venezuelan equine encephalitis virus nsP2 protease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Campos-Gomez, Javier; Ahmad, Fahim; Rodriguez, Efrain

2016-09-15

The encephalitic alphaviruses encode nsP2 protease (nsP2pro), which because of its vital role in virus replication, represents an attractive target for therapeutic intervention. To facilitate the discovery of nsP2 inhibitors we have developed a novel assay for quantitative measurement of nsP2pro activity in a cell-based format. The assay is based on a substrate fusion protein consisting of eGFP and Gaussia luciferase (Gluc) linked together by a small peptide containing a VEEV nsp2pro cleavage sequence. The expression of the substrate protein in cells along with recombinant nsP2pro results in cleavage of the substrate protein resulting in extracellular release of free Gluc.more » The Gluc activity in supernatants corresponds to intracellular nsP2pro-mediated substrate cleavage; thus, providing a simple and convenient way to quantify nsP2pro activity. Here, we demonstrate potential utility of the assay in identification of nsP2pro inhibitors, as well as in investigations related to molecular characterization of nsP2pro. - Highlights: • A novel cell-based assay to measure VEEV nsP2 protease activity was developed. • Assay utility was demonstrated for antiviral screening. • .The assay also proved to be useful in basic mechanistic studies of nsP2 protease.« less

Molecular characterization of the human immunodeficiency virus type 1 among children in Lima, Peru.

PubMed

Carrión, A G; Laguna-Torres, V A; Soto-Castellares, G; Castillo, M; Salazar, E; Negron, E; Kolevic, L; Montano, S M; Sánchez, J L; Bautista, C T; Oberhelman, R A; Kochel, T J

2009-08-01

In Peru, there is a lack of information on molecular analysis in pediatric human immunodeficiency virus (HIV) infection. At present, the mother-to-child transmission rate is estimated at approximately 2-4%. The objective of this study was to assess the molecular epidemiology of HIV-1 in infected children. Children with suspected or confirmed pulmonary tuberculosis were evaluated at two public hospitals between 2002 and 2007. Whole blood samples were obtained from 90 HIV-positive children, who were confirmed to be positive by enzyme-linked immunosorbent assay and Western blot. The specimens were subjected to envelope heteroduplex mobility assay (env HMA) followed by gag and pol gene region sequence analysis. Subtype B was found in 88 (98%) of 90 children and 2 (2%) children were subtype BF recombinants. This is the first report of recombinant HIV strains in HIV-infected children in Peru. Understanding the origin, diversity, and spread of HIV strains worldwide will be necessary for the development of an effective vaccine that targets pediatric populations throughout the world.

Cytotoxicity of iron oxide nanoparticles made from the thermal decomposition of organometallics and aqueous phase transfer with Pluronic F127

PubMed Central

Gonzales, Marcela; Mitsumori, Lee M.; Kushleika, John V.; Rosenfeld, Michael E.; Krishnan, Kannan M.

2010-01-01

Magnetic nanoparticles are promising molecular imaging agents due to their relative high relaxivity and the potential to modify surface functionality to tailor biodistribution. In this work we describe the synthesis of magnetic nanoparticles using organic solvents with organometallic precursors. This method results in nanoparticles that are highly crystalline, and have uniform size and shape. The ability to create a monodispersion of particles of the same size and shape results in unique magnetic properties that can be useful for biomedical applications with MR imaging. Before these nanoparticles can be used in biological applications, however, means are needed to make the nanoparticles soluble in aqueous solutions and the toxicity of these nanoparticles needs to be studied. We have developed two methods to surface modify and transfer these nanoparticles to the aqueous phase using the biocompatible co-polymer, Pluronic F127. Cytotoxicity was found to be dependent on the coating procedure used. Nanoparticle effects on a cell-culture model was quantified using concurrent assaying; a LDH assay to determine cytotoxicity and an MTS assay to determine viability for a 24 hour incubation period. Concurrent assaying was done to insure that nanoparticles did not interfere with the colorimetric assay results. This report demonstrates that a monodispersion of nanoparticles of uniform size and shape can be manufactured. Initial cytotoxicity testing of new molecular imaging agents need to be carefully constructed to avoid interference and erroneous results. PMID:20623517

Use of GeneXpert Remnants for Drug Resistance Profiling and Molecular Epidemiology of Tuberculosis in Libreville, Gabon.

PubMed

Alame-Emane, Amel Kévin; Pierre-Audigier, Catherine; Aboumegone-Biyogo, Oriane Cordelia; Nzoghe-Mveang, Amandine; Cadet-Daniel, Véronique; Sola, Christophe; Djoba-Siawaya, Joël Fleury; Gicquel, Brigitte; Takiff, Howard E

2017-07-01

Multidrug-resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis pose major problems for global health. The GeneXpert MTB/RIF (Xpert) assay rapidly detects resistance to rifampin (RIF r ), but for detection of the additional resistance that defines MDR-TB (MDR tuberculosis) and XDR-TB, and for molecular epidemiology, specimen cultures and a biosafe infrastructure are generally required. We sought to determine whether the remnants of sputa prepared for the Xpert assay could be used directly to find mutations associated with drug resistance and to study molecular epidemiology, thus providing precise characterization of MDR-TB cases in countries lacking biosafety level 3 (BSL3) facilities for M. tuberculosis cultures. After sputa were processed and run on the Xpert instrument, the leftovers of the samples prepared for the Xpert assay were used for PCR amplification and sequencing or for a line probe assay to detect mutations associated with resistance to additional drugs, as well as for molecular epidemiology with spoligotyping and selective mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing. Of 130 sputum samples from Gabon tested with the Xpert assay, 124 yielded interpretable results; 21 (17%) of these were determined to be RIF r Amplification and sequencing or a line probe assay of the Xpert remnants confirmed 18/21 samples as MDR, corresponding to 12/116 (9.5%) new and 6/8 (75%) previously treated TB patients. Spoligotyping and MIRU typing with hypervariable loci identified an MDR Beijing strain present in five samples. We conclude that the remnants of samples processed for the Xpert assay can be used in PCRs to find mutations associated with the resistance to the additional drugs that defines MDR and XDR-TB and to study molecular epidemiology without the need for culturing or a biosafe infrastructure. Copyright © 2017 Alame-Emane et al.

Evaluation of Signature Erosion in Ebola Virus Due to Genomic Drift and Its Impact on the Performance of Diagnostic Assays

PubMed Central

Sozhamannan, Shanmuga; Holland, Mitchell Y.; Hall, Adrienne T.; Negrón, Daniel A.; Ivancich, Mychal; Koehler, Jeffrey W.; Minogue, Timothy D.; Campbell, Catherine E.; Berger, Walter J.; Christopher, George W.; Goodwin, Bruce G.; Smith, Michael A.

2015-01-01

Genome sequence analyses of the 2014 Ebola Virus (EBOV) isolates revealed a potential problem with the diagnostic assays currently in use; i.e., drifting genomic profiles of the virus may affect the sensitivity or even produce false-negative results. We evaluated signature erosion in ebolavirus molecular assays using an in silico approach and found frequent potential false-negative and false-positive results. We further empirically evaluated many EBOV assays, under real time PCR conditions using EBOV Kikwit (1995) and Makona (2014) RNA templates. These results revealed differences in performance between assays but were comparable between the old and new EBOV templates. Using a whole genome approach and a novel algorithm, termed BioVelocity, we identified new signatures that are unique to each of EBOV, Sudan virus (SUDV), and Reston virus (RESTV). Interestingly, many of the current assay signatures do not fall within these regions, indicating a potential drawback in the past assay design strategies. The new signatures identified in this study may be evaluated with real-time reverse transcription PCR (rRT-PCR) assay development and validation. In addition, we discuss regulatory implications and timely availability to impact a rapidly evolving outbreak using existing but perhaps less than optimal assays versus redesign these assays for addressing genomic changes. PMID:26090727

Rapid, highly sensitive detection of herpes simplex virus-1 using multiple antigenic peptide-coated superparamagnetic beads.

PubMed

Ran, Ying-Fen; Fields, Conor; Muzard, Julien; Liauchuk, Viktoryia; Carr, Michael; Hall, William; Lee, Gil U

2014-12-07

A sensitive, rapid, and label free magnetic bead aggregation (MBA) assay has been developed that employs superparamagnetic (SPM) beads to capture, purify, and detect model proteins and the herpes simplex virus (HSV). The MBA assay is based on monitoring the aggregation state of a population of SPM beads using light scattering of individual aggregates. A biotin-streptavidin MBA assay had a femtomolar (fM) level sensitivity for analysis times less than 10 minutes, but the response of the assay becomes nonlinear at high analyte concentrations. A MBA assay for the detection of HSV-1 based on a novel peptide probe resulted in the selective detection of the virus at concentrations as low as 200 viral particles (vp) per mL in less than 30 min. We define the parameters that determine the sensitivity and response of the MBA assay, and the mechanism of enhanced sensitivity of the assay for HSV. The speed, relatively low cost, and ease of application of the MBA assay promise to make it useful for the identification of viral load in resource-limited and point-of-care settings where molecular diagnostics cannot be easily implemented.

Development of an assay for a biomarker of pregnancy and early fetal loss

DOE Office of Scientific and Technical Information (OSTI.GOV)

Canfield, R.E.; O'Connor, J.F.; Birken, S.

1987-10-01

Human chorionic gonadotropin (hCG) is a glycoprotein hormone, secreted by the syncytiotrophoblast cells of the fertilized ovum, that enters the maternal circulation at the time of endometrial implantation. It is composed of two nonidentical subunits; ..cap alpha.. and ..beta.., with molecular weights of 14 kD and 23 kD, respectively. Human chorionic gonadotropin binds to the same receptor as hLH and displays the same biological response, namely, to stimulate the declining function of the corpus luteum to produce progestins and estrogen late in the menstrual cycle. The differences in the structures of hCG and hLH have been exploited to develop antibodiesmore » that can measure hCG specifically in the presence of hLH. Two-site antibody binding assays have been developed, based on a surface immunological concept of hCG epitopes, that involve four distinct regions to which antibodies against hCG can bind simultaneously. Antibody cooperative effects, in conjunction with kinetic advantages derived from the concentration factors by use of the sandwich assay technique (immunoradiometric assay, IRMA), have enabled development of extremely sensitive and specific measurement protocols for urinary hCG. The assay described herein permits the detection of pregnancy on an average 25.4 days after the first day of the preceding menses, as opposed to 29.5 days for conventional radioimmunoassay techniques. In addition, the greater sensitivity and specificity of this assay method has permitted the detection of episodes of fetal loss not detected by radioimmunoassay of urine specimens. A large scale epidemiological study is in progress using this assay technique as a way to identify pregnancies that are lost before becoming clinically apparent.« less

Targeted Peptide Measurements in Biology and Medicine: Best Practices for Mass Spectrometry-based Assay Development Using a Fit-for-Purpose Approach

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carr, Steven A.; Abbateillo, Susan E.; Ackermann, Bradley L.

2014-01-14

Adoption of targeted mass spectrometry (MS) approaches such as multiple reaction monitoring (MRM) to study biological and biomedical questions is well underway in the proteomics community. Successful application depends on the ability to generate reliable assays that uniquely and confidently identify target peptides in a sample. Unfortunately, there is a wide range of criteria being applied to say that an assay has been successfully developed. There is no consensus on what criteria are acceptable and little understanding of the impact of variable criteria on the quality of the results generated. Publications describing targeted MS assays for peptides frequently do notmore » contain sufficient information for readers to establish confidence that the tests work as intended or to be able to apply the tests described in their own labs. Guidance must be developed so that targeted MS assays with established performance can be made widely distributed and applied by many labs worldwide. To begin to address the problems and their solutions, a workshop was held at the National Institutes of Health with representatives from the multiple communities developing and employing targeted MS assays. Participants discussed the analytical goals of their experiments and the experimental evidence needed to establish that the assays they develop work as intended and are achieving the required levels of performance. Using this “fit-for-purpose” approach, the group defined three tiers of assays distinguished by their performance and extent of analytical characterization. Computational and statistical tools useful for the analysis of targeted MS results were described. Participants also detailed the information that authors need to provide in their manuscripts to enable reviewers and readers to clearly understand what procedures were performed and to evaluate the reliability of the peptide or protein quantification measurements reported. This paper presents a summary of the meeting and recommendations. Molecular & Cellular Proteomics 13: 10.1074/mcp.M113.036095, 907–917, 2014.« less

Chemical characterization, antioxidant and antitumor activity of sulfated polysaccharide from Sargassum horneri.

PubMed

Shao, Ping; Chen, Xiaoxiao; Sun, Peilong

2014-05-25

Three water-soluble polysaccharide fractions (SHP30, SHP60, and SHP80) extracted from the Sargassum horneri were obtained by water extraction and radial flow chromatography. The high-performance gel-permeation chromatography analysis showed that the average molecular weight (Mw) of three polysaccharides were approximately 1.58×10(3), 1.92×10(3) and 11.2KDa, respectively. Their in vitro antioxidant activities, antitumor activities were investigated and compared. Among these three polysaccharides, SHP30 with the highest sulfate content and intermediate molecular weight exhibited excellent antioxidant and antitumor activities in the superoxide radical assay, hydroxyl radical assay, reducing power assay, and MTT assay. Then, flow cytometry assay and quantitative real-time reverse transcription-PCR analysis suggested that the accumulation of cells in G0/G1 and S phase effecting apoptosis-associated gene expressions such as Bcl-2 and Bax might account for the growth inhibition of DLD cells by SHP30. Based on these results, we have inferred that sulfate content and molecular weight were the factors influencing antioxidant and antitumor activities. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

Verification of performance specifications of a molecular test: cystic fibrosis carrier testing using the Luminex liquid bead array.

PubMed

Lacbawan, Felicitas L; Weck, Karen E; Kant, Jeffrey A; Feldman, Gerald L; Schrijver, Iris

2012-01-01

The number of clinical laboratories introducing various molecular tests to their existing test menu is continuously increasing. Prior to offering a US Food and Drug Administration-approved test, it is necessary that performance characteristics of the test, as claimed by the company, are verified before the assay is implemented in a clinical laboratory. To provide an example of the verification of a specific qualitative in vitro diagnostic test: cystic fibrosis carrier testing using the Luminex liquid bead array (Luminex Molecular Diagnostics, Inc, Toronto, Ontario). The approach used by an individual laboratory for verification of a US Food and Drug Administration-approved assay is described. Specific verification data are provided to highlight the stepwise verification approach undertaken by a clinical diagnostic laboratory. Protocols for verification of in vitro diagnostic assays may vary between laboratories. However, all laboratories must verify several specific performance specifications prior to implementation of such assays for clinical use. We provide an example of an approach used for verifying performance of an assay for cystic fibrosis carrier screening.

Development and Validation of a Laboratory-Developed Multiplex Real-Time PCR Assay on the BD Max System for Detection of Herpes Simplex Virus and Varicella-Zoster Virus DNA in Various Clinical Specimens.

PubMed

Pillet, Sylvie; Verhoeven, Paul O; Epercieux, Amélie; Bourlet, Thomas; Pozzetto, Bruno

2015-06-01

A multiplex real-time PCR (quantitative PCR [qPCR]) assay detecting herpes simplex virus (HSV) and varicella-zoster virus (VZV) DNA together with an internal control was developed on the BD Max platform combining automated DNA extraction and an open amplification procedure. Its performance was compared to those of PCR assays routinely used in the laboratory, namely, a laboratory-developed test for HSV DNA on the LightCycler instrument and a test using a commercial master mix for VZV DNA on the ABI7500fast system. Using a pool of negative cerebrospinal fluid (CSF) samples spiked with either calibrated controls for HSV-1 and VZV or dilutions of a clinical strain that was previously quantified for HSV-2, the empirical limit of detection of the BD Max assay was 195.65, 91.80, and 414.07 copies/ml for HSV-1, HSV-2, and VZV, respectively. All the samples from HSV and VZV DNA quality control panels (Quality Control for Molecular Diagnostics [QCMD], 2013, Glasgow, United Kingdom) were correctly identified by the BD Max assay. From 180 clinical specimens of various origins, 2 CSF samples were found invalid by the BD Max assay due to the absence of detection of the internal control; a concordance of 100% was observed between the BD Max assay and the corresponding routine tests. The BD Max assay detected the PCR signal 3 to 4 cycles earlier than did the routine methods. With results available within 2 h on a wide range of specimens, this sensitive and fully automated PCR assay exhibited the qualities required for detecting simultaneously HSV and VZV DNA on a routine basis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Evaluation of HER-2/neu gene amplification and overexpression: comparison of frequently used assay methods in a molecularly characterized cohort of breast cancer specimens.

PubMed

Press, Michael F; Slamon, Dennis J; Flom, Kerry J; Park, Jinha; Zhou, Jian-Yuan; Bernstein, Leslie

2002-07-15

To compare and evaluate HER-2/neu clinical assay methods. One hundred seventeen breast cancer specimens with known HER-2/neu amplification and overexpression status were assayed with four different immunohistochemical assays and two different fluorescence in situ hybridization (FISH) assays. The accuracy of the FISH assays for HER-2/neu gene amplification was high, 97.4% for the Vysis PathVision assay (Vysis, Inc, Downers Grove, IL) and 95.7% for the the Ventana INFORM assay (Ventana, Medical Systems, Inc, Tucson, AZ). The immunohistochemical assay with the highest accuracy for HER-2/neu overexpression was obtained with R60 polyclonal antibody (96.6%), followed by immunohistochemical assays performed with 10H8 monoclonal antibody (95.7%), the Ventana CB11 monoclonal antibody (89.7%), and the DAKO HercepTest (88.9%; Dako, Corp, Carpinteria, CA). Only the sensitivities, and therefore, overall accuracy, of the DAKO Herceptest and Ventana CB11 immunohistochemical assays were significantly different from the more sensitive FISH assay. Based on these findings, the FISH assays were highly accurate, with immunohistochemical assays performed with R60 and 10H8 nearly as accurate. The DAKO HercepTest and the Ventana CB11 immunohistochemical assay were statistically significantly different from the Vysis FISH assay in evaluating these previously molecularly characterized breast cancer specimens.

Development of a peptide substrate for detection of Sunn pest damage in wheat flour.

PubMed

Hançerlioğulları, Begüm Zeynep; Köksel, Hamit; Dudak, Fahriye Ceyda

2018-05-07

Since the common protease substrates did not give satisfactory results for the determination of Sunn pest protease activity in damaged wheat, different peptide substrates derived from the repeat sequences of high molecular weight glutenin subunits were synthesized. Hydrolysis of peptides by pest protease was determined by HPLC. Among three peptides having the same consensus motifs, peptide1 (PGQGQQGYYPTSPQQ) showed the best catalytic efficiency. A novel assay was described for monitoring the enzymatic activity of protease extracted from damaged wheat flour. The selected peptide was labeled with a fluorophore (EDANS) and quencher (Dabcyl) to display fluorescence resonance energy transfer (FRET). The proteolytic activity was measured by the change in fluorescence intensity that occurred when the protease cleaved the peptide substrate. Furthermore, the developed assay was modified for rapid and easy detection of bug damage in flour. Flour samples were suspended in water and mixed with fluorescence peptide substrate. After centrifugation, the fluorescence intensities of the supernatants were determined which is proportional with the protease content of the flour. The total analysis time for the developed assay is estimated as 15 minutes. The developed assay permits a significant decrease in time and labor, offering sensitive detection of Sunn pest damage in wheat flour. This article is protected by copyright. All rights reserved.

Species-specific multiplex PCR for the diagnosis of Brucella ovis, Actinobacillus seminis, and Histophilus somni infection in rams.

PubMed

Moustacas, Valéria S; Silva, Teane M A; Costa, Luciana F; Xavier, Mariana N; Carvalho, Custódio A; Costa, Érica A; Paixão, Tatiane A; Santos, Renato L

2013-03-21

Infectious ovine epididymitis results in substantial economic losses worldwide due to reproductive failure and culling of breeders. The most common causative agents of these infections are Brucella ovis, Actinobacillus seminis, and Histophilus somni. The aim of this study was to develop a multiplex PCR assay for simultaneous detection of Brucella ovis, Actinobacillus seminis, and Histophilus somni with species-specific primers applied to biological samples for molecular diagnosis of these infections. The multiplex assay was capable of detecting B. ovis, A. seminis, and H. somni DNA simultaneously from genomic bacterial DNA samples and pool of semen samples from experimentally infected rams. The method was highly specific since it did not amplify DNA from other bacterial species that can potentially cause epididymitis in rams as well as species phylogenetically related to B. ovis. All negative control samples were negative in PCR multiplex assay. Urine can be used as an alternative to semen samples. The species-specific multiplex PCR assay developed in this study can be successfully used for the detection of three of the most common bacterial causes of ovine epididymitis.

Replication Competent Molecular Clones of HIV-1 Expressing Renilla Luciferase Facilitate the Analysis of Antibody Inhibition in PBMC

PubMed Central

Edmonds, Tara G.; Ding, Haitao; Yuan, Xing; Wei, Qing; Smith, Kendra S.; Conway, Joan A.; Wieczorek, Lindsay; Brown, Bruce; Polonis, Victoria; West, John T.; Montefiori, David C.; Kappes, John C.; Ochsenbauer, Christina

2010-01-01

Effective vaccine development for human immunodeficiency virus type 1 (HIV-1) will require assays that ascertain the capacity of vaccine immunogens to elicit neutralizing antibodies (NAb) to diverse HIV-1 strains. To facilitate NAb assessment in peripheral blood mononuclear cell (PBMC)-based assays, we developed an assay-adaptable platform based on a Renilla luciferase (LucR) expressing HIV-1 proviral backbone. LucR was inserted into pNL4-3 DNA, preserving all viral open reading frames. The proviral genome was engineered to facilitate expression of diverse HIV-1 env sequences, allowing analysis in an isogenic background. The resulting Env-IMC-LucR viruses are infectious, and LucR is stably expressed over multiple replications in PBMC. HIV-1 neutralization, targeting TZM-bl cells, was highly correlative comparing virus (LucR) and cell (firefly luciferase) readouts. In PBMC, NAb activity can be analyzed either within a single or multiple cycles of replication. These results represent advancement toward a standardizable PBMC-based neutralization assay for assessing HIV-1 vaccine immunogen efficacy. PMID:20863545

Replication competent molecular clones of HIV-1 expressing Renilla luciferase facilitate the analysis of antibody inhibition in PBMC.

PubMed

Edmonds, Tara G; Ding, Haitao; Yuan, Xing; Wei, Qing; Smith, Kendra S; Conway, Joan A; Wieczorek, Lindsay; Brown, Bruce; Polonis, Victoria; West, John T; Montefiori, David C; Kappes, John C; Ochsenbauer, Christina

2010-12-05

Effective vaccine development for human immunodeficiency virus type 1 (HIV-1) will require assays that ascertain the capacity of vaccine immunogens to elicit neutralizing antibodies (NAb) to diverse HIV-1 strains. To facilitate NAb assessment in peripheral blood mononuclear cell (PBMC)-based assays, we developed an assay-adaptable platform based on a Renilla luciferase (LucR) expressing HIV-1 proviral backbone. LucR was inserted into pNL4-3 DNA, preserving all viral open reading frames. The proviral genome was engineered to facilitate expression of diverse HIV-1 env sequences, allowing analysis in an isogenic background. The resulting Env-IMC-LucR viruses are infectious, and LucR is stably expressed over multiple replications in PBMC. HIV-1 neutralization, targeting TZM-bl cells, was highly correlative comparing virus (LucR) and cell (firefly luciferase) readouts. In PBMC, NAb activity can be analyzed either within a single or multiple cycles of replication. These results represent advancement toward a standardizable PBMC-based neutralization assay for assessing HIV-1 vaccine immunogen efficacy. Copyright © 2010 Elsevier Inc. All rights reserved.

A novel one-step real-time multiplex PCR assay to detect Streptococcus agalactiae presence and serotypes Ia, Ib, and III.

PubMed

Furfaro, Lucy L; Chang, Barbara J; Payne, Matthew S

2017-09-01

Streptococcus agalactiae is the leading cause of early-onset neonatal sepsis. Culture-based screening methods lack the sensitivity of molecular assays and do not indicate serotype; a potentially important virulence marker. We aimed to develop a multiplex PCR to detect S. agalactiae while simultaneously identifying serotypes Ia, Ib, and III; commonly associated with infant disease. Primers were designed to target S. agalactiae serotype-specific cps genes and the dltS gene. The assay was validated with 512 vaginal specimens from pregnant women. 112 (21.9%) were dltS positive, with 14.3%, 0.9%, and 6.3% of these identified as cps Ia, Ib, and III, respectively. Our assay is a specific and sensitive method to simultaneously detect S. agalactiae and serotypes Ia, Ib, and III in a single step. It is of high significance for clinical diagnostic applications and also provides epidemiological data on serotype, information that may be important for vaccine development and other targeted non-antibiotic therapies. Copyright © 2017 Elsevier Inc. All rights reserved.

Human Cell Assays for Synthesis-Dependent Strand Annealing and Crossing over During Double-Strand Break Repair.

PubMed

Zapotoczny, Grzegorz; Sekelsky, Jeff

2017-04-03

DNA double-strand breaks (DSBs) are one of the most deleterious types of lesions to the genome. Synthesis-dependent strand annealing (SDSA) is thought to be a major pathway of DSB repair, but direct tests of this model have only been conducted in budding yeast and Drosophila To better understand this pathway, we developed an SDSA assay for use in human cells. Our results support the hypothesis that SDSA is an important DSB repair mechanism in human cells. We used siRNA knockdown to assess the roles of a number of helicases suggested to promote SDSA. None of the helicase knockdowns reduced SDSA, but knocking down BLM or RTEL1 increased SDSA. Molecular analysis of repair products suggests that these helicases may prevent long-tract repair synthesis. Since the major alternative to SDSA (repair involving a double-Holliday junction intermediate) can lead to crossovers, we also developed a fluorescent assay that detects crossovers generated during DSB repair. Together, these assays will be useful in investigating features and mechanisms of SDSA and crossover pathways in human cells. Copyright © 2017 Zapotoczny and Sekelsky.

Human Cell Assays for Synthesis-Dependent Strand Annealing and Crossing over During Double-Strand Break Repair

PubMed Central

Zapotoczny, Grzegorz; Sekelsky, Jeff

2017-01-01

DNA double-strand breaks (DSBs) are one of the most deleterious types of lesions to the genome. Synthesis-dependent strand annealing (SDSA) is thought to be a major pathway of DSB repair, but direct tests of this model have only been conducted in budding yeast and Drosophila. To better understand this pathway, we developed an SDSA assay for use in human cells. Our results support the hypothesis that SDSA is an important DSB repair mechanism in human cells. We used siRNA knockdown to assess the roles of a number of helicases suggested to promote SDSA. None of the helicase knockdowns reduced SDSA, but knocking down BLM or RTEL1 increased SDSA. Molecular analysis of repair products suggests that these helicases may prevent long-tract repair synthesis. Since the major alternative to SDSA (repair involving a double-Holliday junction intermediate) can lead to crossovers, we also developed a fluorescent assay that detects crossovers generated during DSB repair. Together, these assays will be useful in investigating features and mechanisms of SDSA and crossover pathways in human cells. PMID:28179392

USE OF MOLECULAR PROBES TO ASSESS GEOGRAPHIC DISTRIBUTION OF PFIESTERIA SPECIES. (R827084)

EPA Science Inventory

We have developed multiple polymerase chain reaction (PCR)-based methods for the
detection of Pfiesteria sp. in cultures and environmental samples. More than 2,100 water and
sediment samples from estuarine sites of the U.S. Atlantic and gulf coasts were assayed for the
p...

Do online prognostication tools represent a valid alternative to genomic profiling in the context of adjuvant treatment of early breast cancer? A systematic review of the literature.

PubMed

El Hage Chehade, Hiba; Wazir, Umar; Mokbel, Kinan; Kasem, Abdul; Mokbel, Kefah

2018-01-01

Decision-making regarding adjuvant chemotherapy has been based on clinical and pathological features. However, such decisions are seldom consistent. Web-based predictive models have been developed using data from cancer registries to help determine the need for adjuvant therapy. More recently, with the recognition of the heterogenous nature of breast cancer, genomic assays have been developed to aid in the therapeutic decision-making. We have carried out a comprehensive literature review regarding online prognostication tools and genomic assays to assess whether online tools could be used as valid alternatives to genomic profiling in decision-making regarding adjuvant therapy in early breast cancer. Breast cancer has been recently recognized as a heterogenous disease based on variations in molecular characteristics. Online tools are valuable in guiding adjuvant treatment, especially in resource constrained countries. However, in the era of personalized therapy, molecular profiling appears to be superior in predicting clinical outcome and guiding therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparative genome analysis identifies novel nucleic acid diagnostic targets for use in the specific detection of Haemophilus influenzae.

PubMed

Coughlan, Helena; Reddington, Kate; Tuite, Nina; Boo, Teck Wee; Cormican, Martin; Barrett, Louise; Smith, Terry J; Clancy, Eoin; Barry, Thomas

2015-10-01

Haemophilus influenzae is recognised as an important human pathogen associated with invasive infections, including bloodstream infection and meningitis. Currently used molecular-based diagnostic assays lack specificity in correctly detecting and identifying H. influenzae. As such, there is a need to develop novel diagnostic assays for the specific identification of H. influenzae. Whole genome comparative analysis was performed to identify putative diagnostic targets, which are unique in nucleotide sequence to H. influenzae. From this analysis, we identified 2H. influenzae putative diagnostic targets, phoB and pstA, for use in real-time PCR diagnostic assays. Real-time PCR diagnostic assays using these targets were designed and optimised to specifically detect and identify all 55H. influenzae strains tested. These novel rapid assays can be applied to the specific detection and identification of H. influenzae for use in epidemiological studies and could also enable improved monitoring of invasive disease caused by these bacteria. Copyright © 2015 Elsevier Inc. All rights reserved.

Development of a One-Step Multiplex PCR Assay for Differential Detection of Major Mycobacterium Species

PubMed Central

Chae, Hansong; Han, Seung Jung; Kim, Su-Young; Ki, Chang-Seok; Huh, Hee Jae; Yong, Dongeun

2017-01-01

ABSTRACT The prevalence of tuberculosis continues to be high, and nontuberculous mycobacterial (NTM) infection has also emerged worldwide. Moreover, differential and accurate identification of mycobacteria to the species or subspecies level is an unmet clinical need. Here, we developed a one-step multiplex PCR assay using whole-genome analysis and bioinformatics to identify novel molecular targets. The aims of this assay were to (i) discriminate between the Mycobacterium tuberculosis complex (MTBC) and NTM using rv0577 or RD750, (ii) differentiate M. tuberculosis (M. tuberculosis) from MTBC using RD9, (iii) selectively identify the widespread M. tuberculosis Beijing genotype by targeting mtbk_20680, and (iv) simultaneously detect five clinically important NTM (M. avium, M. intracellulare, M. abscessus, M. massiliense, and M. kansasii) by targeting IS1311, DT1, mass_3210, and mkan_rs12360. An initial evaluation of the multiplex PCR assay using reference strains demonstrated 100% specificity for the targeted Mycobacterium species. Analytical sensitivity ranged from 1 to 10 pg for extracted DNA and was 103 and 104 CFU for pure cultures and nonhomogenized artificial sputum cultures, respectively, of the targeted species. The accuracy of the multiplex PCR assay was further evaluated using 55 reference strains and 94 mycobacterial clinical isolates. Spoligotyping, multilocus sequence analysis, and a commercial real-time PCR assay were employed as standard assays to evaluate the multiplex PCR assay with clinical M. tuberculosis and NTM isolates. The PCR assay displayed 100% identification agreement with the standard assays. Our multiplex PCR assay is a simple, convenient, and reliable technique for differential identification of MTBC, M. tuberculosis, M. tuberculosis Beijing genotype, and major NTM species. PMID:28659320

Development of a One-Step Multiplex PCR Assay for Differential Detection of Major Mycobacterium Species.

PubMed

Chae, Hansong; Han, Seung Jung; Kim, Su-Young; Ki, Chang-Seok; Huh, Hee Jae; Yong, Dongeun; Koh, Won-Jung; Shin, Sung Jae

2017-09-01

The prevalence of tuberculosis continues to be high, and nontuberculous mycobacterial (NTM) infection has also emerged worldwide. Moreover, differential and accurate identification of mycobacteria to the species or subspecies level is an unmet clinical need. Here, we developed a one-step multiplex PCR assay using whole-genome analysis and bioinformatics to identify novel molecular targets. The aims of this assay were to (i) discriminate between the Mycobacterium tuberculosis complex (MTBC) and NTM using rv0577 or RD750, (ii) differentiate M. tuberculosis ( M. tuberculosis ) from MTBC using RD9, (iii) selectively identify the widespread M. tuberculosis Beijing genotype by targeting mtbk_20680 , and (iv) simultaneously detect five clinically important NTM ( M. avium , M. intracellulare , M. abscessus , M. massiliense , and M. kansasii ) by targeting IS 1311 , DT1, mass_3210 , and mkan_rs12360 An initial evaluation of the multiplex PCR assay using reference strains demonstrated 100% specificity for the targeted Mycobacterium species. Analytical sensitivity ranged from 1 to 10 pg for extracted DNA and was 10 3 and 10 4 CFU for pure cultures and nonhomogenized artificial sputum cultures, respectively, of the targeted species. The accuracy of the multiplex PCR assay was further evaluated using 55 reference strains and 94 mycobacterial clinical isolates. Spoligotyping, multilocus sequence analysis, and a commercial real-time PCR assay were employed as standard assays to evaluate the multiplex PCR assay with clinical M. tuberculosis and NTM isolates. The PCR assay displayed 100% identification agreement with the standard assays. Our multiplex PCR assay is a simple, convenient, and reliable technique for differential identification of MTBC, M. tuberculosis , M. tuberculosis Beijing genotype, and major NTM species. Copyright © 2017 American Society for Microbiology.

In silico study of in vitro GPCR assays by QSAR modeling ...

EPA Pesticide Factsheets

The U.S. EPA is screening thousands of chemicals of environmental interest in hundreds of in vitro high-throughput screening (HTS) assays (the ToxCast program). One goal is to prioritize chemicals for more detailed analyses based on activity in molecular initiating events (MIE) of adverse outcome pathways (AOPs). However, the chemical space of interest for environmental exposure is much wider than this set of chemicals. Thus, there is a need to fill data gaps with in silico methods, and quantitative structure-activity relationships (QSARs) are a proven and cost effective approach to predict biological activity. ToxCast in turn provides relatively large datasets that are ideal for training and testing QSAR models. The overall goal of the study described here was to develop QSAR models to fill the data gaps in a larger environmental database of ~32k structures. The specific aim of the current work was to build QSAR models for 18 G-Protein Coupled Receptor (GPCR) assays, part of the aminergic category. Two QSAR modeling strategies were adopted: classification models were developed to separate chemicals into active/non-active classes, and then regression models were built to predict the potency values of the bioassays for the active chemicals. Multiple software programs were used to calculate constitutional, topological and substructural molecular descriptors from two-dimensional (2D) chemical structures. Model-fitting methods included PLSDA (partial least squares d

Molecular biomarkers for chronological age in animal ecology.

PubMed

Jarman, Simon N; Polanowski, Andrea M; Faux, Cassandra E; Robbins, Jooke; De Paoli-Iseppi, Ricardo; Bravington, Mark; Deagle, Bruce E

2015-10-01

The chronological age of an individual animal predicts many of its biological characteristics, and these in turn influence population-level ecological processes. Animal age information can therefore be valuable in ecological research, but many species have no external features that allow age to be reliably determined. Molecular age biomarkers provide a potential solution to this problem. Research in this area of molecular ecology has so far focused on a limited range of age biomarkers. The most commonly tested molecular age biomarker is change in average telomere length, which predicts age well in a small number of species and tissues, but performs poorly in many other situations. Epigenetic regulation of gene expression has recently been shown to cause age-related modifications to DNA and to cause changes in abundance of several RNA types throughout animal lifespans. Age biomarkers based on these epigenetic changes, and other new DNA-based assays, have already been applied to model organisms, humans and a limited number of wild animals. There is clear potential to apply these marker types more widely in ecological studies. For many species, these new approaches will produce age estimates where this was previously impractical. They will also enable age information to be gathered in cross-sectional studies and expand the range of demographic characteristics that can be quantified with molecular methods. We describe the range of molecular age biomarkers that have been investigated to date and suggest approaches for developing the newer marker types as age assays in nonmodel animal species. © 2015 John Wiley & Sons Ltd.

Comparative Evaluation of Serological and Molecular Methods for the Diagnosis of Scrub Typhus in Indian Settings.

PubMed

Gupta, Nitin; Chaudhry, Rama; Kabra, Sushil K; Lodha, Rakesh; Mirdha, Bijay Ranjan; Das, Bimal Kumar; Dey, Aparajit Ballay; Sreenivas, Vishnubhatla

2017-03-24

The major brunt of scrub typhus is borne by developing countries like India but the disease remains neglected. The rapid progression of this infection to serious complications and associated mortality calls for improved diagnostics. The immunoglobulin (Ig)M immunofluorescence assay (IFA), with all of its limitations remains the gold standard. Of 218 suspected cases from patients enrolled in a 2-year study, 30 cases of scrub typhus were detected by IgM IFA, using a 1:64 dilution. The sensitivities of the IgM enzyme-linked immunosorbent assay, IgM rapid flow assay (RFA), and IgG RFA were found to be 97%, 87%, and 77%, respectively. Their respective specificities were 100%, 100%, and 92%, respectively. The sensitivity and specificity of a nested PCR assay targeting a gene encoding a 56-kDa protein were found to be 50% and 100%, respectively.

Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays.

PubMed

Abras, Alba; Ballart, Cristina; Llovet, Teresa; Roig, Carme; Gutiérrez, Cristina; Tebar, Silvia; Berenguer, Pere; Pinazo, María-Jesús; Posada, Elizabeth; Gascón, Joaquim; Schijman, Alejandro G; Gállego, Montserrat; Muñoz, Carmen

2018-01-01

Polymerase chain reaction (PCR) has become a useful tool for the diagnosis of Trypanosoma cruzi infection. The development of automated DNA extraction methodologies and PCR systems is an important step toward the standardization of protocols in routine diagnosis. To date, there are only two commercially available Real-Time PCR assays for the routine laboratory detection of T. cruzi DNA in clinical samples: TCRUZIDNA.CE (Diagnostic Bioprobes Srl) and RealCycler CHAG (Progenie Molecular). Our aim was to evaluate the RealCycler CHAG assay taking into account the whole process. We assessed the usefulness of an automated DNA extraction system based on magnetic particles (EZ1 Virus Mini Kit v2.0, Qiagen) combined with a commercially available Real-Time PCR assay targeting satellite DNA (SatDNA) of T. cruzi (RealCycler CHAG), a methodology used for routine diagnosis in our hospital. It was compared with a well-known strategy combining a commercial DNA isolation kit based on silica columns (High Pure PCR Template Preparation Kit, Roche Diagnostics) with an in-house Real-Time PCR targeting SatDNA. The results of the two methodologies were in almost perfect agreement, indicating they can be used interchangeably. However, when variations in protocol factors were applied (sample treatment, extraction method and Real-Time PCR), the results were less convincing. A comprehensive fine-tuning of the whole procedure is the key to successful results. Guanidine EDTA-blood (GEB) samples are not suitable for DNA extraction based on magnetic particles due to inhibition, at least when samples are not processed immediately. This is the first study to evaluate the RealCycler CHAG assay taking into account the overall process, including three variables (sample treatment, extraction method and Real-Time PCR). Our findings may contribute to the harmonization of protocols between laboratories and to a wider application of Real-Time PCR in molecular diagnostic laboratories associated with health centers.

Molecular Phenotyping Combines Molecular Information, Biological Relevance, and Patient Data to Improve Productivity of Early Drug Discovery.

PubMed

Drawnel, Faye Marie; Zhang, Jitao David; Küng, Erich; Aoyama, Natsuyo; Benmansour, Fethallah; Araujo Del Rosario, Andrea; Jensen Zoffmann, Sannah; Delobel, Frédéric; Prummer, Michael; Weibel, Franziska; Carlson, Coby; Anson, Blake; Iacone, Roberto; Certa, Ulrich; Singer, Thomas; Ebeling, Martin; Prunotto, Marco

2017-05-18

Today, novel therapeutics are identified in an environment which is intrinsically different from the clinical context in which they are ultimately evaluated. Using molecular phenotyping and an in vitro model of diabetic cardiomyopathy, we show that by quantifying pathway reporter gene expression, molecular phenotyping can cluster compounds based on pathway profiles and dissect associations between pathway activities and disease phenotypes simultaneously. Molecular phenotyping was applicable to compounds with a range of binding specificities and triaged false positives derived from high-content screening assays. The technique identified a class of calcium-signaling modulators that can reverse disease-regulated pathways and phenotypes, which was validated by structurally distinct compounds of relevant classes. Our results advocate for application of molecular phenotyping in early drug discovery, promoting biological relevance as a key selection criterion early in the drug development cascade. Copyright © 2017 Elsevier Ltd. All rights reserved.

Current and future molecular diagnostics in non-small-cell lung cancer.

PubMed

Li, Chun Man; Chu, Wing Ying; Wong, Di Lun; Tsang, Hin Fung; Tsui, Nancy Bo Yin; Chan, Charles Ming Lok; Xue, Vivian Wei Wen; Siu, Parco Ming Fai; Yung, Benjamin Yat Ming; Chan, Lawrence Wing Chi; Wong, Sze Chuen Cesar

2015-01-01

The molecular investigation of lung cancer has opened up an advanced area for the diagnosis and therapeutic management of lung cancer patients. Gene alterations in cancer initiation and progression provide not only information on molecular changes in lung cancer but also opportunities in advanced therapeutic regime by personalized targeted therapy. EGFR mutations and ALK rearrangement are important predictive biomarkers for the efficiency of tyrosine kinase inhibitor treatment in lung cancer patients. Moreover, epigenetic aberration and microRNA dysregulation are recent advances in the early detection and monitoring of lung cancer. Although a wide range of molecular tests are available, standardization and validation of assay protocols are essential for the quality of the test outcome. In this review, current and new advancements of molecular biomarkers for non-small-cell lung cancer will be discussed. Recommendations on future development of molecular diagnostic services will also be explored.

Application of computer-assisted molecular modeling for immunoassay of low molecular weight food contaminants: A review.

PubMed

Xu, Zhen-Lin; Shen, Yu-Dong; Beier, Ross C; Yang, Jin-Yi; Lei, Hong-Tao; Wang, Hong; Sun, Yuan-Ming

2009-08-11

Immunoassay for low molecular weight food contaminants, such as pesticides, veterinary drugs, and mycotoxins is now a well-established technique which meets the demand for a rapid, reliable, and cost-effective analytical method. However, due to limited understanding of the molecular structure of antibody binding sites and antigenic epitopes, as well as the intermolecular binding forces that come into play, the traditional 'trial and error' method used to develop antibodies still remains the method of choice. Therefore, development of enhanced immunochemical techniques for specific- and generic-assays, requires new approaches for antibody design that will improve affinity and specificity of the antibody in a more rapid and economic manner. Computer-assisted molecular modeling (CAMM) has been demonstrated to be a useful tool to help the immunochemist develop immunoassays. CAMM methods can be used to help direct improvements to important antibody features, and can provide insights into the effects of molecular structure on biological activity that are difficult or impossible to obtain in any other way. In this review, we briefly summarize applications of CAMM in immunoassay development, including assisting in hapten design, explaining cross-reactivity, modeling antibody-antigen interactions, and providing insights into the effects of the mouse body temperature on the three-dimensional conformation of a hapten during antibody production. The fundamentals and theory, programs and software, limitations, and prospects of CAMM in immunoassay development were also discussed.

Molecular allergy diagnostics using multiplex assays: methodological and practical considerations for use in research and clinical routine: Part 21 of the Series Molecular Allergology.

PubMed

Jakob, Thilo; Forstenlechner, Peter; Matricardi, Paolo; Kleine-Tebbe, Jörg

The availability of single allergens and their use in microarray technology enables the simultaneous determination of specific IgE (sIgE) to a multitude of different allergens (> 100) in a multiplex procedure requiring only minute amounts of serum. This allows extensive individual sensitization profiles to be determined from a single analysis. Combined with a patient's medical history, these profiles simplify identification of cross-reactivity; permit a more accurate estimation of the risk of severe reactions; and enable the indication for specific immunotherapy to be more precisely established, particularly in cases of polysensitization. Strictly speaking, a multiplex assay is not a single test, but instead more than 100 simultaneous tests. This places considerable demands on the production, quality assurance, and interpretation of data. The following chapter describes the multiplex test systems currently available and discusses their characteristics. Performance data are presented and the sIgE values obtained from multiplex and singleplex assays are compared. Finally, the advantages and limitations of molecular allergy diagnostics using multiplex assays in clinical routine are discussed, and innovative possibilities for clinical research are described. The multiplex diagnostic tests available for clinical routine have now become well established. The interpretation of test results is demanding, particularly since all individual results need to be checked for their plausibility and clinical relevance on the basis of previous history (patient history, clinical symptoms, challenge test results). There is still room for improvement in certain areas, for example with respect to the overall test sensitivity of the method, as well as the availability and quality of particular allergens. The current test systems are just the beginning of a continuous development that will influence and most likely change clinical allergology in the coming years.

MicroRNA Detection Using a Double Molecular Beacon Approach: Distinguishing Between miRNA and Pre-miRNA.

PubMed

James, Amanda Marie; Baker, Meredith B; Bao, Gang; Searles, Charles D

2017-01-01

MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally regulate gene expression and are recognized for their roles both as modulators of disease progression and as biomarkers of disease activity, including neurological diseases, cancer, and cardiovascular disease (CVD). Commonly, miRNA abundance is assessed using quantitative real-time PCR (qRT-PCR), however, qRT-PCR for miRNA can be labor intensive, time consuming, and may lack specificity for detection of mature versus precursor forms of miRNA. Here, we describe a novel double molecular beacon approach to miRNA assessment that can distinguish and quantify mature versus precursor forms of miRNA in a single assay, an essential feature for use of miRNAs as biomarkers for disease. Using this approach, we found that molecular beacons with DNA or combined locked nucleic acid (LNA)-DNA backbones can detect mature and precursor miRNAs (pre-miRNAs) of low (< 1 nM) abundance in vitro . The double molecular beacon assay was accurate in assessing miRNA abundance in a sample containing a mixed population of mature and precursor miRNAs. In contrast, qRT-PCR and the single molecular beacon assay overestimated miRNA abundance. Additionally, the double molecular beacon assay was less labor intensive than traditional qRT-PCR and had 10-25% increased specificity. Our data suggest that the double molecular beacon-based approach is more precise and specific than previous methods, and has the promise of being the standard for assessing miRNA levels in biological samples.

MicroRNA Detection Using a Double Molecular Beacon Approach: Distinguishing Between miRNA and Pre-miRNA

PubMed Central

James, Amanda Marie; Baker, Meredith B.; Bao, Gang; Searles, Charles D.

2017-01-01

MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally regulate gene expression and are recognized for their roles both as modulators of disease progression and as biomarkers of disease activity, including neurological diseases, cancer, and cardiovascular disease (CVD). Commonly, miRNA abundance is assessed using quantitative real-time PCR (qRT-PCR), however, qRT-PCR for miRNA can be labor intensive, time consuming, and may lack specificity for detection of mature versus precursor forms of miRNA. Here, we describe a novel double molecular beacon approach to miRNA assessment that can distinguish and quantify mature versus precursor forms of miRNA in a single assay, an essential feature for use of miRNAs as biomarkers for disease. Using this approach, we found that molecular beacons with DNA or combined locked nucleic acid (LNA)-DNA backbones can detect mature and precursor miRNAs (pre-miRNAs) of low (< 1 nM) abundance in vitro. The double molecular beacon assay was accurate in assessing miRNA abundance in a sample containing a mixed population of mature and precursor miRNAs. In contrast, qRT-PCR and the single molecular beacon assay overestimated miRNA abundance. Additionally, the double molecular beacon assay was less labor intensive than traditional qRT-PCR and had 10-25% increased specificity. Our data suggest that the double molecular beacon-based approach is more precise and specific than previous methods, and has the promise of being the standard for assessing miRNA levels in biological samples. PMID:28255356

Development of internally controlled duplex real-time NASBA diagnostics assays for the detection of microorganisms associated with bacterial meningitis.

PubMed

Clancy, Eoin; Coughlan, Helena; Higgins, Owen; Boo, Teck Wee; Cormican, Martin; Barrett, Louise; Smith, Terry J; Reddington, Kate; Barry, Thomas

2016-08-01

Three duplex molecular beacon based real-time Nucleic Acid Sequence Based Amplification (NASBA) assays have been designed and experimentally validated targeting RNA transcripts for the detection and identification of Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae respectively. Each real-time NASBA diagnostics assay includes an endogenous non-competitive Internal Amplification Control (IAC) to amplify the splice variant 1 mRNA of the Homo sapiens TBP gene from human total RNA. All three duplex real-time NASBA diagnostics assays were determined to be 100% specific for the target species tested for. Also the Limits of Detection (LODs) for the H. influenzae, N. meningitidis and S. pneumoniae duplex real-time NASBA assays were 55.36, 0.99, and 57.24 Cell Equivalents (CE) respectively. These robust duplex real-time NASBA diagnostics assays have the potential to be used in a clinical setting for the rapid (<60min) specific detection and identification of the most prominent microorganisms associated with bacterial meningitis in humans. Copyright © 2016 Elsevier B.V. All rights reserved.

Total internal reflection ellipsometry and SPR detection of low molecular weight environmental toxins

NASA Astrophysics Data System (ADS)

Nabok, A. V.; Tsargorodskaya, A.; Hassan, A. K.; Starodub, N. F.

2005-06-01

The environmental toxins, such as herbicides simazine and atrazine, and T2 mycotoxin were registered with the optical methods of surface plasmon resonance (SPR) and recently developed total internal reflection ellipsometry (TIRE). The immune assay approach was exploited for in situ registration of the above low molecular weight toxins with specific antibodies immobilised onto the gold surface via (poly)allylamine hydrochloride layer using electrostatic self-assembly (ESA) technique. The comparison of two methods of SPR and TIRE shows a higher sensitivity of the latter.

Genetic sex determination assays in 53 mammalian species: Literature analysis and guidelines for reporting standardization.

PubMed

Hrovatin, Karin; Kunej, Tanja

2018-01-01

Erstwhile, sex was determined by observation, which is not always feasible. Nowadays, genetic methods are prevailing due to their accuracy, simplicity, low costs, and time-efficiency. However, there is no comprehensive review enabling overview and development of the field. The studies are heterogeneous, lacking a standardized reporting strategy. Therefore, our aim was to collect genetic sexing assays for mammals and assemble them in a catalogue with unified terminology. Publications were extracted from online databases using key words such as sexing and molecular. The collected data were supplemented with species and gene IDs and the type of sex-specific sequence variant (SSSV). We developed a catalogue and graphic presentation of diagnostic tests for molecular sex determination of mammals, based on 58 papers published from 2/1991 to 10/2016. The catalogue consists of five categories: species, genes, SSSVs, methods, and references. Based on the analysis of published literature, we propose minimal requirements for reporting, consisting of: species scientific name and ID, genetic sequence with name and ID, SSSV, methodology, genomic coordinates (e.g., restriction sites, SSSVs), amplification system, and description of detected amplicon and controls. The present study summarizes vast knowledge that has up to now been scattered across databases, representing the first step toward standardization regarding molecular sexing, enabling a better overview of existing tests and facilitating planned designs of novel tests. The project is ongoing; collecting additional publications, optimizing field development, and standardizing data presentation are needed.

A novel PCR-RFLP assay for molecular characterization of Echinococcus granulosus sensu lato and closely related species in developing countries.

PubMed

Chaâbane-Banaoues, Raja; Oudni-M'rad, Myriam; M'rad, Selim; Amani, Hizem; Mezhoud, Habib; Babba, Hamouda

2016-10-01

Cystic echinococcosis, due to Echinococcus granulosus sensu lato (s. l.), currently affects three million people, especially in low-income countries and results in high livestock production loss. DNA-based methods demonstrated genetic variability of E. granulosus s. l., and five species were recognized to belong to the complex, including E. granulosus sensu stricto (s.s) (genotypes G1-G3), Echinococcus equinus (genotype G4), Echinococcus ortleppi (genotype G5), Echinococcus canadensis (genotypes G6-G10), and the lion strain Echinococcus felidis. The characterization of Echinococcus species responsible for human and animal echinococcosis is crucial to adapt the preventive measures against this parasitic disease. The sequencing approach is the gold standard for genotyping assays. Unfortunately, developing countries do not often have access to these techniques. Based on in silico RFLP tools, we described an accurate PCR-RFLP method for Echinococcus spp. characterization. The double digestion with the HaeIII and HinfI restriction enzymes of the PCR product from nad1 gene (1071 bp) led to a clear discrimination between E. granulosus s. l. and most closely related species (Echinococcus shiquicus and Echinococcus multilocularis).Molecular procedures and phylogenetic analysis confirmed the efficiency and the reproducibility of this simple and fast PCR-RFLP method. This technique is proved useful for fresh/unfixed and FF-PET tissues and enables large-scale molecular epidemiological screening in developing countries.

Tiered High-Throughput Screening Approach to Identify ...

EPA Pesticide Factsheets

High-throughput screening (HTS) for potential thyroid–disrupting chemicals requires a system of assays to capture multiple molecular-initiating events (MIEs) that converge on perturbed thyroid hormone (TH) homeostasis. Screening for MIEs specific to TH-disrupting pathways is limited in the US EPA ToxCast screening assay portfolio. To fill one critical screening gap, the Amplex UltraRed-thyroperoxidase (AUR-TPO) assay was developed to identify chemicals that inhibit TPO, as decreased TPO activity reduces TH synthesis. The ToxCast Phase I and II chemical libraries, comprised of 1,074 unique chemicals, were initially screened using a single, high concentration to identify potential TPO inhibitors. Chemicals positive in the single concentration screen were retested in concentration-response. Due to high false positive rates typically observed with loss-of-signal assays such as AUR-TPO, we also employed two additional assays in parallel to identify possible sources of nonspecific assay signal loss, enabling stratification of roughly 300 putative TPO inhibitors based upon selective AUR-TPO activity. A cell-free luciferase inhibition assay was used to identify nonspecific enzyme inhibition among the putative TPO inhibitors, and a cytotoxicity assay using a human cell line was used to estimate the cellular tolerance limit. Additionally, the TPO inhibition activities of 150 chemicals were compared between the AUR-TPO and an orthogonal peroxidase oxidation assay using

Inhibition profiles of phosphatidylinositol 3-kinase inhibitors against PI3K superfamily and human cancer cell line panel JFCR39.

PubMed

Kong, Dexin; Dan, Shingo; Yamazaki, Kanami; Yamori, Takao

2010-04-01

As accumulating evidences suggest close involvement of phosphatidylinositol 3-kinase (PI3K) in various diseases particularly cancer, considerable competition occurs in development of PI3K inhibitors. Consequently, novel PI3K inhibitors such as ZSTK474, GDC-0941 and NVP-BEZ235 have been developed. Even though all these inhibitors were reported to inhibit class I PI3K but not dozens of protein kinases, whether they have different molecular targets remained unknown. To investigate such molecular target specificity, we have determined the inhibitory effects of these novel inhibitors together with classical PI3K inhibitor LY294002 on PI3K superfamily (including classes I, II, and III PI3Ks, PI4K and PI3K-related kinases) by using several novel non-radioactive biochemical assays. As a result, ZSTK474 and GDC-0941 indicated highly similar inhibition profiles for PI3K superfamily, with class I PI3K specificity much higher than NVP-BEZ235 and LY294002. We further investigated their growth inhibition effects on JFCR39, a human cancer cell line panel which we established for molecular target identification, and analysed their cell growth inhibition profiles (fingerprints) by using COMPARE analysis programme. Interestingly, we found ZSTK474 exhibited a highly similar fingerprint with GDC-0941 (r=0.863), more similar than with that of either NVP-BEZ235 or LY294002, suggesting that ZSTK474 shares more in molecular targets with GDC-0941 than with either of the other two PI3K inhibitors, consistent with the biochemical assay result. The biological implication of the difference in molecular target specificity of these PI3K inhibitors is under investigation. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Collaborative study for the establishment of replacement batches of heparin low- molecular-mass for assay biological reference preparations.

PubMed

Terao, E; Daas, A; Rautmann, G; Buchheit, K-H

2010-10-01

A collaborative study was run by the European Directorate for the Quality of Medicines & HealthCare (EDQM) in the context of the Biological Standardisation Programme (BSP), under the aegis of the Council of Europe and the European Commission, to establish replacement batches for the dwindling stocks of the Heparin low-molecular-mass for assay European Pharmacopoeia Biological Reference Preparation (BRP). The replacement batches of BRP are intended to be used in the assays for anti-Xa and anti-IIa activities, as described in the European Pharmacopoeia (Ph. Eur.) monograph Heparins, low-molecular-mass (0828). Three freeze-dried candidate batches were calibrated against the current International Standard (IS) for Heparin, lowmolecular- weight (2nd IS, 01/608). For the purpose of the continuity check between subsequent BRP batches, the current Heparin low-molecular-mass for assay BRP (batch 5) was also included in the test panel. Thirteen official medicines control and manufacturers laboratories from European and non-European countries contributed data. A central statistical analysis of the datasets was performed at the EDQM. On the basis of the results, the 3 candidate materials were assigned a potency of 104 IU/vial for the anti-Xa activity and 31 IU/vial for the anti-IIa activity. Taken into account the preliminary stability data and the results of this collaborative study, the 3 batches of candidate BRP were adopted in June 2010 by the Commission of the Ph. Eur. as Heparin low-molecular-mass for assay BRP batches 6, 7 and 8.

Unveiling molecular events in the brain by noninvasive imaging.

PubMed

Klohs, Jan; Rudin, Markus

2011-10-01

Neuroimaging allows researchers and clinicians to noninvasively assess structure and function of the brain. With the advances of imaging modalities such as magnetic resonance, nuclear, and optical imaging; the design of target-specific probes; and/or the introduction of reporter gene assays, these technologies are now capable of visualizing cellular and molecular processes in vivo. Undoubtedly, the system biological character of molecular neuroimaging, which allows for the study of molecular events in the intact organism, will enhance our understanding of physiology and pathophysiology of the brain and improve our ability to diagnose and treat diseases more specifically. Technical/scientific challenges to be faced are the development of highly sensitive imaging modalities, the design of specific imaging probe molecules capable of penetrating the CNS and reporting on endogenous cellular and molecular processes, and the development of tools for extracting quantitative, biologically relevant information from imaging data. Today, molecular neuroimaging is still an experimental approach with limited clinical impact; this is expected to change within the next decade. This article provides an overview of molecular neuroimaging approaches with a focus on rodent studies documenting the exploratory state of the field. Concepts are illustrated by discussing applications related to the pathophysiology of Alzheimer's disease.

Design and Construction of a Single-Tube, LATE-PCR, Multiplex Endpoint Assay with Lights-On/Lights-Off Probes for the Detection of Pathogens Associated with Sepsis

PubMed Central

Carver-Brown, Rachel K.; Reis, Arthur H.; Rice, Lisa M.; Czajka, John W.; Wangh, Lawrence J.

2012-01-01

Aims. The goal of this study was to construct a single tube molecular diagnostic multiplex assay for the detection of microbial pathogens commonly associated with septicemia, using LATE-PCR and Lights-On/Lights-Off probe technology. Methods and Results. The assay described here identified pathogens associated with sepsis by amplification and analysis of the 16S ribosomal DNA gene sequence for bacteria and specific gene sequences for fungi. A sequence from an unidentified gene in Lactococcus lactis subsp. cremoris served as a positive control for assay function. LATE-PCR was used to generate single-stranded amplicons that were then analyzed at endpoint over a wide temperature range in a specific fluorescent color. Each bacterial target was identified by its pattern of hybridization to Lights-On/Lights-Off probes derived from molecular beacons. Complex mixtures of targets were also detected. Conclusions. All microbial targets were identified in samples containing low starting copy numbers of pathogen genomic DNA, both as individual targets and in complex mixtures. Significance and Impact of the Study. This assay uses new technology to achieve an advance in the field of molecular diagnostics: a single-tube multiplex assay for identification of pathogens commonly associated with sepsis. PMID:23326668

High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization

PubMed Central

Rodríguez-Escribano, David; de Salas, Felipe; Camarero, Susana

2017-01-01

Lignin valorization is a pending issue for the integrated conversion of lignocellulose in consumer goods. Lignosulfonates (LS) are the main technical lignins commercialized today. However, their molecular weight should be enlarged to meet application requirements as additives or dispersing agents. Oxidation of lignosulfonates with fungal oxidoreductases, such as laccases, can increase the molecular weight of lignosulfonates by the cross-linking of lignin phenols. To advance in this direction, we describe here the development of a high-throughput screening (HTS) assay for the directed evolution of laccases, with lignosulfonate as substrate and the Folin–Ciocalteau reagent (FCR), to detect the decrease in phenolic content produced upon polymerization of lignosulfonate by the enzyme. Once the reaction conditions were adjusted to the 96-well-plate format, the enzyme for validating the assay was selected from a battery of high-redox-potential laccase variants functionally expressed in S. cerevisiae (the preferred host for the directed evolution of fungal oxidoreductases). The colorimetric response (absorbance at 760 nm) correlated with laccase activity secreted by the yeast. The HTS assay was reproducible (coefficient of variation (CV) = 15%) and sensitive enough to detect subtle differences in activity among yeast clones expressing a laccase mutant library obtained by error-prone PCR (epPCR). The method is therefore feasible for screening thousands of clones during the precise engineering of laccases toward valorization of lignosulfonates. PMID:28820431

Direct Molecular Detection and Genotyping of Borrelia burgdorferi from Whole Blood of Patients with Early Lyme Disease

PubMed Central

Eshoo, Mark W.; Crowder, Christopher C.; Rebman, Alison W.; Rounds, Megan A.; Matthews, Heather E.; Picuri, John M.; Soloski, Mark J.; Ecker, David J.; Schutzer, Steven E.; Aucott, John N.

2012-01-01

Direct molecular tests in blood for early Lyme disease can be insensitive due to low amount of circulating Borrelia burgdorferi DNA. To address this challenge, we have developed a sensitive strategy to both detect and genotype B. burgdorferi directly from whole blood collected during the initial patient visit. This strategy improved sensitivity by employing 1.25 mL of whole blood, a novel pre-enrichment of the entire specimen extract for Borrelia DNA prior to a multi-locus PCR and electrospray ionization mass spectrometry detection assay. We evaluated the assay on blood collected at the initial presentation from 21 endemic area patients who had both physician-diagnosed erythema migrans (EM) and positive two-tiered serology either at the initial visit or at a follow-up visit after three weeks of antibiotic therapy. Results of this DNA analysis showed detection of B. burgdorferi in 13 of 21 patients (62%). In most cases the new assay also provided the B. burgdorferi genotype. The combined results of our direct detection assay with initial physician visit serology resulted in the detection of early Lyme disease in 19 of 21 (90%) of patients at the initial visit. In 5 of 21 cases we demonstrate the ability to detect B. burgdorferi in early Lyme disease directly from whole blood specimens prior to seroconversion. PMID:22590620

High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodriguez-Escribano, David; de Salas, Felipe; Pardo, Isabel

Lignin valorization is a pending issue for the integrated conversion of lignocellulose in consumer goods. Lignosulfonates (LS) are the main technical lignins commercialized today. However, their molecular weight should be enlarged to meet application requirements as additives or dispersing agents. Oxidation of lignosulfonates with fungal oxidoreductases, such as laccases, can increase the molecular weight of lignosulfonates by the cross-linking of lignin phenols. To advance in this direction, we describe here the development of a high-throughput screening (HTS) assay for the directed evolution of laccases, with lignosulfonate as substrate and the Folin-Ciocalteau reagent (FCR), to detect the decrease in phenolic contentmore » produced upon polymerization of lignosulfonate by the enzyme. Once the reaction conditions were adjusted to the 96-well-plate format, the enzyme for validating the assay was selected from a battery of high-redox-potential laccase variants functionally expressed in S. cerevisiae (the preferred host for the directed evolution of fungal oxidoreductases). The colorimetric response (absorbance at 760 nm) correlated with laccase activity secreted by the yeast. The HTS assay was reproducible (coefficient of variation (CV) = 15%) and sensitive enough to detect subtle differences in activity among yeast clones expressing a laccase mutant library obtained by error-prone PCR (epPCR). As a result, the method is therefore feasible for screening thousands of clones during the precise engineering of laccases toward valorization of lignosulfonates.« less

High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization

DOE PAGES

Rodriguez-Escribano, David; de Salas, Felipe; Pardo, Isabel; ...

2017-08-18

Lignin valorization is a pending issue for the integrated conversion of lignocellulose in consumer goods. Lignosulfonates (LS) are the main technical lignins commercialized today. However, their molecular weight should be enlarged to meet application requirements as additives or dispersing agents. Oxidation of lignosulfonates with fungal oxidoreductases, such as laccases, can increase the molecular weight of lignosulfonates by the cross-linking of lignin phenols. To advance in this direction, we describe here the development of a high-throughput screening (HTS) assay for the directed evolution of laccases, with lignosulfonate as substrate and the Folin-Ciocalteau reagent (FCR), to detect the decrease in phenolic contentmore » produced upon polymerization of lignosulfonate by the enzyme. Once the reaction conditions were adjusted to the 96-well-plate format, the enzyme for validating the assay was selected from a battery of high-redox-potential laccase variants functionally expressed in S. cerevisiae (the preferred host for the directed evolution of fungal oxidoreductases). The colorimetric response (absorbance at 760 nm) correlated with laccase activity secreted by the yeast. The HTS assay was reproducible (coefficient of variation (CV) = 15%) and sensitive enough to detect subtle differences in activity among yeast clones expressing a laccase mutant library obtained by error-prone PCR (epPCR). As a result, the method is therefore feasible for screening thousands of clones during the precise engineering of laccases toward valorization of lignosulfonates.« less

Principles of quantitation of viral loads using nucleic acid sequence-based amplification in combination with homogeneous detection using molecular beacons.

PubMed

Weusten, Jos J A M; Carpay, Wim M; Oosterlaken, Tom A M; van Zuijlen, Martien C A; van de Wiel, Paul A

2002-03-15

For quantitative NASBA-based viral load assays using homogeneous detection with molecular beacons, such as the NucliSens EasyQ HIV-1 assay, a quantitation algorithm is required. During the amplification process there is a constant growth in the concentration of amplicons to which the beacon can bind while generating a fluorescence signal. The overall fluorescence curve contains kinetic information on both amplicon formation and beacon binding, but only the former is relevant for quantitation. In the current paper, mathematical modeling of the relevant processes is used to develop an equation describing the fluorescence curve as a function of the amplification time and the relevant kinetic parameters. This equation allows reconstruction of RNA formation, which is characterized by an exponential increase in concentrations as long as the primer concentrations are not rate limiting and by linear growth over time after the primer pool is depleted. During the linear growth phase, the actual quantitation is based on assessing the amplicon formation rate from the viral RNA relative to that from a fixed amount of calibrator RNA. The quantitation procedure has been successfully applied in the NucliSens EasyQ HIV-1 assay.

Detection and Identification of Probiotic Lactobacillus plantarum Strains by Multiplex PCR Using RAPD-Derived Primers

PubMed Central

Galanis, Alex; Kourkoutas, Yiannis; Tassou, Chrysoula C.; Chorianopoulos, Nikos

2015-01-01

Lactobacillus plantarum 2035 and Lactobacillus plantarum ACA-DC 2640 are two lactic acid bacteria (LAB) strains that have been isolated from Feta cheese. Both display significant potential for the production of novel probiotic food products. The aim of the present study was the development of an accurate and efficient method for the molecular detection and identification of the above strains in a single reaction. A multiplex PCR assay was designed for each strain, based on specific primers derived from Random Amplified Polymorphic DNA (RAPD) Sequenced Characterized Amplified Region (SCAR) analysis. The specificity of the assay was tested with a total of 23 different LAB strains, for L. plantarum 2035 and L. plantarum ACA-DC 2640. The multiplex PCR assay was also successfully applied for the detection of the above cultures in yogurt samples prepared in our lab. The proposed methodology may be applied for monitoring the presence of these strains in food products, thus evaluating their probiotic character. Moreover, our strategy may be adapted for other novel LAB strains with probiotic potential, thus providing a powerful tool for molecular discrimination that could be invaluable to the food industry. PMID:26506345

Detection and Identification of Probiotic Lactobacillus plantarum Strains by Multiplex PCR Using RAPD-Derived Primers.

PubMed

Galanis, Alex; Kourkoutas, Yiannis; Tassou, Chrysoula C; Chorianopoulos, Nikos

2015-10-22

Lactobacillus plantarum 2035 and Lactobacillus plantarum ACA-DC 2640 are two lactic acid bacteria (LAB) strains that have been isolated from Feta cheese. Both display significant potential for the production of novel probiotic food products. The aim of the present study was the development of an accurate and efficient method for the molecular detection and identification of the above strains in a single reaction. A multiplex PCR assay was designed for each strain, based on specific primers derived from Random Amplified Polymorphic DNA (RAPD) Sequenced Characterized Amplified Region (SCAR) analysis. The specificity of the assay was tested with a total of 23 different LAB strains, for L. plantarum 2035 and L. plantarum ACA-DC 2640. The multiplex PCR assay was also successfully applied for the detection of the above cultures in yogurt samples prepared in our lab. The proposed methodology may be applied for monitoring the presence of these strains in food products, thus evaluating their probiotic character. Moreover, our strategy may be adapted for other novel LAB strains with probiotic potential, thus providing a powerful tool for molecular discrimination that could be invaluable to the food industry.

Discovery of Highly Potent Tyrosinase Inhibitor, T1, with Significant Anti-Melanogenesis Ability by zebrafish in vivo Assay and Computational Molecular Modeling

NASA Astrophysics Data System (ADS)

Chen, Wang-Chuan; Tseng, Tien-Sheng; Hsiao, Nai-Wan; Lin, Yun-Lian; Wen, Zhi-Hong; Tsai, Chin-Chuan; Lee, Yu-Ching; Lin, Hui-Hsiung; Tsai, Keng-Chang

2015-01-01

Tyrosinase is involved in melanin biosynthesis and the abnormal accumulation of melanin pigments leading to hyperpigmentation disorders that can be treated with depigmenting agents. A natural product T1, bis(4-hydroxybenzyl)sulfide, isolated from the Chinese herbal plant, Gastrodia elata, is a strong competitive inhibitor against mushroom tyrosinase (IC50 = 0.53 μM, Ki = 58 +/- 6 nM), outperforms than kojic acid. The cell viability and melanin quantification assay demonstrate that 50 μM of T1 apparently attenuates 20% melanin content of human normal melanocytes without significant cell toxicity. Moreover, the zebrafish in vivo assay reveals that T1 effectively reduces melanogenesis with no adverse side effects. The acute oral toxicity study evidently confirms that T1 molecule is free of discernable cytotoxicity in mice. Furthermore, the molecular modeling demonstrates that the sulfur atom of T1 coordinating with the copper ions in the active site of tyrosinase is essential for mushroom tyrosinase inhibition and the ability of diminishing the human melanin synthesis. These results evident that T1 isolated from Gastrodia elata is a promising candidate in developing pharmacological and cosmetic agents of great potency in skin-whitening.

The comparison of pyrosequencing molecular Gram stain, culture, and conventional Gram stain for diagnosing orthopaedic infections.

PubMed

Kobayashi, Naomi; Bauer, Thomas W; Tuohy, Marion J; Lieberman, Isador H; Krebs, Viktor; Togawa, Daisuke; Fujishiro, Takaaki; Procop, Gary W

2006-08-01

We have developed a combined real-time PCR and pyrosequencing assay that successfully differentiated the vast majority of gram-positive and gram-negative bacteria when bacterial isolates were tested. The purpose of this study was to evaluate this assay on clinical specimens obtained from orthopedic surgeries, and to prospectively compare the results of "molecular Gram stain" with culture and conventional direct Gram stain. Forty-five surgical specimens were obtained from patients who underwent orthopedic surgery procedures. The DNA was extracted and a set of broad-range PCR primers that targeted a part of the 16S rDNA gene was used for pan-bacterial PCR. The amplicons were submitted for pyrosequencing and the resulting molecular Gram stain characteristics were recorded. Culture and direct Gram staining were performed using standard methods for all cases. Surgical specimens were reviewed histologically for all cases that had a discrepancy between culture and molecular results. There was an 86.7% (39/45) agreement between the traditional and molecular methods. In 12/14 (85.7%) culture-proven cases of bacterial infection, molecular Gram stain characteristics were in agreement with the culture results, while the conventional Gram stain result was in agreement only for five cases (35.7%). In the 31 culture negative cases, 27 cases were also PCR negative, whereas 4 were PCR positive. Three of these were characterized as gram negative and one as gram positive by this molecular method. Molecular determination of the Gram stain characteristics of bacteria that cause orthopedic infections may be achieved, in most instances, by this method. Further studies are necessary to understand the clinical importance of PCR-positive/culture-negative results.

Development of one-step quantitative reverse transcription PCR for the rapid detection of flaviviruses.

PubMed

Patel, Pranav; Landt, Olfert; Kaiser, Marco; Faye, Oumar; Koppe, Tanja; Lass, Ulrich; Sall, Amadou A; Niedrig, Matthias

2013-02-14

The genus Flavivirus includes several pathogenic agents that cause severe illness in humans. Re-emergence of West Nile virus in Europe and continuous spread of certain flaviviruses such as dengue, yellow fever and Japanese encephalitis viruses represent a global danger to public health. Therefore, a rapid and accurate molecular method is required for diagnostics and epidemiological surveillance of flaviviruses. A Pan-Flavi quantitative RT-PCR assay using a Locked-Nucleic Acid probe targeting the flavivirus NS5 gene was developed and optimized to detect a wide range of flaviviruses simultaneously. The specificity and sensitivity of the Pan-Flavi assay were tested using RNA of different flaviviruses and non-flaviviruses. Furthermore, the assay was compared directly to flavivirus species-specific assays for the ability to detect flaviviruses sensitively. Two degenerate primers and one Locked-Nucleic Acids probe were designed to amplify most of the flaviviruses. To increase the specificity and fluorescence signal of the Pan-Flavi assay for detection of yellow fever virus and dengue virus 4, additional primers and probes were included. Viral RNA of thirty different flaviviruses was detected, verifying the broad range specificity. The testing of this assay was successful, using standard plasmid and RNA dilutions of yellow fever virus vaccine strain, dengue virus 1 and tick-borne encephalitis virus, with a sensitivity limit of 10-100 genome copies/reaction. Also comparatively good results were achieved for detecting different flaviviruses by the Pan-Flavi assay when compared to the flavivirus species-specific assays. The assay is rapid, broad-range flavivirus-specific and highly sensitive making it a valuable tool for rapid detection of flaviviruses in livestock samples, epidemiological studies or as useful complement to single flavivirus-specific assays for clinical diagnosis.

Detection of Streptococcus pyogenes using rapid visual molecular assay.

PubMed

Zhao, Xiangna; He, Xiaoming; Li, Huan; Zhao, Jiangtao; Huang, Simo; Liu, Wei; Wei, Xiao; Ding, Yiwei; Wang, Zhaoyan; Zou, Dayang; Wang, Xuesong; Dong, Derong; Yang, Zhan; Yan, Xiabei; Huang, Liuyu; Du, Shuangkui; Yuan, Jing

2015-09-01

Streptococcus pyogenes is an increasingly important pathogen in many parts of the world. Rapid and accurate detection of S. pyogenes aids in the control of the infection. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed and validated for the specific detection of S. pyogenes. The assay incorporates two methods: a chromogenic analysis using a calcein/Mn(2+) complex and real-time turbidity monitoring to assess the reaction. Both methods detected the target DNA within 60 min under 64°C isothermal conditions. The assay used specifically designed primers to target spy1258, and correctly identified 111 strains of S. pyogenes and 32 non-S. pyogenes strains, including other species of the genus Streptococcus. Tests using reference strains showed that the LAMP assay was highly specific. The sensitivity of the assay, with a detection limit of 1.49 pg DNA, was 10-fold greater than that of PCR. The LAMP assay established in this study is simple, fast and sensitive, and does not rely upon any special equipment; thus, it could be employed in clinical diagnosis. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A European multicenter study on the analytical performance of the VERIS HBV assay.

PubMed

Braun, Patrick; Delgado, Rafael; Drago, Monica; Fanti, Diana; Fleury, Hervé; Izopet, Jacques; Lombardi, Alessandra; Mancon, Alessandro; Marcos, Maria Angeles; Sauné, Karine; O Shea, Siobhan; Pérez-Rivilla, Alfredo; Ramble, John; Trimoulet, Pascale; Vila, Jordi; Whittaker, Duncan; Artus, Alain; Rhodes, Daniel

Hepatitis B viral load monitoring is an essential part of managing patients with chronic Hepatits B infection. Beckman Coulter has developed the VERIS HBV Assay for use on the fully automated Beckman Coulter DxN VERIS Molecular Diagnostics System. 1 OBJECTIVES: To evaluate the analytical performance of the VERIS HBV Assay at multiple European virology laboratories. Precision, analytical sensitivity, negative sample performance, linearity and performance with major HBV genotypes/subtypes for the VERIS HBV Assay was evaluated. Precision showed an SD of 0.15 log 10 IU/mL or less for each level tested. Analytical sensitivity determined by probit analysis was between 6.8-8.0 IU/mL. Clinical specificity on 90 unique patient samples was 100.0%. Performance with 754 negative samples demonstrated 100.0% not detected results, and a carryover study showed no cross contamination. Linearity using clinical samples was shown from 1.23-8.23 log 10 IU/mL and the assay detected and showed linearity with major HBV genotypes/subtypes. The VERIS HBV Assay demonstrated comparable analytical performance to other currently marketed assays for HBV DNA monitoring. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluation of Several Biochemical and Molecular Techniques for Identification of Streptococcus pneumoniae and Streptococcus pseudopneumoniae and Their Detection in Respiratory Samples

PubMed Central

Schelfaut, Jacqueline J. G.; Bernards, Alexandra T.; Claas, Eric C. J.

2012-01-01

The identification and detection of mitis group streptococci, which contain Streptococcus pneumoniae, have been hampered by the lack of sensitive and specific assays. In this study, we evaluated several biochemical and molecular assays for the identification of S. pneumoniae and Streptococcus pseudopneumoniae and their distinction from other mitis group streptococci using a collection of 54 isolates obtained by the routine culturing of 53 respiratory specimens from patients with community-acquired pneumonia. The combined results of the biochemical and molecular assays indicated the presence of 23 S. pneumoniae, 2 S. pseudopneumoniae, and 29 other mitis group streptococcal isolates. The tube bile solubility test that is considered gold standard for the identification of S. pneumoniae showed concordant results with optochin susceptibility testing (CO2 atmosphere) and a real-time multiplex PCR assay targeting the Spn9802 fragment and the autolysin gene. Optochin susceptibility testing upon incubation in an O2 atmosphere, bile solubility testing by oxgall disk, matrix-assisted laser desorption ionization–time of flight mass spectrometry, and sequence analysis of the tuf and rpoB genes resulted in several false-positive, false-negative, or inconclusive results. The S. pseudopneumoniae isolates could be identified only by molecular assays, and the multiplex real-time PCR assay was concluded to be most convenient for the identification of S. pneumoniae and S. pseudopneumoniae isolates. Using this method, S. pneumoniae and S. pseudopneumoniae DNA could be detected in the respiratory samples from which they were isolated and in an additional 11 samples from which only other streptococci were isolated. PMID:22278834

Application of mass spectrometry technologies for the discovery of low-molecular weight modulators of enzymes and protein-protein interactions.

PubMed

Zehender, Hartmut; Mayr, Lorenz M

2007-10-01

In recent years, mass spectrometry has gained widespread use as an assay and screening technology in drug discovery because it enables sensitive, label-free detection of low-molecular weight modulators of biomolecules as well as sensitive and accurate detection of high-molecular weight modifications of biomolecules. Electrospray and matrix-assisted laser desorption ionization are the most widely used ionization techniques to identify chemical compounds interfering with enzymatic function, receptor-ligand binding or molecules modulating a protein-protein interaction of interest. Mass spectrometry based techniques are no longer restricted to screening in biochemical assay systems but have now become also applicable to imaging of biomolecules and chemical compounds in cell-based assay systems and even in highly complex tissue sections.

Modeling systematic errors: polychromatic sources of Beer-Lambert deviations in HPLC/UV and nonchromatographic spectrophotometric assays.

PubMed

Galli, C

2001-07-01

It is well established that the use of polychromatic radiation in spectrophotometric assays leads to excursions from the Beer-Lambert limit. This Note models the resulting systematic error as a function of assay spectral width, slope of molecular extinction coefficient, and analyte concentration. The theoretical calculations are compared with recent experimental results; a parameter is introduced which can be used to estimate the magnitude of the systematic error in both chromatographic and nonchromatographic spectrophotometric assays. It is important to realize that the polychromatic radiation employed in common laboratory equipment can yield assay errors up to approximately 4%, even at absorption levels generally considered 'safe' (i.e. absorption <1). Thus careful consideration of instrumental spectral width, analyte concentration, and slope of molecular extinction coefficient is required to ensure robust analytical methods.

Kinetic analysis using low-molecular mass xyloglucan oligosaccharides defines the catalytic mechanism of a Populus xyloglucan endotransglycosylase

PubMed Central

Saura-Valls, Marc; Fauré, Régis; Ragàs, Sergi; Piens, Kathleen; Brumer, Harry; Teeri, Tuula T.; Cottaz, Sylvain; Driguez, Hugues; Planas, Antoni

2005-01-01

Plant XETs [XG (xyloglucan) endotransglycosylases] catalyse the transglycosylation from a XG donor to a XG or low-molecular-mass XG fragment as the acceptor, and are thought to be important enzymes in the formation and remodelling of the cellulose-XG three-dimensional network in the primary plant cell wall. Current methods to assay XET activity use the XG polysaccharide as the donor substrate, and present limitations for kinetic and mechanistic studies of XET action due to the polymeric and polydisperse nature of the substrate. A novel activity assay based on HPCE (high performance capillary electrophoresis), in conjunction with a defined low-molecular-mass XGO {XG oligosaccharide; (XXXGXXXG, where G=Glcβ1,4- and X=[Xylα1,6]Glcβ1,4-)} as the glycosyl donor and a heptasaccharide derivatized with ANTS [8-aminonaphthalene-1,3,6-trisulphonic acid; (XXXG-ANTS)] as the acceptor substrate was developed and validated. The recombinant enzyme PttXET16A from Populus tremula x tremuloides (hybrid aspen) was characterized using the donor/acceptor pair indicated above, for which preparative scale syntheses have been optimized. The low-molecular-mass donor underwent a single transglycosylation reaction to the acceptor substrate under initial-rate conditions, with a pH optimum at 5.0 and maximal activity between 30 and 40 °C. Kinetic data are best explained by a ping-pong bi-bi mechanism with substrate inhibition by both donor and acceptor. This is the first assay for XETs using a donor substrate other than polymeric XG, enabling quantitative kinetic analysis of different XGO donors for specificity, and subsite mapping studies of XET enzymes. PMID:16356166

Kinetic analysis using low-molecular mass xyloglucan oligosaccharides defines the catalytic mechanism of a Populus xyloglucan endotransglycosylase.

PubMed

Saura-Valls, Marc; Fauré, Régis; Ragàs, Sergi; Piens, Kathleen; Brumer, Harry; Teeri, Tuula T; Cottaz, Sylvain; Driguez, Hugues; Planas, Antoni

2006-04-01

Plant XETs [XG (xyloglucan) endotransglycosylases] catalyse the transglycosylation from a XG donor to a XG or low-molecular-mass XG fragment as the acceptor, and are thought to be important enzymes in the formation and remodelling of the cellulose-XG three-dimensional network in the primary plant cell wall. Current methods to assay XET activity use the XG polysaccharide as the donor substrate, and present limitations for kinetic and mechanistic studies of XET action due to the polymeric and polydisperse nature of the substrate. A novel activity assay based on HPCE (high performance capillary electrophoresis), in conjunction with a defined low-molecular-mass XGO {XG oligosaccharide; (XXXGXXXG, where G=Glcbeta1,4- and X=[Xylalpha1,6]Glcbeta1,4-)} as the glycosyl donor and a heptasaccharide derivatized with ANTS [8-aminonaphthalene-1,3,6-trisulphonic acid; (XXXG-ANTS)] as the acceptor substrate was developed and validated. The recombinant enzyme PttXET16A from Populus tremula x tremuloides (hybrid aspen) was characterized using the donor/acceptor pair indicated above, for which preparative scale syntheses have been optimized. The low-molecular-mass donor underwent a single transglycosylation reaction to the acceptor substrate under initial-rate conditions, with a pH optimum at 5.0 and maximal activity between 30 and 40 degrees C. Kinetic data are best explained by a ping-pong bi-bi mechanism with substrate inhibition by both donor and acceptor. This is the first assay for XETs using a donor substrate other than polymeric XG, enabling quantitative kinetic analysis of different XGO donors for specificity, and subsite mapping studies of XET enzymes.

Using yeast to determine the functional consequences of mutations in the human p53 tumor suppressor gene: An introductory course‐based undergraduate research experience in molecular and cell biology

PubMed Central

Brownell, Sara E.; Seawell, Patricia Chandler; Malladi, Shyamala; Imam, Jamie F. Conklin; Singla, Veena; Bradon, Nicole; Cyert, Martha S.; Stearns, Tim

2016-01-01

Abstract The opportunity to engage in scientific research is an important, but often neglected, component of undergraduate training in biology. We describe the curriculum for an innovative, course‐based undergraduate research experience (CURE) appropriate for a large, introductory cell and molecular biology laboratory class that leverages students′ high level of interest in cancer. The course is highly collaborative and emphasizes the analysis and interpretation of original scientific data. During the course, students work in teams to characterize a collection of mutations in the human p53 tumor suppressor gene via expression and analysis in yeast. Initially, student pairs use both qualitative and quantitative assays to assess the ability of their p53 mutant to activate expression of reporter genes, and they localize their mutation within the p53 structure. Through facilitated discussion, students suggest possible molecular explanations for the transactivation defects displayed by their p53 mutants and propose experiments to test these hypotheses that they execute during the second part of the course. They use a western blot to determine whether mutant p53 levels are reduced, a DNA‐binding assay to test whether recognition of any of three p53 target sequences is compromised, and fluorescence microscopy to assay nuclear localization. Students studying the same p53 mutant periodically convene to discuss and interpret their combined data. The course culminates in a poster session during which students present their findings to peers, instructors, and the greater biosciences community. Based on our experience, we provide recommendations for the development of similar large introductory lab courses. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(2):161–178, 2017. PMID:27873457

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos.

PubMed

Berrun, A C; Stachura, D L

2017-11-30

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell lineages that comprise mature blood. Isolation and identification of these HSPCs is difficult because they are defined ex post facto; they can only be defined after their differentiation into specific cell lineages. Over the past few decades, the zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop ex utero, and by 48 h post-fertilization (hpf) have generated definitive HSPCs. Assays to assess HSPC differentiation and proliferation capabilities have been developed, utilizing transplantation and subsequent reconstitution of the hematopoietic system in addition to visualizing specialized transgenic lines with confocal microscopy. However, these assays are cost prohibitive, technically difficult, and time consuming for many laboratories. Development of an in vitro model to assess HSPCs would be cost effective, quicker, and present fewer difficulties compared to previously described methods, allowing laboratories to quickly assess mutagenesis and drug screens that affect HSPC biology. This novel in vitro assay to assess HSPCs is performed by plating dissociated whole zebrafish embryos and adding exogenous factors that promote only HSPC differentiation and proliferation. Embryos are dissociated into single cells and plated with HSPC-supportive colony stimulating factors that cause them to generate colony forming units (CFUs) that arise from a single progenitor cell. These assays should allow more careful examination of the molecular pathways responsible for HSPC proliferation, differentiation, and regulation, which will allow researchers to understand the underpinnings of vertebrate hematopoiesis and its dysregulation during disease.

Ultra-Sensitive Detection of Plasmodium falciparum by Amplification of Multi-Copy Subtelomeric Targets

PubMed Central

Hofmann, Natalie; Mwingira, Felista; Shekalaghe, Seif; Robinson, Leanne J.; Mueller, Ivo; Felger, Ingrid

2015-01-01

Background Planning and evaluating malaria control strategies relies on accurate definition of parasite prevalence in the population. A large proportion of asymptomatic parasite infections can only be identified by surveillance with molecular methods, yet these infections also contribute to onward transmission to mosquitoes. The sensitivity of molecular detection by PCR is limited by the abundance of the target sequence in a DNA sample; thus, detection becomes imperfect at low densities. We aimed to increase PCR diagnostic sensitivity by targeting multi-copy genomic sequences for reliable detection of low-density infections, and investigated the impact of these PCR assays on community prevalence data. Methods and Findings Two quantitative PCR (qPCR) assays were developed for ultra-sensitive detection of Plasmodium falciparum, targeting the high-copy telomere-associated repetitive element 2 (TARE-2, ∼250 copies/genome) and the var gene acidic terminal sequence (varATS, 59 copies/genome). Our assays reached a limit of detection of 0.03 to 0.15 parasites/μl blood and were 10× more sensitive than standard 18S rRNA qPCR. In a population cross-sectional study in Tanzania, 295/498 samples tested positive using ultra-sensitive assays. Light microscopy missed 169 infections (57%). 18S rRNA qPCR failed to identify 48 infections (16%), of which 40% carried gametocytes detected by pfs25 quantitative reverse-transcription PCR. To judge the suitability of the TARE-2 and varATS assays for high-throughput screens, their performance was tested on sample pools. Both ultra-sensitive assays correctly detected all pools containing one low-density P. falciparum–positive sample, which went undetected by 18S rRNA qPCR, among nine negatives. TARE-2 and varATS qPCRs improve estimates of prevalence rates, yet other infections might still remain undetected when absent in the limited blood volume sampled. Conclusions Measured malaria prevalence in communities is largely determined by the sensitivity of the diagnostic tool used. Even when applying standard molecular diagnostics, prevalence in our study population was underestimated by 8% compared to the new assays. Our findings highlight the need for highly sensitive tools such as TARE-2 and varATS qPCR in community surveillance and for monitoring interventions to better describe malaria epidemiology and inform malaria elimination efforts. PMID:25734259

Characterization of stem-like cells in a new astroblastoma cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Coban, Esra Aydemir; Kasikci, Ezgi; Karatas, Omer Faruk

Cell lines established from tumors are the most commonly used models in cancer research, and their use in recent years has enabled a greater understanding of the biology of cancer and the means to develop effective treatment strategies. Astroblastomas are uncommon neuroepithelial tumors of glial origin, predominantly affecting young people, mainly teenagers and children, predominantly females. To date, only a single study has reported that astroblastomas contain a large number of neural stem-like cells, which had only a partial proliferation capacity and differentiation. Our objective was to establish an astroblastoma cell line to investigate the presence of astroblastic cells andmore » cancer stem-like cells. The migratory and invasion abilities of the cells were quantified with invasion and migration assays and compared to a glioblastoma cell line. The presence of stem cells was detected with surface-marker analysis by using flow cytometry, and measuring the differentiation ability with a differentiation assay and the self-renewal capacity with a sphere-forming assay. These characteristics may determine whether this novel cell line is a model for astroblastomas that may have stem-cell characteristics. With this novel cell line, scientists can investigate the molecular pathways underlying astroblastomas and develop new therapeutic strategies for patients with these tumors. - Highlights: • An establishment of a novel astroblastoma cell line was proposed. • The presence of astroblastic cells and cancer stem-like cells was investigated. • The molecular pathways underlying astroblastomas may be investigated. • New therapeutic strategies for patients with astroblastoma may be developed.« less

Liquid biopsy utility for the surveillance of cutaneous malignant melanoma patients.

PubMed

Huang, Sharon K; Hoon, Dave S B

2016-03-01

Cutaneous melanoma is one of the highest incident-rate cancers with increasing prevalence in Western societies. Despite the advent of new approved therapeutics, the 5-year overall survival rate of stage IV melanoma patients remains below 15%. Current treatments for late stage disease have shown higher efficacy when treated at a lower disease burden. Thus, blood-based biomarkers capable of detecting melanoma prior to clinically evident distant metastasis, will improve the treatment and outcomes for melanoma patients. To that end, effective treatment of melanoma necessitates identification of patients at risk for developing distant metastases. Furthermore, employing blood biomarkers that monitor cancer progression over the course of treatment is a promising solution to post-treatment drug resistance often developed in melanoma patients. Non-invasive blood biomarker assays allow for regular dynamic monitoring of disease. "Liquid Biopsy" of blood, which exploits circulating tumor cells (CTCs), cell-free circulating tumor DNA (ctDNA) and cell-free circulating microRNA (cmiRNA), has been shown to detect prognostic factors for relapse in AJCC stage III and stage IV melanoma patients. Moreover, molecular characterization of CTC and analysis of various forms of ctDNA present promising potential in development of individualized therapy for melanoma patients. New approaches such as massive parallel sequencing (MPS) provide a comprehensive view of the disease progression, allowing for the selection of therapeutic options for individual patients. With advancements of improving molecular assays, liquid biopsy analysis as a powerful, routine clinical assay for melanoma patients, is highly promising prospective. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Functional Genomic Analysis of Cotton Genes with Agrobacterium-Mediated Virus-Induced Gene Silencing

PubMed Central

Gao, Xiquan; Shan, Libo

2015-01-01

Cotton (Gossypium spp.) is one of the most agronomically important crops worldwide for its unique textile fiber production and serving as food and feed stock. Molecular breeding and genetic engineering of useful genes into cotton have emerged as advanced approaches to improve cotton yield, fiber quality, and resistance to various stresses. However, the understanding of gene functions and regulations in cotton is largely hindered by the limited molecular and biochemical tools. Here, we describe the method of an Agrobacterium infiltration-based virus-induced gene silencing (VIGS) assay to transiently silence endogenous genes in cotton at 2-week-old seedling stage. The genes of interest could be readily silenced with a consistently high efficiency. To monitor gene silencing efficiency, we have cloned cotton GrCla1 from G. raimondii, a homolog gene of Arabidopsis Cloroplastos alterados 1 (AtCla1) involved in chloroplast development, and inserted into a tobacco rattle virus (TRV) binary vector pYL156. Silencing of GrCla1 results in albino phenotype on the newly emerging leaves, serving as a visual marker for silencing efficiency. To further explore the possibility of using VIGS assay to reveal the essential genes mediating disease resistance to Verticillium dahliae, a fungal pathogen causing severe Verticillium wilt in cotton, we developed a seedling infection assay to inoculate cotton seedlings when the genes of interest are silenced by VIGS. The method we describe here could be further explored for functional genomic analysis of cotton genes involved in development and various biotic and abiotic stresses. PMID:23386302

Functional genomic analysis of cotton genes with agrobacterium-mediated virus-induced gene silencing.

PubMed

Gao, Xiquan; Shan, Libo

2013-01-01

Cotton (Gossypium spp.) is one of the most agronomically important crops worldwide for its unique textile fiber production and serving as food and feed stock. Molecular breeding and genetic engineering of useful genes into cotton have emerged as advanced approaches to improve cotton yield, fiber quality, and resistance to various stresses. However, the understanding of gene functions and regulations in cotton is largely hindered by the limited molecular and biochemical tools. Here, we describe the method of an Agrobacterium infiltration-based virus-induced gene silencing (VIGS) assay to transiently silence endogenous genes in cotton at 2-week-old seedling stage. The genes of interest could be readily silenced with a consistently high efficiency. To monitor gene silencing efficiency, we have cloned cotton GrCla1 from G. raimondii, a homolog gene of Arabidopsis Cloroplastos alterados 1 (AtCla1) involved in chloroplast development, and inserted into a tobacco rattle virus (TRV) binary vector pYL156. Silencing of GrCla1 results in albino phenotype on the newly emerging leaves, serving as a visual marker for silencing efficiency. To further explore the possibility of using VIGS assay to reveal the essential genes mediating disease resistance to Verticillium dahliae, a fungal pathogen causing severe Verticillium wilt in cotton, we developed a seedling infection assay to inoculate cotton seedlings when the genes of interest are silenced by VIGS. The method we describe here could be further explored for functional genomic analysis of cotton genes involved in development and various biotic and abiotic stresses.

PROLIFERATION AS A KEY EVENT IN DEVELOPMENTAL TOXICITY: "CHEMICAL SCREENING IN HUMAN NEURAL STEM CELLS USING HIGH CONTENT IMAGING

EPA Science Inventory

New toxicity testing approaches will rely on in vitro assays to assess chemical effects at the cellular and molecular level. Cell proliferation is imperative to normal development, and chemical disruption of this process can be detrimental to the organism. As part of an effort to...

Probe molecules (PrM) approach in adverse outcome pathway (AOP) based high throughput screening (HTS): in vivo discovery for developing in vitro target methods

EPA Science Inventory

Efficient and accurate adverse outcome pathway (AOP) based high-throughput screening (HTS) methods use a systems biology based approach to computationally model in vitro cellular and molecular data for rapid chemical prioritization; however, not all HTS assays are grounded by rel...

A novel DANP-coupled hairpin RT-PCR for rapid detection of Chikungunya virus.

PubMed

Chen, Huixin; Takei, Fumie; Koay, Evelyn Siew-Chuan; Nakatani, Kazuhiko; Chu, Justin Jang Hann

2013-03-01

Chikungunya has re-emerged as an important arboviral infection of global health significance. Because of lack of a vaccine and effective treatment, rapid diagnosis plays an important role in early clinical management of patients. In this study, we have developed a novel molecular diagnostic platform that ensures a rapid and cost-effective one-step RT-PCR assay, with high sensitivity and specificity, for the early detection of the Chikungunya virus (CHIKV). It uses 2,7-diamino-1,8-naphthyridine derivative (DANP)-labeled cytosine-bulge hairpin primers to amplify the nsP2 region of the CHIKV genome, followed by measurement of the fluorescence emitted from DANP-primer complexes after PCRs. The detection limit of our assay was 0.01 plaque-forming units per reaction of CHIKV. Furthermore, the HP-nsP2 primers were highly specific in detecting CHIKV, without any cross-reactivity with the panel of RNA viruses validated in this study. The feasibility of the DANP-coupled hairpin RT-PCR for clinical diagnosis was evaluated using clinical serum samples from CHIKV-infected patients, and the specificity and sensitivity were 100% (95% CI, 80.0% to 100%) and 95.5% (95% CI, 75.1% to 99.8%), respectively. These findings confirmed its potential as a point-of-care clinical molecular diagnostic assay for CHIKV in acute-phase patient serum samples. Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Novel real-time polymerase chain reaction assay for simultaneous detection of recurrent fusion genes in acute myeloid leukemia.

PubMed

Dolz, Sandra; Barragán, Eva; Fuster, Óscar; Llop, Marta; Cervera, José; Such, Esperanza; De Juan, Inmaculada; Palanca, Sarai; Murria, Rosa; Bolufer, Pascual; Luna, Irene; Gómez, Inés; López, María; Ibáñez, Mariam; Sanz, Miguel A

2013-09-01

The recent World Health Organization classification recognizes different subtypes of acute myeloid leukemia (AML) according to the presence of several recurrent genetic abnormalities. Detection of these abnormalities and other molecular changes is of increasing interest because it contributes to a refined diagnosis and prognostic assessment in AML and enables monitoring of minimal residual disease. These genetic abnormalities can be detected using single RT-PCR, although the screening is still labor intensive and costly. We have developed a novel real-time RT-PCR assay to simultaneously detect 15 AML-associated rearrangements that is a simple and easily applicable method for use in clinical diagnostic laboratories. This method showed 100% specificity and sensitivity (95% confidence interval, 91% to 100% and 92% to 100%, respectively). The procedure was validated in a series of 105 patients with AML. The method confirmed all translocations detected using standard cytogenetics and fluorescence in situ hybridization and some additional undetected rearrangements. Two patients demonstrated two molecular rearrangements simultaneously, with BCR-ABL1 implicated in both, in addition to RUNX1-MECOM in one patient and PML-RARA in another. In conclusion, this novel real-time RT-PCR assay for simultaneous detection of multiple AML-associated fusion genes is a versatile and sensitive method for reliable screening of recurrent rearrangements in AML. Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

A New Single-Step PCR Assay for the Detection of the Zoonotic Malaria Parasite Plasmodium knowlesi

PubMed Central

Lucchi, Naomi W.; Poorak, Mitra; Oberstaller, Jenna; DeBarry, Jeremy; Srinivasamoorthy, Ganesh; Goldman, Ira; Xayavong, Maniphet; da Silva, Alexandre J.; Peterson, David S.; Barnwell, John W.; Kissinger, Jessica; Udhayakumar, Venkatachalam

2012-01-01

Background Recent studies in Southeast Asia have demonstrated substantial zoonotic transmission of Plasmodium knowlesi to humans. Microscopically, P. knowlesi exhibits several stage-dependent morphological similarities to P. malariae and P. falciparum. These similarities often lead to misdiagnosis of P. knowlesi as either P. malariae or P. falciparum and PCR-based molecular diagnostic tests are required to accurately detect P. knowlesi in humans. The most commonly used PCR test has been found to give false positive results, especially with a proportion of P. vivax isolates. To address the need for more sensitive and specific diagnostic tests for the accurate diagnosis of P. knowlesi, we report development of a new single-step PCR assay that uses novel genomic targets to accurately detect this infection. Methodology and Significant Findings We have developed a bioinformatics approach to search the available malaria parasite genome database for the identification of suitable DNA sequences relevant for molecular diagnostic tests. Using this approach, we have identified multi-copy DNA sequences distributed in the P. knowlesi genome. We designed and tested several novel primers specific to new target sequences in a single-tube, non-nested PCR assay and identified one set of primers that accurately detects P. knowlesi. We show that this primer set has 100% specificity for the detection of P. knowlesi using three different strains (Nuri, H, and Hackeri), and one human case of malaria caused by P. knowlesi. This test did not show cross reactivity with any of the four human malaria parasite species including 11 different strains of P. vivax as well as 5 additional species of simian malaria parasites. Conclusions The new PCR assay based on novel P. knowlesi genomic sequence targets was able to accurately detect P. knowlesi. Additional laboratory and field-based testing of this assay will be necessary to further validate its utility for clinical diagnosis of P. knowlesi. PMID:22363751

A luminescence assay for natural product inhibitors of the Mycobacterium tuberculosis proteasome.

PubMed

Gunderwala, Amber; Porter, John

2016-01-01

Mycobacterium tuberculosis (Mtb) causes a large global burden of disease, with a high mortality rate in healthy and immuno-compromised patients. A number of molecular targets have been identified for treatment of this disease, including the Mtb proteasome. The Mtb proteasome enhances Mtb survival during nitrosative and oxidative stress in the latent, non-replicative phase. Therefore, Mtb proteasome inhibition could help to combat Mtb infections that do not respond to current therapies. To develop and validate a novel biochemical assay to assess Mtb proteasome activity in the presence of organic and aqueous plant test extracts. Fluorescence (photoluminescence) and luminescence (chemiluminescence) assays were investigated as potential methods to determine the robustness and repeatability for use in screening natural product extracts for Mtb proteasome inhibitors. The fluorescence assay, used widely for Mtb proteasome activity assays, was subject to interference due to the natural fluorescence of compounds in many of the extracts; there is little interference with the luminescence approach. As proof of principle, we used the luminescence assay to screen a small set of plant test extracts. Luminescence is the more suitable assay for assay of plant natural product extracts. The sensitivities of the luminescence and fluorescence assays are comparable. A Z'-factor of 0.58 for the luminescence assay makes it suitable for medium-to-high throughput screening efforts. Copyright © 2016 John Wiley & Sons, Ltd.

Analysis of Histone Deacetylase-Dependent Effects on Cell Migration Using the Stripe Assay.

PubMed

Mertsch, Sonja; Thanos, Solon

2017-01-01

For normal embryonic development/morphogenesis, cell migration and homing are well-orchestrated and important events requiring specific cellular mechanisms. In diseases such as cancer deregulated cell migration represents a major problem. Therefore, numerous efforts are under way to understand the molecular mechanisms of tumor cell migration and to generate more efficient tumor therapies. Cell migration assays are one of the most commonly used functional assays. The wound-healing assay or the Boyden chamber assay are variations of these assays. Nearly all of them are two-dimensional assays and the cells can only migrate on one substrate at a time. This is in contrast to the in vivo situation where the cells are faced simultaneously with different surfaces and interact with different cell types. To approach this in vivo situation we used a modified version of the stripe assay designed by Bonhoeffer and colleagues to examine mechanisms of axonal guidance. The design of this assay allows cells to decide between two different substrates offered at the same time. Utilizing alternating neuronal substrates for migration analyses we can partially mimic the complex in vivo situation for brain tumor cells. Here we describe the detailed protocol to perform a modified version of the stripe assay in order to observe substrate-dependent migration effects in vitro, to analyze the effect of Rho-dependent kinases (ROCKS), of histone deacetylases (HDACs) and of other molecules on glioma cells.

Improving statistical inference on pathogen densities estimated by quantitative molecular methods: malaria gametocytaemia as a case study.

PubMed

Walker, Martin; Basáñez, María-Gloria; Ouédraogo, André Lin; Hermsen, Cornelus; Bousema, Teun; Churcher, Thomas S

2015-01-16

Quantitative molecular methods (QMMs) such as quantitative real-time polymerase chain reaction (q-PCR), reverse-transcriptase PCR (qRT-PCR) and quantitative nucleic acid sequence-based amplification (QT-NASBA) are increasingly used to estimate pathogen density in a variety of clinical and epidemiological contexts. These methods are often classified as semi-quantitative, yet estimates of reliability or sensitivity are seldom reported. Here, a statistical framework is developed for assessing the reliability (uncertainty) of pathogen densities estimated using QMMs and the associated diagnostic sensitivity. The method is illustrated with quantification of Plasmodium falciparum gametocytaemia by QT-NASBA. The reliability of pathogen (e.g. gametocyte) densities, and the accompanying diagnostic sensitivity, estimated by two contrasting statistical calibration techniques, are compared; a traditional method and a mixed model Bayesian approach. The latter accounts for statistical dependence of QMM assays run under identical laboratory protocols and permits structural modelling of experimental measurements, allowing precision to vary with pathogen density. Traditional calibration cannot account for inter-assay variability arising from imperfect QMMs and generates estimates of pathogen density that have poor reliability, are variable among assays and inaccurately reflect diagnostic sensitivity. The Bayesian mixed model approach assimilates information from replica QMM assays, improving reliability and inter-assay homogeneity, providing an accurate appraisal of quantitative and diagnostic performance. Bayesian mixed model statistical calibration supersedes traditional techniques in the context of QMM-derived estimates of pathogen density, offering the potential to improve substantially the depth and quality of clinical and epidemiological inference for a wide variety of pathogens.

Molecular karyotyping of the 2La inversion in Anopheles gambiae.

PubMed

White, Bradley J; Santolamazza, Federica; Kamau, Luna; Pombi, Marco; Grushko, Olga; Mouline, Karine; Brengues, Cecile; Guelbeogo, Wamdaogo; Coulibaly, Mamadou; Kayondo, Jonathan K; Sharakhov, Igor; Simard, Frederic; Petrarca, Vincenzo; Della Torre, Alessandra; Besansky, Nora J

2007-02-01

The African malaria vector Anopheles gambiae is polymorphic for alternative arrangements on the left arm of chromosome 2 (2La and 2L+(a)) that are non-randomly distributed with respect to degree of aridity. Detailed studies on the ecological role of inversion 2La have been hindered by the technical demands of traditional karyotype analysis and by sex- and stage-specific limitations on the availability of polytene chromosomes favorable for analysis. Recent molecular characterization of both inversion breakpoints presented the opportunity to develop a polymerase chain reaction (PCR)-based method for karyotype analysis. Here we report the development of this molecular diagnostic assay and the results of extensive field validation. When tested on 765 An. gambiae specimens sampled across Africa, the molecular approach compared favorably with traditional cytologic methods, correctly scoring > 94% of these specimens. By providing ready access to the 2La karyotype, this tool lays groundwork for future studies of the ecological genomics of this medically important species.

[Development of indel markers for molecular authentication of Panax ginseng and P. quinquefolius].

PubMed

Wang, Rong-Bo; Tian, Hui-Li; Wang, Hong-Tao; Li, Gui-Sheng

2018-04-01

Panax ginseng and P. quinquefolius are two kinds of important medicinal herbs. They are morphologically similar but have different pharmacological effects. Therefore, botanical origin authentication of these two ginsengs is of great importance for ensuring pharmaceutical efficacy and food safety. Based on the fact that intron position in orthologous genes is highly conserved across plant species, intron length polymorphisms were exploited from unigenes of ginseng. Specific primers were respectively designed for these two species based on their insertion/deletion sequences of cytochrome P450 and glyceraldehyde 3-phosphate dehydrogenase, and multiplex PCR was conducted for molecular authentication of P.ginseng and P. quinquefolius. The results showed that the developed multiplex PCR assay was effective for molecular authentication of P.ginseng and P. quinquefolius without strict PCR condition and the optimization of reaction system.This study provides a preferred ideal marker system for molecular authentication of ginseng,and the presented method can be employed in origin authentication of other herbal preparations. Copyright© by the Chinese Pharmaceutical Association.

Smart point-of-care systems for molecular diagnostics based on nanotechnology: whole blood glucose analysis

NASA Astrophysics Data System (ADS)

Devadhasan, Jasmine P.; Kim, Sanghyo

2015-07-01

Complementary metal oxide semiconductor (CMOS) image sensors are received great attention for their high efficiency in biological applications. The present work describes a CMOS image sensor-based whole blood glucose monitoring system through a point-of-care (POC) approach. A simple poly-ethylene terephthalate (PET) film chip was developed to carry out the enzyme kinetic reaction at various concentrations of blood glucose. In this technique, assay reagent was adsorbed onto amine functionalized silica (AFSiO2) nanoparticles in order to achieve glucose oxidation on the PET film chip. The AFSiO2 nanoparticles can immobilize the assay reagent with an electrostatic attraction and eased to develop the opaque platform which was technically suitable chip to analyze by the camera module. The oxidized glucose then produces a green color according to the glucose concentration and is analyzed by the camera module as a photon detection technique. The photon number decreases with increasing glucose concentration. The simple sensing approach, utilizing enzyme immobilized AFSiO2 nanoparticle chip and assay detection method was developed for quantitative glucose measurement.

Development of a pentaplex PCR assay for the simultaneous detection of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, L. helveticus, L. fermentum in whey starter for Grana Padano cheese.

PubMed

Cremonesi, Paola; Vanoni, Laura; Morandi, Stefano; Silvetti, Tiziana; Castiglioni, Bianca; Brasca, Milena

2011-03-30

A pentaplex PCR assay for the rapid, selective and simultaneous detection of Lactobacillus helveticus, L. delbrueckii subsp. lactis, L. delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and L. fermentum, was developed. The target sequences were a group of genes coding for beta-galactosidase production (S. thermophilus and L. delbrueckii subsp. bulgaricus), for cell-enveloped associated proteinase synthesis (L. helveticus), for dipeptide transport system production (L. delbrueckii subsp. lactis) and for arginine-ornithine antiporter protein production (L. fermentum). The analytical specificity of the assay was evaluated with 5 reference strains and 140 lactic acid bacterial strains derived from raw milk cheeses and belonging to the Lactobacillus, Streptococcus, Lactococcus and Enterococcus genera. The identification limit for each target strain was 10(3)CFU/ml. This new molecular assay was used to investigate the LAB population by direct extraction of DNA from the 12 whey cultures for Grana Padano. The pentaplex PCR assay revealed a good correspondence with microbiological analyses and allowed to identify even minor LAB community members which, can be out-competed in vitro by numerically more abundant microbial species. Copyright © 2011 Elsevier B.V. All rights reserved.

Liquid biopsy and its role in an advanced clinical trial for lung cancer.

PubMed

Johann, Donald J; Steliga, Mathew; Shin, Ik J; Yoon, Donghoon; Arnaoutakis, Konstantinos; Hutchins, Laura; Liu, Meeiyueh; Liem, Jason; Walker, Karl; Pereira, Andy; Yang, Mary; Jeffus, Susanne K; Peterson, Erich; Xu, Joshua

2018-02-01

Liquid biopsy methodologies, for the purpose of plasma genotyping of cell-free DNA (cfDNA) of solid tumors, are a new class of novel molecular assays. Such assays are rapidly entering the clinical sphere of research-based monitoring in translational oncology, especially for thoracic malignancies. Potential applications for these blood-based cfDNA assays include: (i) initial diagnosis, (ii) response to therapy and follow-up, (iii) tumor evolution, and (iv) minimal residual disease evaluation. Precision medicine will benefit from cutting-edge molecular diagnostics, especially regarding treatment decisions in the adjuvant setting, where avoiding over-treatment and unnecessary toxicity are paramount. The use of innovative genetic analysis techniques on individual patient tumor samples is being pursued in several advanced clinical trials. Rather than using a categorical treatment plan, the next critical step of therapeutic decision making is providing the "right" cancer therapy for an individual patient, including correct dose and timeframe based on the molecular analysis of the tumor in question. Per the 21st Century Cures Act, innovative clinical trials are integral for biomarker and drug development. This will include advanced clinical trials utilizing: (i) innovative assays, (ii) molecular profiling with cutting-edge bioinformatics, and (iii) clinically relevant animal or tissue models. In this paper, a mini-review addresses state-of-the-art liquid biopsy approaches. Additionally, an on-going advanced clinical trial for lung cancer with novelty through synergizing liquid biopsies, co-clinical trials, and advanced bioinformatics is also presented. Impact statement Liquid biopsy technology is providing a new source for cancer biomarkers, and adds new dimensions in advanced clinical trials. Utilizing a non-invasive routine blood draw, the liquid biopsy provides abilities to address perplexing issues of tumor tissue heterogeneity by identifying mutations in both primary and metastatic lesions. Regarding the assessment of response to cancer therapy, the liquid biopsy is not ready to replace medical imaging, but adds critical new information; for instance, through a temporal assessment of quantitative circulating tumor DNA (ctDNA) assay results, and importantly, the ability to monitor for signs of resistance, via emerging clones. Adjuvant therapy may soon be considered based on a quantitative cfDNA assay. As sensitivity and specificity of the technology continue to progress, cancer screening and prevention will improve and save countless lives by finding the cancer early, so that a routine surgery may be all that is required for a definitive cure.

European Multicenter Study on Analytical Performance of Veris HIV-1 Assay.

PubMed

Braun, Patrick; Delgado, Rafael; Drago, Monica; Fanti, Diana; Fleury, Hervé; Hofmann, Jörg; Izopet, Jacques; Kalus, Ulrich; Lombardi, Alessandra; Marcos, Maria Angeles; Mileto, Davide; Sauné, Karine; O'Shea, Siobhan; Pérez-Rivilla, Alfredo; Ramble, John; Trimoulet, Pascale; Vila, Jordi; Whittaker, Duncan; Artus, Alain; Rhodes, Daniel W

2017-07-01

The analytical performance of the Veris HIV-1 assay for use on the new, fully automated Beckman Coulter DxN Veris molecular diagnostics system was evaluated at 10 European virology laboratories. The precision, analytical sensitivity, performance with negative samples, linearity, and performance with HIV-1 groups/subtypes were evaluated. The precision for the 1-ml assay showed a standard deviation (SD) of 0.14 log 10 copies/ml or less and a coefficient of variation (CV) of ≤6.1% for each level tested. The 0.175-ml assay showed an SD of 0.17 log 10 copies/ml or less and a CV of ≤5.2% for each level tested. The analytical sensitivities determined by probit analysis were 19.3 copies/ml for the 1-ml assay and 126 copies/ml for the 0.175-ml assay. The performance with 1,357 negative samples demonstrated 99.2% with not detected results. Linearity using patient samples was shown from 1.54 to 6.93 log 10 copies/ml. The assay performed well, detecting and showing linearity with all HIV-1 genotypes tested. The Veris HIV-1 assay demonstrated analytical performance comparable to that of currently marketed HIV-1 assays. (DxN Veris products are Conformité Européenne [CE]-marked in vitro diagnostic products. The DxN Veris product line has not been submitted to the U.S. FDA and is not available in the U.S. market. The DxN Veris molecular diagnostics system is also known as the Veris MDx molecular diagnostics system and the Veris MDx system.). Copyright © 2017 American Society for Microbiology.

Correlation of cholinergic drug induced quenching of acetylcholinesterase bound thioflavin-T fluorescence with their inhibition activity

NASA Astrophysics Data System (ADS)

Islam, Mullah Muhaiminul; Rohman, Mostofa Ataur; Gurung, Arun Bahadur; Bhattacharjee, Atanu; Aguan, Kripamoy; Mitra, Sivaprasad

2018-01-01

The development of new acetylcholinesterase inhibitors (AChEIs) and subsequent assay of their inhibition efficiency is considered to be a key step for AD treatment. The fluorescence intensity of thioflavin-T (ThT) bound in the active site of acetylcholinesterase (AChE) quenches substantially in presence of standard AChEI drugs due to the dynamic replacement of the fluorophore from the AChE active site as confirmed from steady state emission as well as time-resolved fluorescence anisotropy measurement and molecular dynamics simulation in conjunction with docking calculation. The parametrized % quenching data for individual system shows excellent correlation with enzyme inhibition activity measured independently by standard Ellman AChE assay method in a high throughput plate reader system. The results are encouraging towards design of a fluorescence intensity based AChE inhibition assay method and may provide a better toolset to rapidly evaluate as well as develop newer AChE-inhibitors for AD treatment.

Indole-3-Carbonitriles as DYRK1A Inhibitors by Fragment-Based Drug Design.

PubMed

Meine, Rosanna; Becker, Walter; Falke, Hannes; Preu, Lutz; Loaëc, Nadège; Meijer, Laurent; Kunick, Conrad

2018-01-24

Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a potential drug target because of its role in the development of Down syndrome and Alzheimer's disease. The selective DYRK1A inhibitor 10-iodo-11 H -indolo[3,2- c ]quinoline-6-carboxylic acid (KuFal194), a large, flat and lipophilic molecule, suffers from poor water solubility, limiting its use as chemical probe in cellular assays and animal models. Based on the structure of KuFal194, 7-chloro-1 H -indole-3-carbonitrile was selected as fragment template for the development of smaller and less lipophilic DYRK1A inhibitors. By modification of this fragment, a series of indole-3-carbonitriles was designed and evaluated as potential DYRK1A ligands by molecular docking studies. Synthesis and in vitro assays on DYRK1A and related protein kinases identified novel double-digit nanomolar inhibitors with submicromolar activity in cell culture assays.

Diagnosis and Management of Hepatitis C Virus Infection.

PubMed

Mukherjee, Ronita; Burns, Andrew; Rodden, Diane; Chang, Frances; Chaum, Manita; Garcia, Nancy; Bollipalli, Nikitha; Niemz, Angelika

2015-10-01

The hepatitis C virus (HCV) infects more than 200 million people globally, with increasing incidence, especially in developing countries. HCV infection frequently progresses to chronic liver disease, creating a heavy economic burden on resource-poor countries and lowering patient quality of life. Effective HCV diagnosis, treatment selection, and treatment monitoring are important in stopping disease progression. Serological assays, which detect anti-HCV antibodies in the patient after seroconversion, are used for initial HCV diagnosis. Qualitative and quantitative molecular assays are used to confirm initial diagnosis, determine viral load, and genotype the dominant strain. Viral load and genotype information are used to guide appropriate treatment. Various other biomarker assays are performed to assess liver function and enable disease staging. Most of these diagnostic methods are mature and routinely used in high-resource countries with well-developed laboratory infrastructure. Few technologies, however, are available that address the needs of low-resource areas with high HCV prevalence, such as Africa and Southeast Asia. © 2015 Society for Laboratory Automation and Screening.

Development and evaluation of a quantitative PCR assay for detection of Hepatozoon sp.

PubMed

Criado-Fornelio, A; Buling, A; Cunha-Filho, N A; Ruas, J L; Farias, N A R; Rey-Valeiron, C; Pingret, J L; Etievant, M; Barba-Carretero, J C

2007-12-25

With the aim to improve current molecular diagnostic techniques of Hepatozoon sp. in carnivore mammals, we developed a quantitative PCR (qPCR) assay with SYBR Green I((R)). The method, consisting of amplification of a 235bp fragment of the 18S rRNA gene, is able to detect at least 0.1fg of parasite DNA. Reproducible quantitative results were obtained over a range of 0.1ng-0.1fg of Hepatozoon sp. DNA. To assess the performance of the qPCR assay, DNA samples from dogs (140) and cats (50) were tested with either standard PCR or qPCR. Positive samples were always confirmed by partial sequencing of the 18S rRNA gene. Quantitative PCR was 15.8% more sensitive than standard PCR to detect H. canis in dogs. In cats, no infections were detected by standard PCR, compared to two positives by qPCR (which were infected by H. canis as shown by sequencing).

Development of a rapid assay to detect the dinoflagellate Amyloodinium ocellatum using loop-mediated isothermal amplification (LAMP).

PubMed

Picón-Camacho, Sara M; Thompson, William P; Blaylock, Reginald B; Lotz, Jeffrey M

2013-09-23

Amyloodinium ocellatum is a highly pathogenic dinoflagellate parasite with global distribution that causes high mortalities in the culture of tropical and sub-tropical marine and estuarine fishes. Diagnosis typically occurs through gross examination following the onset of morbidity, at which point treatment is of limited benefit. In the present study, a new molecular diagnostic tool for the rapid detection of A. ocellatum (AO) was developed using the loop-mediated isothermal amplification method (LAMP). The AO-LAMP assay designed is highly specific using a set of four primers - two outer and two inner primers targeting six different regions on the 5' end of the Small Subunit rDNA region (SSU rDNA) of A. ocellatum. The AO-LAMP assay, optimized for 25-30 min at 62°C, amplified the DNA from A. ocellatum extracted from both water and gill tissue samples and did not amplify DNA from four closely related dinoflagellate sp ecies. The detection limit of the AO-LAMP assay was 10 fg, exceptionally higher than the conventional PCR (1 pg). In addition, the standardized AO-LAMP assay was capable of detecting single tomonts and trophonts; the assay was not affected by the presence of possible inhibitory substances present in environmental water samples or gill samples. The AO-LAMP assay developed in the present study provides a novel useful tool for the simple, rapid and sensitive detection of A. ocellatum in water and gill tissue samples, which could assist in the early detection and improved control of A. ocellatum infections in aquaculture systems. Copyright © 2013 Elsevier B.V. All rights reserved.

A Pentaplex PCR Assay for the Detection and Differentiation of Shigella Species

PubMed Central

Ojha, Suvash Chandra; Yean Yean, Chan; Ismail, Asma; Banga Singh, Kirnpal-Kaur

2013-01-01

The magnitude of shigellosis in developing countries is largely unknown because an affordable detection method is not available. Current laboratory diagnosis of Shigella spp. is laborious and time consuming and has low sensitivity. Hence, in the present study, a molecular-based diagnostic assay which amplifies simultaneously four specific genes to identify invC for Shigella genus, rfc for S. flexneri, wbgZ for S. sonnei, and rfpB for S. dysenteriae, as well as one internal control (ompA) gene, was developed in a single reaction to detect and differentiate Shigella spp. Validation with 120 Shigella strains and 37 non-Shigella strains yielded 100% specificity. The sensitivity of the PCR was 100 pg of genomic DNA, 5.4 × 104 CFU/ml, or approximately 120 CFU per reaction mixture of bacteria. The sensitivity of the pentaplex PCR assay was further improved following preincubation of the stool samples in Gram-negative broth. A preliminary study with 30 diarrhoeal specimens resulted in no cross-reaction with other non-Shigella strains tested. We conclude that the developed pentaplex PCR assay is robust and can provide information about the four target genes that are essential for the identification of the Shigella genus and the three Shigella species responsible for the majority of shigellosis cases. PMID:23509722

Isothermal Recombinase Polymerase amplification (RPA) of Schistosoma haematobium DNA and oligochromatographic lateral flow detection.

PubMed

Rosser, A; Rollinson, D; Forrest, M; Webster, B L

2015-09-04

Accurate diagnosis of urogenital schistosomiasis is vital for surveillance/control programs. Amplification of schistosome DNA in urine by PCR is sensitive and specific but requires infrastructure, financial resources and skilled personnel, often not available in endemic areas. Recombinase Polymerase Amplification (RPA) is an isothermal DNA amplification/detection technology that is simple, rapid, portable and needs few resources. Here a Schistosoma haematobium RPA assay was developed and adapted so that DNA amplicons could be detected using oligochromatographic Lateral Flow (LF) strips. The assay successfully amplified S. haematobium DNA at 30-45 °C in 10 mins and was sensitive to a lower limit of 100 fg of DNA. The assay was also successful with the addition of crude urine, up to 5% of the total reaction volume. Cross amplification occurred with other schistosome species but not with other common urine microorganisms. The LF-RPA assay developed here can amplify and detect low levels of S. haematobium DNA. Reactions are rapid, require low temperatures and positive reactions are interpreted using lateral flow strips, reducing the need for infrastructure and resources. This together with an ability to withstand inhibitors within urine makes RPA a promising technology for further development as a molecular diagnostic tool for urogenital schistosomiasis.

Metabolism Retrofit Strategies for ToxCast Assays (BOSC)

EPA Science Inventory

The EPA’s ToxCast program utilizes a wide variety of high-throughput screening assays (HTS) to assess chemical perturbations of molecular and cellular endpoints. A limitation of many HTS assays used for toxicity assessment is the lack of xenobiotic metabolism (XM) which precludes...

Development of a recombinase polymerase amplification assay for rapid detection of Francisella noatunensis subsp. orientalis

PubMed Central

Gustavo Ramirez-Paredes, Jose; Harold, Graham; Lopez-Jimena, Benjamin; Adams, Alexandra; Weidmann, Manfred

2018-01-01

Francisella noatunensis subsp. orientalis (Fno) is the causative agent of piscine francisellosis in warm water fish including tilapia. The disease induces chronic granulomatous inflammation with high morbidity and can result in high mortality. Early and accurate detection of Fno is crucial to set appropriate outbreak control measures in tilapia farms. Laboratory detection of Fno mainly depends on bacterial culture and molecular techniques. Recombinase polymerase amplification (RPA) is a novel isothermal technology that has been widely used for the molecular diagnosis of various infectious diseases. In this study, a recombinase polymerase amplification (RPA) assay for rapid detection of Fno was developed and validated. The RPA reaction was performed at a constant temperature of 42°C for 20 min. The RPA assay was performed using a quantitative plasmid standard containing a unique Fno gene sequence. Validation of the assay was performed not only by using DNA from Fno, closely related Francisella species and other common bacterial pathogens in tilapia farms, but also by screening 78 Nile tilapia and 5 water samples. All results were compared with those obtained by previously established real-time qPCR. The developed RPA showed high specificity in detection of Fno with no cross-detection of either the closely related Francisella spp. or the other tested bacteria. The Fno-RPA performance was highly comparable to the published qPCR with detection limits at 15 and 11 DNA molecules detected, respectively. The RPA gave quicker results in approximately 6 min in contrast to the qPCR that needed about 90 min to reach the same detection limit, taking only 2.7–3 min to determine Fno in clinical samples. Moreover, RPA was more tolerant to reaction inhibitors than qPCR when tested with field samples. The fast reaction, simplicity, cost-effectiveness, sensitivity and specificity make the RPA an attractive diagnostic tool that will contribute to controlling the infection through prompt on-site detection of Fno. PMID:29444148

Development of a recombinase polymerase amplification assay for rapid detection of Francisella noatunensis subsp. orientalis.

PubMed

Shahin, Khalid; Gustavo Ramirez-Paredes, Jose; Harold, Graham; Lopez-Jimena, Benjamin; Adams, Alexandra; Weidmann, Manfred

2018-01-01

Francisella noatunensis subsp. orientalis (Fno) is the causative agent of piscine francisellosis in warm water fish including tilapia. The disease induces chronic granulomatous inflammation with high morbidity and can result in high mortality. Early and accurate detection of Fno is crucial to set appropriate outbreak control measures in tilapia farms. Laboratory detection of Fno mainly depends on bacterial culture and molecular techniques. Recombinase polymerase amplification (RPA) is a novel isothermal technology that has been widely used for the molecular diagnosis of various infectious diseases. In this study, a recombinase polymerase amplification (RPA) assay for rapid detection of Fno was developed and validated. The RPA reaction was performed at a constant temperature of 42°C for 20 min. The RPA assay was performed using a quantitative plasmid standard containing a unique Fno gene sequence. Validation of the assay was performed not only by using DNA from Fno, closely related Francisella species and other common bacterial pathogens in tilapia farms, but also by screening 78 Nile tilapia and 5 water samples. All results were compared with those obtained by previously established real-time qPCR. The developed RPA showed high specificity in detection of Fno with no cross-detection of either the closely related Francisella spp. or the other tested bacteria. The Fno-RPA performance was highly comparable to the published qPCR with detection limits at 15 and 11 DNA molecules detected, respectively. The RPA gave quicker results in approximately 6 min in contrast to the qPCR that needed about 90 min to reach the same detection limit, taking only 2.7-3 min to determine Fno in clinical samples. Moreover, RPA was more tolerant to reaction inhibitors than qPCR when tested with field samples. The fast reaction, simplicity, cost-effectiveness, sensitivity and specificity make the RPA an attractive diagnostic tool that will contribute to controlling the infection through prompt on-site detection of Fno.

Detection, differentiation, and VP1 sequencing of duck hepatitis A virus type 1 and type 3 by a 1-step duplex reverse-transcription PCR assay.

PubMed

Wen, X J; Cheng, A C; Wang, M S; Jia, R Y; Zhu, D K; Chen, S; Liu, M F; Liu, F; Chen, X Y

2014-09-01

Duck hepatitis A virus (DHAV) is an infectious pathogen causing fatal duck viral hepatitis in ducklings. Although both the inactivated vaccines and live attenuated vaccines have been used to protect ducklings, DHAV-1 and DHAV-3 still cause significant serious damage to the duck industry in China and South Korea. For rapid detection, differentiation, and epidemic investigation of DHAV in China, a genotype-specific 1-step duplex reverse-transcription (RT) PCR assay was established in this study. The sensitivity and specificity of the developed RT-PCR assay was evaluated with nucleic acids extracted from 2 DHAV reference strains, and 9 other infectious viruses and bacteria. The genotype-specific primers amplified different size DNA fragments encompassing the complete VP1 gene of the DHAV-1 or DHAV-3. The assay detected the liver samples collected from experimentally infected ducklings and dead ducklings collected from different regions of China. Sequence analysis of these DNA fragments indicated that VP1 sequences of DHAV-1 can be used to distinguish wild type and vaccine strains. The phylogenetic analysis of VP1 sequences indicated that the developed RT-PCR assay can be used for epidemic investigation of DHAV-1 and DHAV-3. The developed RT-PCR assay can be used as a specific molecular tool for simultaneous detection, differentiation, and sequencing the VP1 gene of DHAV-1 and DHAV-3, which can be used for understanding the epidemiology and evolution of DHAV. © 2014 Poultry Science Association Inc.

Hepatitis E Virus (HEV) Detection and Quantification by a Real-Time Reverse Transcription-PCR Assay Calibrated to the World Health Organization Standard for HEV RNA

PubMed Central

Germer, Jeffrey J.; Ankoudinova, Irina; Belousov, Yevgeniy S.; Mahoney, Walt; Dong, Chen; Meng, Jihong; Mandrekar, Jayawant N.

2017-01-01

ABSTRACT Hepatitis E virus (HEV) has emerged as a cause of chronic hepatitis among immunocompromised patients. Molecular assays have become important tools for the diagnosis and management of these chronically infected patients. A real-time reverse transcription-quantitative PCR (RT-qPCR) assay utilizing Pleiades probe chemistry and an RNA internal control for the simultaneous detection and quantification of HEV RNA in human serum was developed based on an adaptation of a previously described and broadly reactive primer set targeting the overlapping open reading frame 2/3 (ORF2/3) nucleotide sequence of HEV. A chimeric bovine viral diarrhea virus construct containing an HEV RNA insert (SynTura HEV) was developed, value assigned with the first World Health Organization (WHO) international standard for HEV RNA (code 6329/10), and used to prepare working assay calibrators and controls, which supported an assay quantification range of 100 to 5,000,000 IU/ml. The analytical sensitivity (95% detection rate) of this assay was 25.2 IU/ml (95% confidence interval [CI], 19.2 to 44.1 IU/ml). The assay successfully amplified 16 different HEV sequences with significant nucleotide mismatching in primer/probe binding regions, while evaluation of a WHO international reference panel for HEV genotypes (code 8578/13) showed viral load results falling within the result ranges generated by WHO collaborative study participants for all panel members (genotypes 1 to 4). Broadly reactive RT-qPCR primers targeting HEV ORF2/3 were successfully adapted for use in an assay based on Pleiades probe chemistry. The availability of secondary standards calibrated to the WHO HEV international standard can improve the standardization and performance of assays for the detection and quantification of HEV RNA. PMID:28228493

Formalization, Annotation and Analysis of Diverse Drug and Probe Screening Assay Datasets Using the BioAssay Ontology (BAO)

PubMed Central

Vempati, Uma D.; Przydzial, Magdalena J.; Chung, Caty; Abeyruwan, Saminda; Mir, Ahsan; Sakurai, Kunie; Visser, Ubbo; Lemmon, Vance P.; Schürer, Stephan C.

2012-01-01

Huge amounts of high-throughput screening (HTS) data for probe and drug development projects are being generated in the pharmaceutical industry and more recently in the public sector. The resulting experimental datasets are increasingly being disseminated via publically accessible repositories. However, existing repositories lack sufficient metadata to describe the experiments and are often difficult to navigate by non-experts. The lack of standardized descriptions and semantics of biological assays and screening results hinder targeted data retrieval, integration, aggregation, and analyses across different HTS datasets, for example to infer mechanisms of action of small molecule perturbagens. To address these limitations, we created the BioAssay Ontology (BAO). BAO has been developed with a focus on data integration and analysis enabling the classification of assays and screening results by concepts that relate to format, assay design, technology, target, and endpoint. Previously, we reported on the higher-level design of BAO and on the semantic querying capabilities offered by the ontology-indexed triple store of HTS data. Here, we report on our detailed design, annotation pipeline, substantially enlarged annotation knowledgebase, and analysis results. We used BAO to annotate assays from the largest public HTS data repository, PubChem, and demonstrate its utility to categorize and analyze diverse HTS results from numerous experiments. BAO is publically available from the NCBO BioPortal at http://bioportal.bioontology.org/ontologies/1533. BAO provides controlled terminology and uniform scope to report probe and drug discovery screening assays and results. BAO leverages description logic to formalize the domain knowledge and facilitate the semantic integration with diverse other resources. As a consequence, BAO offers the potential to infer new knowledge from a corpus of assay results, for example molecular mechanisms of action of perturbagens. PMID:23155465

Phenotypic Screening Approaches to Develop Aurora Kinase Inhibitors: Drug Discovery Perspectives.

PubMed

Marugán, Carlos; Torres, Raquel; Lallena, María José

2015-01-01

Targeting mitotic regulators as a strategy to fight cancer implies the development of drugs against key proteins, such as Aurora-A and -B. Current drugs, which target mitosis through a general mechanism of action (stabilization/destabilization of microtubules), have several side effects (neutropenia, alopecia, and emesis). Pharmaceutical companies aim at avoiding these unwanted effects by generating improved and selective drugs that increase the quality of life of the patients. However, the development of these drugs is an ambitious task that involves testing thousands of compounds through biochemical and cell-based assays. In addition, molecules usually target complex biological processes, involving several proteins and different molecular pathways, further emphasizing the need for high-throughput screening techniques and multiplexing technologies in order to identify drugs with the desired phenotype. We will briefly describe two multiplexing technologies [high-content imaging (HCI) and flow cytometry] and two key processes for drug discovery research (assay development and validation) following our own published industry quality standards. We will further focus on HCI as a useful tool for phenotypic screening and will provide a concrete example of HCI assay to detect Aurora-A or -B selective inhibitors discriminating the off-target effects related to the inhibition of other cell cycle or non-cell cycle key regulators. Finally, we will describe other assays that can help to characterize the in vitro pharmacology of the inhibitors.

Differentiation and identification of Shigella spp. and enteroinvasive Escherichia coli in environmental waters by a molecular method and biochemical test.

PubMed

Hsu, Bing-Mu; Wu, Shu-Fen; Huang, Shih-Wei; Tseng, Yu-Jung; Ji, Dar-Der; Chen, Jung-Sheng; Shih, Feng-Cheng

2010-02-01

Both Shigella spp. and enteroinvasive Escherichia coli (EIEC) are important human pathogens that are responsible for the majority of cases of endemic bacillary dysentery. However, they are difficult to identify and differentiate by biochemical tests or molecular methods alone. In this study, we developed a procedure to detect Shigella spp. and EIEC from environmental water samples using membrane filtration followed by nutrient broth enrichment, isolation using selective culture plates, and identification of the invasion plasmid antigen H (ipaH) gene by PCR amplification and DNA sequencing. Finally, we used a biochemical test and a serological assay to differentiate between Shigella and EIEC. Among the 93 water samples from nine reservoirs and one watershed, 76 (81.7%) water samples of culture plates had candidate colonies of Shigella and EIEC and 5 water samples were positive (5.4%) for a Shigella- and EIEC-specific polymerase chain reaction targeting the ipaH gene. Guided by the molecular method, the biochemical test, and the serological assay, 11 ipaH gene-positive isolates from 5 water samples were all identified as EIEC. (c) 2009 Elsevier Ltd. All rights reserved.

The persistence of bifidobacteria populations in a river measured by molecular and culture techniques.

PubMed

Bonjoch, X; Lucena, F; Blanch, A R

2009-10-01

To determine relative to faecal coliforms (FC) and sulfite-reducing clostridia (SRC), the environmental persistence of natural populations of Bifidobacterium spp. enumerated by culturing and quantitative polymerase chain reaction (q-PCR). Dialysis tubing containing river supplemented with overnight cultures of Bifidobacterium adolescentis (BA) and Bifidobacterium dentium (BD) or urban wastewater were suspended in a river for up to 10 days. At intervals, the contents of each dialysis tube were assayed using q-PCR assays for BA and BD, and selective culture media for FC, SRC, total bifidobacteria (TB), sorbitol-fermenting bifidobacteria (SFB) and cultivable BA. Mean summer T(90) values were 251 h for SRC, 92 h for FC, 48 h for BA and BD by q-PCR, and 9 h for TB. Bifidobacterium spp. was the population with the lowest persistence, showing seasonal differences in T(90) when measured by culture techniques or by q-PCR. This difference in relative persistence is because of a longer persistence of molecular targets than cultivable cells. The persistence of a viable bifidobacteria cells is shorter, but the longest persistence of molecular targets. This factor could be used for origin the faecal pollution in water for the development of microbial source tracking (MST).

Random amplified polymorphic DNA PCR in the teaching of molecular epidemiology.

PubMed

Reinoso, Elina B; Bettera, Susana G

2016-07-08

In this article, we describe a basic practical laboratory designed for fifth-year undergraduate students of Microbiology as part of the Epidemiology course. This practice provides the students with the tools for molecular epidemiological analysis of pathogenic microorganisms using a rapid and simple PCR technique. The aim of this work was to assay RAPD-PCR technique in order to infer possible epidemiological relationships. The activity gives students an appreciation of the value of applying a simple molecular biological method as RAPD-PCR to a discipline-specific question. It comprises a three-session laboratory module to genetically assay DNAs from strains isolated from a food outbreak. © 2016 by The International Union of Biochemistry and Molecular Biology, 44(4):391-396, 2016. © 2016 The International Union of Biochemistry and Molecular Biology.

Advances in PCR technology.

PubMed

Lauerman, Lloyd H

2004-12-01

Since the discovery of the polymerase chain reaction (PCR) 20 years ago, an avalanche of scientific publications have reported major developments and changes in specialized equipment, reagents, sample preparation, computer programs and techniques, generated through business, government and university research. The requirement for genetic sequences for primer selection and validation has been greatly facilitated by the development of new sequencing techniques, machines and computer programs. Genetic libraries, such as GenBank, EMBL and DDBJ continue to accumulate a wealth of genetic sequence information for the development and validation of molecular-based diagnostic procedures concerning human and veterinary disease agents. The mechanization of various aspects of the PCR assay, such as robotics, microfluidics and nanotechnology, has made it possible for the rapid advancement of new procedures. Real-time PCR, DNA microarray and DNA chips utilize these newer techniques in conjunction with computer and computer programs. Instruments for hand-held PCR assays are being developed. The PCR and reverse transcription-PCR (RT-PCR) assays have greatly accelerated the speed and accuracy of diagnoses of human and animal disease, especially of the infectious agents that are difficult to isolate or demonstrate. The PCR has made it possible to genetically characterize a microbial isolate inexpensively and rapidly for identification, typing and epidemiological comparison.

The reference strain Aeromonas hydrophicla CIP 57.50 should be reclassified as Aeromonas salmonicida CIP 57.50.

PubMed

Miñana-Galbis, David; Farfàn, Maribel; Lorén, J Gaspar; Fusté, M Carmen

2010-03-01

The use of reference strains is a critical element for the quality control of different assays, from the development of molecular methods to the evaluation of antimicrobial activities. Most of the strains used in these assays are not type strains and some of them are cited erroneously because of subsequent reclassifications and descriptions of novel species. In this study, we propose that the reference strain Aeromonas hydrophila CIP 57.50 be reclassified as Aeromonas salmonicida CIP 57.50 based on phenotypic characterization and sequence analyses of the cpn60, dnaJ, gyrB and rpoD genes.

Review on enzyme-linked immunosorbent assays for sulfonamide residues in edible animal products.

PubMed

Zhang, Hongyan; Wang, Shuo

2009-10-31

The current status of enzyme-linked immunosorbent assays (ELISAs) for sulfonamides in edible animal products is reviewed. The attention was focused on the design and synthesis of haptens, conjugation to carrier protein, production of antibody, application of homologous and heterologous systems, as well as the molecular modeling of the haptens and sulfonamides. Researches have shown that sulfonamides seem to be particularly resistant to attempts to produce broad specificity antibodies. By summarizing the available research on sulfonamide ELISAs, it is hoped that it can be considered as a basis for further investigation aimed at developing the most efficient approaches for detection.

Retrofit Strategies for Incorporating Xenobiotic Metabolism into High Throughput Screening Assays (EMGS)

EPA Science Inventory

The US EPA’s ToxCast program is designed to assess chemical perturbations of molecular and cellular endpoints using a variety of high-throughput screening (HTS) assays. However, existing HTS assays have limited or no xenobiotic metabolism which could lead to a mischaracterization...

BRAF mutation testing in solid tumors: a methodological comparison.

PubMed

Weyant, Grace W; Wisotzkey, Jeffrey D; Benko, Floyd A; Donaldson, Keri J

2014-09-01

Solid tumor genotyping has become standard of care for the characterization of proto-oncogene mutational status, which has traditionally been accomplished with Sanger sequencing. However, companion diagnostic assays and comparable laboratory-developed tests are becoming increasingly popular, such as the cobas 4800 BRAF V600 Mutation Test and the INFINITI KRAS-BRAF assay, respectively. This study evaluates and validates the analytical performance of the INFINITI KRAS-BRAF assay and compares concordance of BRAF status with two reference assays, the cobas test and Sanger sequencing. DNA extraction from FFPE tissue specimens was performed followed by multiplex PCR amplification and fluorescent label incorporation using allele-specific primer extension. Hybridization to a microarray, signal detection, and analysis were then performed. The limits of detection were determined by testing dilutions of mutant BRAF alleles within wild-type background DNA, and accuracy was calculated based on these results. The INFINITI KRAS-BRAF assay produced 100% concordance with the cobas test and Sanger sequencing and had sensitivity equivalent to the cobas assay. The INFINITI assay is repeatable with at least 95% accuracy in the detection of mutant and wild-type BRAF alleles. These results confirm that the INFINITI KRAS-BRAF assay is comparable to traditional sequencing and the Food and Drug Administration-approved companion diagnostic assay for the detection of BRAF mutations. Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Species-specific multiplex PCR for the diagnosis of Brucella ovis, Actinobacillus seminis, and Histophilus somni infection in rams

PubMed Central

2013-01-01

Background Infectious ovine epididymitis results in substantial economic losses worldwide due to reproductive failure and culling of breeders. The most common causative agents of these infections are Brucella ovis, Actinobacillus seminis, and Histophilus somni. The aim of this study was to develop a multiplex PCR assay for simultaneous detection of Brucella ovis, Actinobacillus seminis, and Histophilus somni with species-specific primers applied to biological samples for molecular diagnosis of these infections. Results The multiplex assay was capable of detecting B. ovis, A. seminis, and H. somni DNA simultaneously from genomic bacterial DNA samples and pool of semen samples from experimentally infected rams. The method was highly specific since it did not amplify DNA from other bacterial species that can potentially cause epididymitis in rams as well as species phylogenetically related to B. ovis. All negative control samples were negative in PCR multiplex assay. Urine can be used as an alternative to semen samples. Conclusions The species-specific multiplex PCR assay developed in this study can be successfully used for the detection of three of the most common bacterial causes of ovine epididymitis. PMID:23514236

Nonaminoglycoside compounds induce readthrough of nonsense mutations

PubMed Central

Damoiseaux, Robert; Nahas, Shareef; Gao, Kun; Hu, Hailiang; Pollard, Julianne M.; Goldstine, Jimena; Jung, Michael E.; Henning, Susanne M.; Bertoni, Carmen

2009-01-01

Large numbers of genetic disorders are caused by nonsense mutations for which compound-induced readthrough of premature termination codons (PTCs) might be exploited as a potential treatment strategy. We have successfully developed a sensitive and quantitative high-throughput screening (HTS) assay, protein transcription/translation (PTT)–enzyme-linked immunosorbent assay (ELISA), for identifying novel PTC-readthrough compounds using ataxia-telangiectasia (A-T) as a genetic disease model. This HTS PTT-ELISA assay is based on a coupled PTT that uses plasmid templates containing prototypic A-T mutated (ATM) mutations for HTS. The assay is luciferase independent. We screened ∼34,000 compounds and identified 12 low-molecular-mass nonaminoglycosides with potential PTC-readthrough activity. From these, two leading compounds consistently induced functional ATM protein in ATM-deficient cells containing disease-causing nonsense mutations, as demonstrated by direct measurement of ATM protein, restored ATM kinase activity, and colony survival assays for cellular radiosensitivity. The two compounds also demonstrated readthrough activity in mdx mouse myotube cells carrying a nonsense mutation and induced significant amounts of dystrophin protein. PMID:19770270

Development and systematic validation of qPCR assays for rapid and reliable differentiation of Xylella fastidiosa strains causing citrus variegated chlorosis.

PubMed

Li, Wenbin; Teixeira, Diva C; Hartung, John S; Huang, Qi; Duan, Yongping; Zhou, Lijuan; Chen, Jianchi; Lin, Hong; Lopes, Silvio; Ayres, A Juliano; Levy, Laurene

2013-01-01

The xylem-limited, Gram-negative, fastidious plant bacterium Xylella fastidiosa is the causal agent of citrus variegated chlorosis (CVC), a destructive disease affecting approximately half of the citrus plantations in the State of São Paulo, Brazil. The disease was recently found in Central America and is threatening the multi-billion U.S. citrus industry. Many strains of X. fastidiosa are pathogens or endophytes in various plants growing in the U.S., and some strains cross infect several host plants. In this study, a TaqMan-based assay targeting the 16S rDNA signature region was developed for the identification of X. fastidiosa at the species level. Another TaqMan-based assay was developed for the specific identification of the CVC strains. Both new assays have been systematically validated in comparison with the primer/probe sets from four previously published assays on one platform and under similar PCR conditions, and shown to be superior. The species specific assay detected all X. fastidiosa strains and did not amplify any other citrus pathogen or endophyte tested. The CVC-specific assay detected all CVC strains but did not amplify any non-CVC X. fastidiosa nor any other citrus pathogen or endophyte evaluated. Both sets were multiplexed with a reliable internal control assay targeting host plant DNA, and their diagnostic specificity and sensitivity remained unchanged. This internal control provides quality assurance for DNA extraction, performance of PCR reagents, platforms and operators. The limit of detection for both assays was equivalent to 2 to 10 cells of X. fastidiosa per reaction for field citrus samples. Petioles and midribs of symptomatic leaves of sweet orange harbored the highest populations of X. fastidiosa, providing the best materials for detection of the pathogen. These new species specific assay will be invaluable for molecular identification of X. fastidiosa at the species level, and the CVC specific assay will be very powerful for the specific identification of X. fastidiosa strains that cause citrus variegated chlorosis. Published by Elsevier B.V.

Improving molecular tools for global surveillance of measles virus.

PubMed

Bankamp, Bettina; Byrd-Leotis, Lauren A; Lopareva, Elena N; Woo, Gibson K S; Liu, Chunyu; Jee, Youngmee; Ahmed, Hinda; Lim, Wilina W; Ramamurty, Nalini; Mulders, Mick N; Featherstone, David; Bellini, William J; Rota, Paul A

2013-09-01

The genetic characterization of wild-type measles viruses plays an important role in the description of viral transmission pathways and the verification of measles elimination. The 450 nucleotides that encode the carboxyl-terminus of the nucleoprotein (N-450) are routinely sequenced for genotype analysis. The objectives of this study were to develop improved primers and controls for RT-PCR reactions used for genotyping of measles samples and to develop a method to provide a convenient, safe, and inexpensive means to distribute measles RNA for RT-PCR assays and practice panels. A newly designed, genetically defined synthetic RNA and RNA isolated from cells infected with currently circulating genotypes were used to compare the sensitivity of primer pairs in RT-PCR and nested PCR. FTA® cards loaded with lysates of measles infected cells were tested for their ability to preserve viral RNA and destroy virus infectivity. A new primer pair, MeV216/MeV214, was able to amplify N-450 from viruses representing 10 currently circulating genotypes and a genotype A vaccine strain and demonstrated 100-fold increased sensitivity compared to the previously used primer set. A nested PCR assay further increased the sensitivity of detection from patient samples. A synthetic positive control RNA was developed that produced PCR products that are distinguishable by size from PCR products amplified from clinical samples. FTA® cards completely inactivated measles virus and stabilized RNA for at least six months. These improved molecular tools will advance molecular characterization of circulating measles viruses globally and provide enhanced quality control measures. Published by Elsevier B.V.

Improving molecular tools for global surveillance of measles virus⋆

PubMed Central

Bankamp, Bettina; Byrd-Leotis, Lauren A.; Lopareva, Elena N.; Woo, Gibson K.S.; Liu, Chunyu; Jee, Youngmee; Ahmed, Hinda; Lim, Wilina W.; Ramamurty, Nalini; Mulders, Mick N.; Featherstone, David; Bellini, William J.; Rota, Paul A.

2017-01-01

Background The genetic characterization of wild-type measles viruses plays an important role in the description of viral transmission pathways and the verification of measles elimination. The 450 nucleotides that encode the carboxyl-terminus of the nucleoprotein (N-450) are routinely sequenced for genotype analysis. Objectives The objectives of this study were to develop improved primers and controls for RT-PCR reactions used for genotyping of measles samples and to develop a method to provide a convenient, safe, and inexpensive means to distribute measles RNA for RT-PCR assays and practice panels. Study design A newly designed, genetically defined synthetic RNA and RNA isolated from cells infected with currently circulating genotypes were used to compare the sensitivity of primer pairs in RT-PCR and nested PCR. FTA® cards loaded with lysates of measles infected cells were tested for their ability to preserve viral RNA and destroy virus infectivity. Results A new primer pair, MeV216/MeV214, was able to amplify N-450 from viruses representing 10 currently circulating genotypes and a genotype A vaccine strain and demonstrated 100-fold increased sensitivity compared to the previously used primer set. A nested PCR assay further increased the sensitivity of detection from patient samples. A synthetic positive control RNA was developed that produced PCR products that are distinguishable by size from PCR products amplified from clinical samples. FTA® cards completely inactivated measles virus and stabilized RNA for at least six months. Conclusions These improved molecular tools will advance molecular characterization of circulating measles viruses globally and provide enhanced quality control measures. PMID:23806666

[Inventive activity of the Department of Molecular Immunology of the Palladin Institute of Biochemistry of NAS of Ukraine].

PubMed

Danilova, V M; Vynogradova, R P; Torkhova, S G

2016-01-01

The article is devoted to the inventive activity of the Department of Molecular Immunology of the Palladin Institute of Biochemistry of NAS of Ukraine in the context of the history of its inception, development and in the context of scholarly and organizational activities of Sergii Vasyl’ovych Komisarenko. This autumn marks 50th anniversary since young Sergii Komisarenko (now – Academician of NAS and NAMS of Ukraine, Dr. Biol. Sci., Professor) has joined the Palladin Institute of Biochemistry, has completed all stages of the academic carrier from PhD student to Head of the Institute. He is the first in Ukraine who started the new branch of research – molecular immunology, created a strong scientific school, which earned worldwide acclaim and made significant contribution to finding solutions to current problems in human health sciences. S.V. Komisarenko was among those, who were first in the USSR to use immunoenzyme and flow cytofluometric assays, hybridoma technology for producing monoclonal antibodies and immunochemical assay of proteins, which became the basis for development of highly sensitive and highly specific immunodiagnostic systems, which are of high necessity in medicine, veterinary, development of immunotechnologies, environment monitoring, etc. Under his leadership the Department has made a series of important discoveries and developments including relating to antitumour immunotoxins, effects of low dose radiation on the immune system of Chernobyl liquidators, immunochemical structure of neurotoxin apamine, cytochrom c, fibrinogen and fibrin molecules at different stages of polymerization, diphtheria toxin and its receptor, tuberculosis causing micobacterium, roles of protease-activated receptors (PARs) and nicotinic acetylcholine receptors of lymphocytes, nature of polyreactive immunoglobulins (PRIGs), among other important scientific contributions. S.V. Komisarenko and his colleagues also hold numerous (more than 80) author’s certificates and patents in Ukraine and USSR.

Measuring T cell-mediated cytotoxicity using fluorogenic caspase substrates.

PubMed

Chahroudi, A; Silvestri, G; Feinberg, M B

2003-10-01

Cytotoxic T lymphocytes (CTLs) play a major role in the immune response against viruses and other intracellular pathogens. In addition, CTLs are implicated in the control of tumor cells in certain settings. Accurate measures of CTL function are of critical importance to study the pathogenesis of infectious diseases and to evaluate the efficacy of new vaccines and immunotherapies. To this end, we have recently developed a flow cytometry-based CTL (FCC) assay that measures the CTL-induced caspase activation within target cells using cell permeable fluorogenic caspase substrates. This novel assay reliably detects, by flow cytometry or fluorescence/confocal microscopy, antigen-specific CTLs in a wide variety of human and murine systems, and is safer and more informative than the standard 51Cr-release assay. In addition, the flow cytometric CTL (FCC) assay provides an alternative method that is often more sensitive and physiologically informative when compared to previously described FCC assays, as it measures a biological indicator of apoptosis within the target cell. The FCC assay may thus represent a useful tool to further understand the molecular and cellular mechanisms that underlie CTL-mediated killing during tumorigenesis or following infection with viruses or other intracellular pathogens.

Target-Specific Assay for Rapid and Quantitative Detection of Mycobacterium chimaera DNA

PubMed Central

Zozaya-Valdés, Enrique; Porter, Jessica L.; Coventry, John; Fyfe, Janet A. M.; Carter, Glen P.; Gonçalves da Silva, Anders; Schultz, Mark B.; Seemann, Torsten; Johnson, Paul D. R.; Stewardson, Andrew J.; Bastian, Ivan; Roberts, Sally A.; Howden, Benjamin P.; Williamson, Deborah A.

2017-01-01

ABSTRACT Mycobacterium chimaera is an opportunistic environmental mycobacterium belonging to the Mycobacterium avium-M. intracellulare complex. Although most commonly associated with pulmonary disease, there has been growing awareness of invasive M. chimaera infections following cardiac surgery. Investigations suggest worldwide spread of a specific M. chimaera clone, associated with contaminated hospital heater-cooler units used during the surgery. Given the global dissemination of this clone, its potential to cause invasive disease, and the laboriousness of current culture-based diagnostic methods, there is a pressing need to develop rapid and accurate diagnostic assays specific for M. chimaera. Here, we assessed 354 mycobacterial genome sequences and confirmed that M. chimaera is a phylogenetically coherent group. In silico comparisons indicated six DNA regions present only in M. chimaera. We targeted one of these regions and developed a TaqMan quantitative PCR (qPCR) assay for M. chimaera with a detection limit of 100 CFU/ml in whole blood spiked with bacteria. In vitro screening against DNA extracted from 40 other mycobacterial species and 22 bacterial species from 21 diverse genera confirmed the in silico-predicted specificity for M. chimaera. Screening 33 water samples from heater-cooler units with this assay highlighted the increased sensitivity of PCR compared to culture, with 15 of 23 culture-negative samples positive by M. chimaera qPCR. We have thus developed a robust molecular assay that can be readily and rapidly deployed to screen clinical and environmental specimens for M. chimaera. PMID:28381604

Target-Specific Assay for Rapid and Quantitative Detection of Mycobacterium chimaera DNA.

PubMed

Zozaya-Valdés, Enrique; Porter, Jessica L; Coventry, John; Fyfe, Janet A M; Carter, Glen P; Gonçalves da Silva, Anders; Schultz, Mark B; Seemann, Torsten; Johnson, Paul D R; Stewardson, Andrew J; Bastian, Ivan; Roberts, Sally A; Howden, Benjamin P; Williamson, Deborah A; Stinear, Timothy P

2017-06-01

Mycobacterium chimaera is an opportunistic environmental mycobacterium belonging to the Mycobacterium avium - M. intracellulare complex. Although most commonly associated with pulmonary disease, there has been growing awareness of invasive M. chimaera infections following cardiac surgery. Investigations suggest worldwide spread of a specific M. chimaera clone, associated with contaminated hospital heater-cooler units used during the surgery. Given the global dissemination of this clone, its potential to cause invasive disease, and the laboriousness of current culture-based diagnostic methods, there is a pressing need to develop rapid and accurate diagnostic assays specific for M. chimaera Here, we assessed 354 mycobacterial genome sequences and confirmed that M. chimaera is a phylogenetically coherent group. In silico comparisons indicated six DNA regions present only in M. chimaera We targeted one of these regions and developed a TaqMan quantitative PCR (qPCR) assay for M. chimaera with a detection limit of 100 CFU/ml in whole blood spiked with bacteria. In vitro screening against DNA extracted from 40 other mycobacterial species and 22 bacterial species from 21 diverse genera confirmed the in silico -predicted specificity for M. chimaera Screening 33 water samples from heater-cooler units with this assay highlighted the increased sensitivity of PCR compared to culture, with 15 of 23 culture-negative samples positive by M. chimaera qPCR. We have thus developed a robust molecular assay that can be readily and rapidly deployed to screen clinical and environmental specimens for M. chimaera . Copyright © 2017 American Society for Microbiology.

Simple fluorescence-based detection of protein kinase A activity using a molecular beacon probe.

PubMed

Ma, Changbei; Lv, Xiaoyuan; Wang, Kemin; Jin, Shunxin; Liu, Haisheng; Wu, Kefeng; Zeng, Weimin

2017-11-02

Protein kinase A was detected by quantifying the amount of ATP used after a protein kinase reaction. The ATP assay was performed using the T4 DNA ligase and a molecular beacon (MB). In the presence of ATP, DNA ligase catalyzed the ligation of short DNA. The ligation product then hybridized to MB, resulting in a fluorescence enhancement of the MB. This assay was capable of determining protein kinase A in the range of 12.5∼150 nM, with a detection limit of 1.25 nM. Furthermore, this assay could also be used to investigate the effect of genistein on protein kinase A. It was a universal, non-radioisotopic, and homogeneous method for assaying protein kinase A.

Use of chemical flocculation and nested PCR for Heterodera glycines detection in DNA extracts from field soils with low population densities

USDA-ARS?s Scientific Manuscript database

The soybean cyst nematode (SCN) Heterodera glycines is a major pathogen of soybean world-wide. Distinction between SCN and other members of H. schachtii sensu stricto group based on morphology is a tedious task. A molecular assay was developed to detect SCN in field soils with low population densiti...

INDUCTION OF GENE EXPRESSION IN SHEEPSHEAD MINNOWS (CYPRINODON VARIEGATUS) TREATED WITH 17B-ESTRADIOL, DIETHYLSTILBESTROL, OR ETHINYLESTRADIOL: THE USE OF MRNA FINGERPRINTS AS AN INDICATOR OF GENE REGULATION

EPA Science Inventory

The recent interest in hormonally active environmental contaminants has sparked a drive to find sensitive methods to measure their effects on wildlife. A molecular-based assay has been developed to measure the induction of gene expression in sheepshead minnows (Cyprinodon variega...

MHC class I target recognition, immunophenotypes and proteomic profiles of natural killer cells within the spleens of day-14 chick embryos

USDA-ARS?s Scientific Manuscript database

Chicken natural killer (NK) cells are not well defined, so little is known about the molecular interactions controlling their activity. At day 14 of embryonic development, chick spleens are a rich source of T-cellfree CD8aa+, CD3_ cells with natural killing activity. Cell-mediated cytotoxicity assay...

Quick detection of Leifsonia xyli subsp. xyli by PCR and necleotide sequence analysis of PCR amplicons from Chinese Leifsonia xyli subsp. xyli isolates

USDA-ARS?s Scientific Manuscript database

A quick polymerase chain reaction (PCR) assay was developed for the detection of Leifsonia xyli subsp. xyli (Lxx), the bacterial causal agent of ratoon stunting disease (RSD) of sugarcane, in crude juice samples from stalks. After removal of abiotic impurities and large molecular weight microorgani...

Preanalytical considerations in detection of colorectal cancer in blood serum using Raman molecular imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Treado, Patrick J.; Stewart, Shona D.; Smith, Aaron; Kirschner, Heather; Post, Christopher; Overholt, Bergein F.

2016-03-01

Colorectal cancer (CRC) is the third most common cancer in men and women in the United States. Raman Molecular Imaging (RMI) is an effective technique to evaluate human tissue, cells and bodily fluids, including blood serum for disease diagnosis. ChemImage Corporation, in collaboration with clinicians, has been engaged in development of an in vitro diagnostic Raman assay focused on CRC detection. The Raman Assay for Colorectal Cancer (RACC) exploits the high specificity of Raman imaging to distinguish diseased from normal dried blood serum droplets without additional reagents. Pilot Study results from testing of hundreds of biobank patient samples have demonstrated that RACC detects CRC with high sensitivity and specificity. However, expanded clinical trials, which are ongoing, are revealing a host of important preanalytical considerations associated with sample collection, sample storage and stability, sample shipping, sample preparation and sample interferents, which impact detection performance. Results from recent clinical studies will be presented.

Turning tryptophanase into odor-generating biosensors.

PubMed

Xu, Yaqin; Zhang, Zhuyuan; Ali, M Monsur; Sauder, Joanna; Deng, Xudong; Giang, Karen; Aguirre, Sergio D; Pelton, Robert; Li, Yingfu; Filipe, Carlos D M

2014-03-03

An odor-based sensor system that exploits the metabolic enzyme tryptophanase (TPase) as the key component is reported. This enzyme is able to convert an odorless substrate like S-methyl-L-cysteine or L-tryptophan into the odorous products methyl mercaptan or indole. To make a biosensor, TPase was biotinylated so that it could be coupled with a molecular recognition element, such as an antibody, to develop an ELISA-like assay. This method was used for the detection of an antibody present in nM concentrations by the human nose. TPase can also be combined with the enzyme pyridoxal kinase (PKase) for use in a coupled assay to detect adenosine 5'-triphosphate (ATP). When ATP is present in the low μM concentration range, the coupled enzymatic system generates an odor that is easily detectable by the human nose. Biotinylated TPase can be combined with various biotin-labeled molecular recognition elements, thereby enabling a broad range of applications for this odor-based reporting system. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of an alkaline protease associated with a granulosis virus of Plodia interpunctella.

PubMed

Tweeten, K A; Bulla, L A; Consigli, R A

1978-06-01

An alkaline protease was found to be associated with the granulosis virus of the Indian meal moth. Plodia interpunctella. The protease was located within the protein matrix of the occluded virus and hydrolyzed the major constituent of this matrix, a 28,000-dalton protein (granulin), to a mixture of polypeptides ranging in molecular weight from 10,000 to 27,000. A rapid, sensitive assay for the protease was developed using radioactively labeled granulosis virus as substrate. With this assay, the proteolytic activity could be detected by measuring the release of acid-soluble peptides from the labeled virus. The protease had a pH optimum of 10.5 and a temperature optimum of 40 degrees C and was inhibited by diisopropyl phosphorofluoridate, phenylmethylsulfonyl fluoride, and L-(1-tosylamido-2-phenyl) ethyl chloromethyl ketone. Purification of the protease from matrix protein was achieved by anion-exchange and gel permeation chromatography. The molecular weight of the isolated protease, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, was approximately 14,000.

Approaches to developing alternative and predictive toxicology based on PBPK/PD and QSAR modeling.

PubMed Central

Yang, R S; Thomas, R S; Gustafson, D L; Campain, J; Benjamin, S A; Verhaar, H J; Mumtaz, M M

1998-01-01

Systematic toxicity testing, using conventional toxicology methodologies, of single chemicals and chemical mixtures is highly impractical because of the immense numbers of chemicals and chemical mixtures involved and the limited scientific resources. Therefore, the development of unconventional, efficient, and predictive toxicology methods is imperative. Using carcinogenicity as an end point, we present approaches for developing predictive tools for toxicologic evaluation of chemicals and chemical mixtures relevant to environmental contamination. Central to the approaches presented is the integration of physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) and quantitative structure--activity relationship (QSAR) modeling with focused mechanistically based experimental toxicology. In this development, molecular and cellular biomarkers critical to the carcinogenesis process are evaluated quantitatively between different chemicals and/or chemical mixtures. Examples presented include the integration of PBPK/PD and QSAR modeling with a time-course medium-term liver foci assay, molecular biology and cell proliferation studies. Fourier transform infrared spectroscopic analyses of DNA changes, and cancer modeling to assess and attempt to predict the carcinogenicity of the series of 12 chlorobenzene isomers. Also presented is an ongoing effort to develop and apply a similar approach to chemical mixtures using in vitro cell culture (Syrian hamster embryo cell transformation assay and human keratinocytes) methodologies and in vivo studies. The promise and pitfalls of these developments are elaborated. When successfully applied, these approaches may greatly reduce animal usage, personnel, resources, and time required to evaluate the carcinogenicity of chemicals and chemical mixtures. Images Figure 6 PMID:9860897

Implementation of a Multiplex and Quantitative Proteomics Platform for Assessing Protein Lysates Using DNA-Barcoded Antibodies.

PubMed

Lee, Jinho; Geiss, Gary K; Demirkan, Gokhan; Vellano, Christopher P; Filanoski, Brian; Lu, Yiling; Ju, Zhenlin; Yu, Shuangxing; Guo, Huifang; Bogatzki, Lisa Y; Carter, Warren; Meredith, Rhonda K; Krishnamurthy, Savitri; Ding, Zhiyong; Beechem, Joseph M; Mills, Gordon B

2018-06-01

Molecular analysis of tumors forms the basis for personalized cancer medicine and increasingly guides patient selection for targeted therapy. Future opportunities for personalized medicine are highlighted by the measurement of protein expression levels via immunohistochemistry, protein arrays, and other approaches; however, sample type, sample quantity, batch effects, and "time to result" are limiting factors for clinical application. Here, we present a development pipeline for a novel multiplexed DNA-labeled antibody platform which digitally quantifies protein expression from lysate samples. We implemented a rigorous validation process for each antibody and show that the platform is amenable to multiple protocols covering nitrocellulose and plate-based methods. Results are highly reproducible across technical and biological replicates, and there are no observed "batch effects" which are common for most multiplex molecular assays. Tests from basal and perturbed cancer cell lines indicate that this platform is comparable to orthogonal proteomic assays such as Reverse-Phase Protein Array, and applicable to measuring the pharmacodynamic effects of clinically-relevant cancer therapeutics. Furthermore, we demonstrate the potential clinical utility of the platform with protein profiling from breast cancer patient samples to identify molecular subtypes. Together, these findings highlight the potential of this platform for enhancing our understanding of cancer biology in a clinical translation setting. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Advanced yellow fever virus genome detection in point-of-care facilities and reference laboratories.

PubMed

Domingo, Cristina; Patel, Pranav; Yillah, Jasmin; Weidmann, Manfred; Méndez, Jairo A; Nakouné, Emmanuel Rivalyn; Niedrig, Matthias

2012-12-01

Reported methods for the detection of the yellow fever viral genome are beset by limitations in sensitivity, specificity, strain detection spectra, and suitability to laboratories with simple infrastructure in areas of endemicity. We describe the development of two different approaches affording sensitive and specific detection of the yellow fever genome: a real-time reverse transcription-quantitative PCR (RT-qPCR) and an isothermal protocol employing the same primer-probe set but based on helicase-dependent amplification technology (RT-tHDA). Both assays were evaluated using yellow fever cell culture supernatants as well as spiked and clinical samples. We demonstrate reliable detection by both assays of different strains of yellow fever virus with improved sensitivity and specificity. The RT-qPCR assay is a powerful tool for reference or diagnostic laboratories with real-time PCR capability, while the isothermal RT-tHDA assay represents a useful alternative to earlier amplification techniques for the molecular diagnosis of yellow fever by field or point-of-care laboratories.

Drosophila hemocyte migration: an in vivo assay for directional cell migration.

PubMed

Moreira, Carolina G A; Regan, Jennifer C; Zaidman-Rémy, Anna; Jacinto, Antonio; Prag, Soren

2011-01-01

This protocol describes an in vivo assay for random and directed hemocyte migration in Drosophila. Drosophila is becoming an increasingly powerful model system for in vivo cell migration analysis, combining unique genetic tools with translucency of the embryo and pupa, which allows direct imaging and traceability of different cell types. In the assay we present here, we make use of the hemocyte response to epithelium wounding to experimentally induce a transition from random to directed migration. Time-lapse confocal microscopy of hemocyte migration in untreated conditions provides a random cell migration assay that allows identification of molecular mechanisms involved in this complex process. Upon laser-induced wounding of the thorax epithelium, a rapid chemotactic response changes hemocyte migratory behavior into a directed migration toward the wound site. This protocol provides a direct comparison of cells during both types of migration in vivo, and combined with recently developed resources such as transgenic RNAi, is ideal for forward genetic screens.

Development of loop-mediated isothermal amplification with Plasmodium falciparum unique genes for molecular diagnosis of human malaria.

PubMed

Zhang, Yijing; Yao, Yi; Du, Weixing; Wu, Kai; Xu, Wenyue; Lin, Min; Tan, Huabing; Li, Jian

2017-07-01

In order to achieve better outcomes for treatment and in the prophylaxis of malaria, it is imperative to develop a sensitive, specific, and accurate assay for early diagnosis of Plasmodium falciparum infection, which is the major cause of malaria. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP) assay with P. falciparum unique genes for sensitive, specific, and accurate detection of P. falciparum infection. The unique genes of P. falciparum were randomly selected from PlasmoDB. The LAMP primers of the unique genes were designed using PrimerExplorer V4. LAMP assays with primers from unique genes of P. falciparum and conserved 18S rRNA gene were developed and their sensitivity was assessed. The specificity of the most sensitive LAMP assay was further examined using genomic DNA from Plasmodium vivax, Plasmodium yoelii and Toxoplasma gondii. Finally, the unique gene-based LAMP assay was validated using clinical samples of P. falciparum infection cases. A total of 31 sets of top-scored LAMP primers from nine unique genes were selected from the pools of designed primers. The LAMP assay with PF3D7_1253300-5 was the most sensitive with the detection limit 5 parasites/μl, and it displayed negative LAMP assay with the genomic DNA samples of P. vivax, P. yoelii, and T. gondii. The LAMP assay with PF3D7_0112300 (18S rRNA) was less sensitive with the detection limit 50 parasites/μl, and it displayed negative LAMP assay with the genomic DNA samples of P. yoelii and T. gondii, but displayed positive LAMP detection with P. vivax. The positive detection rate of the LAMP assay with PF3D7_1253300-5 was 90% (27/30), higher than that (80%, 24/30) of the positive rate of PF3D7_0112300 (18S rRNA) in examining clinical samples of P. falciparum infection cases. The LAMP assay with the primer set PF3D7_1253300-5 was more sensitive, specific, and accurate than those with PF3D7_0112300 (18S rRNA) in examining P. falciparum infection, and therefore it is a promising tool for diagnosis of P. falciparum infection.

Sequencing of the variable region of rpsB to discriminate between Streptococcus pneumoniae and other streptococcal species.

PubMed

Wyllie, Anne L; Pannekoek, Yvonne; Bovenkerk, Sandra; van Engelsdorp Gastelaars, Jody; Ferwerda, Bart; van de Beek, Diederik; Sanders, Elisabeth A M; Trzciński, Krzysztof; van der Ende, Arie

2017-09-01

The vast majority of streptococci colonizing the human upper respiratory tract are commensals, only sporadically implicated in disease. Of these, the most pathogenic is Mitis group member, Streptococcus pneumoniae Phenotypic and genetic similarities between streptococci can cause difficulties in species identification. Using ribosomal S2-gene sequences extracted from whole-genome sequences published from 501 streptococci, we developed a method to identify streptococcal species. We validated this method on non-pneumococcal isolates cultured from cases of severe streptococcal disease ( n = 101) and from carriage ( n = 103), and on non-typeable pneumococci from asymptomatic individuals ( n = 17) and on whole-genome sequences of 1157 pneumococcal isolates from meningitis in the Netherlands. Following this, we tested 221 streptococcal isolates in molecular assays originally assumed specific for S. pneumoniae , targeting cpsA , lytA , piaB , ply , Spn9802, zmpC and capsule-type-specific genes. Cluster analysis of S2-sequences showed grouping according to species in line with published phylogenies of streptococcal core genomes. S2-typing convincingly distinguished pneumococci from non-pneumococcal species (99.2% sensitivity, 100% specificity). Molecular assays targeting regions of lytA and piaB were 100% specific for S. pneumoniae , whereas assays targeting cpsA , ply , Spn9802, zmpC and selected serotype-specific assays (but not capsular sequence typing) showed a lack of specificity. False positive results were over-represented in species associated with carriage, although no particular confounding signal was unique for carriage isolates. © 2017 The Authors.

Sequencing of the variable region of rpsB to discriminate between Streptococcus pneumoniae and other streptococcal species

PubMed Central

Pannekoek, Yvonne; Bovenkerk, Sandra; van Engelsdorp Gastelaars, Jody; Ferwerda, Bart; van de Beek, Diederik; Sanders, Elisabeth A. M.; Trzciński, Krzysztof; van der Ende, Arie

2017-01-01

The vast majority of streptococci colonizing the human upper respiratory tract are commensals, only sporadically implicated in disease. Of these, the most pathogenic is Mitis group member, Streptococcus pneumoniae. Phenotypic and genetic similarities between streptococci can cause difficulties in species identification. Using ribosomal S2-gene sequences extracted from whole-genome sequences published from 501 streptococci, we developed a method to identify streptococcal species. We validated this method on non-pneumococcal isolates cultured from cases of severe streptococcal disease (n = 101) and from carriage (n = 103), and on non-typeable pneumococci from asymptomatic individuals (n = 17) and on whole-genome sequences of 1157 pneumococcal isolates from meningitis in the Netherlands. Following this, we tested 221 streptococcal isolates in molecular assays originally assumed specific for S. pneumoniae, targeting cpsA, lytA, piaB, ply, Spn9802, zmpC and capsule-type-specific genes. Cluster analysis of S2-sequences showed grouping according to species in line with published phylogenies of streptococcal core genomes. S2-typing convincingly distinguished pneumococci from non-pneumococcal species (99.2% sensitivity, 100% specificity). Molecular assays targeting regions of lytA and piaB were 100% specific for S. pneumoniae, whereas assays targeting cpsA, ply, Spn9802, zmpC and selected serotype-specific assays (but not capsular sequence typing) showed a lack of specificity. False positive results were over-represented in species associated with carriage, although no particular confounding signal was unique for carriage isolates. PMID:28931649

Optimising molecular diagnostic capacity for effective control of tuberculosis in high-burden settings.

PubMed

Sabiiti, W; Mtafya, B; Kuchaka, D; Azam, K; Viegas, S; Mdolo, A; Farmer, E C W; Khonga, M; Evangelopoulos, D; Honeyborne, I; Rachow, A; Heinrich, N; Ntinginya, N E; Bhatt, N; Davies, G R; Jani, I V; McHugh, T D; Kibiki, G; Hoelscher, M; Gillespie, S H

2016-08-01

The World Health Organization's 2035 vision is to reduce tuberculosis (TB) associated mortality by 95%. While low-burden, well-equipped industrialised economies can expect to see this goal achieved, it is challenging in the low- and middle-income countries that bear the highest burden of TB. Inadequate diagnosis leads to inappropriate treatment and poor clinical outcomes. The roll-out of the Xpert(®) MTB/RIF assay has demonstrated that molecular diagnostics can produce rapid diagnosis and treatment initiation. Strong molecular services are still limited to regional or national centres. The delay in implementation is due partly to resources, and partly to the suggestion that such techniques are too challenging for widespread implementation. We have successfully implemented a molecular tool for rapid monitoring of patient treatment response to anti-tuberculosis treatment in three high TB burden countries in Africa. We discuss here the challenges facing TB diagnosis and treatment monitoring, and draw from our experience in establishing molecular treatment monitoring platforms to provide practical insights into successful optimisation of molecular diagnostic capacity in resource-constrained, high TB burden settings. We recommend a holistic health system-wide approach for molecular diagnostic capacity development, addressing human resource training, institutional capacity development, streamlined procurement systems, and engagement with the public, policy makers and implementers of TB control programmes.

Evaluation of qPCR-Based Assays for Leprosy Diagnosis Directly in Clinical Specimens

PubMed Central

Sarno, Euzenir Nunes; Moraes, Milton Ozório

2011-01-01

The increased reliability and efficiency of the quantitative polymerase chain reaction (qPCR) makes it a promising tool for performing large-scale screening for infectious disease among high-risk individuals. To date, no study has evaluated the specificity and sensitivity of different qPCR assays for leprosy diagnosis using a range of clinical samples that could bias molecular results such as difficult-to-diagnose cases. In this study, qPCR assays amplifying different M. leprae gene targets, sodA, 16S rRNA, RLEP and Ag 85B were compared for leprosy differential diagnosis. qPCR assays were performed on frozen skin biopsy samples from a total of 62 patients: 21 untreated multibacillary (MB), 26 untreated paucibacillary (PB) leprosy patients, as well as 10 patients suffering from other dermatological diseases and 5 healthy donors. To develop standardized protocols and to overcome the bias resulted from using chromosome count cutoffs arbitrarily defined for different assays, decision tree classifiers were used to estimate optimum cutoffs and to evaluate the assays. As a result, we found a decreasing sensitivity for Ag 85B (66.1%), 16S rRNA (62.9%), and sodA (59.7%) optimized assay classifiers, but with similar maximum specificity for leprosy diagnosis. Conversely, the RLEP assay showed to be the most sensitive (87.1%). Moreover, RLEP assay was positive for 3 samples of patients originally not diagnosed as having leprosy, but these patients developed leprosy 5–10 years after the collection of the biopsy. In addition, 4 other samples of patients clinically classified as non-leprosy presented detectable chromosome counts in their samples by the RLEP assay suggesting that those patients either had leprosy that was misdiagnosed or a subclinical state of leprosy. Overall, these results are encouraging and suggest that RLEP assay could be useful as a sensitive diagnostic test to detect M. leprae infection before major clinical manifestations. PMID:22022631

Clinical evaluation of the new Roche platform of serological and molecular cytomegalovirus-specific assays in the diagnosis and prognosis of congenital cytomegalovirus infection.

PubMed

Chiereghin, Angela; Pavia, Claudia; Gabrielli, Liliana; Piccirilli, Giulia; Squarzoni, Diego; Turello, Gabriele; Gibertoni, Dino; Simonazzi, Giuliana; Capretti, Maria Grazia; Lanari, Marcello; Lazzarotto, Tiziana

2017-10-01

Clinical evaluation of the Elecsys ® CMV IgM, IgG, IgG Avidity and COBAS AmpliPrep/COBAS TaqMan CMV (COBAS CMV) assays (Roche Diagnostics AG) in the diagnosis and prognosis of congenital CMV infection was performed. In this study, 150 preselected clinical samples (50 primary infection sera, 50 amniotic fluid [AF] and 50 newborn urine) were processed using Roche serological/molecular CMV-specific tests. Results were compared with those obtained by routine assays (comparator assays). The Elecsys ® CMV IgM and IgG assays showed a perfect agreement (100%) with the comparator assays. Using the combination of the Elecsys ® CMV IgM and IgG Avidity assays results, a primary infection was identified in 100% of cases. Inappropriate avidity CMV IgG values in two samples with very low IgG values (<6 AU/mL) were observed. COBAS CMV assay showed an agreement equal to 98% and 100% with comparator assays by processing AF and urine samples, respectively. Among AF with quantitative results, Lin's concordance correlation was 0.933 and comparator-COBAS CMV assays gave CMV-DNA loads differing by <0.5 log 10 DNA. Finally, higher CMV-DNA levels in AF samples were associated with a symptomatic outcome (p=0.003). The Roche CMV-specific assays compared well with the comparator assays, thus providing to be suitable for clinical use. Copyright © 2017 Elsevier B.V. All rights reserved.

Dissection and Downstream Analysis of Zebra Finch Embryos at Early Stages of Development

PubMed Central

Murray, Jessica R.; Stanciauskas, Monika E.; Aralere, Tejas S.; Saha, Margaret S.

2014-01-01

The zebra finch (Taeniopygiaguttata) has become an increasingly important model organism in many areas of research including toxicology1,2, behavior3, and memory and learning4,5,6. As the only songbird with a sequenced genome, the zebra finch has great potential for use in developmental studies; however, the early stages of zebra finch development have not been well studied. Lack of research in zebra finch development can be attributed to the difficulty of dissecting the small egg and embryo. The following dissection method minimizes embryonic tissue damage, which allows for investigation of morphology and gene expression at all stages of embryonic development. This permits both bright field and fluorescence quality imaging of embryos, use in molecular procedures such as in situ hybridization (ISH), cell proliferation assays, and RNA extraction for quantitative assays such as quantitative real-time PCR (qtRT-PCR). This technique allows investigators to study early stages of development that were previously difficult to access. PMID:24999108

Multigene assays and molecular markers in breast cancer: systematic review of health economic analyses.

PubMed

Rouzier, Roman; Pronzato, Paolo; Chéreau, Elisabeth; Carlson, Josh; Hunt, Barnaby; Valentine, William J

2013-06-01

Breast cancer is the most common female cancer and is associated with a significant clinical and economic burden. Multigene assays and molecular markers represent an opportunity to direct chemotherapy only to patients likely to have significant benefit. This systematic review examines published health economic analyses to assess the support for adjuvant therapy decision making. Literature searches of PubMed, the Cochrane Library, and congress databases were carried out to identify economic evaluations of multigene assays and molecular markers published between 2002 and 2012. After screening and data extraction, study quality was assessed using the Quality of Health Economic Studies instrument. The review identified 29 publications that reported evaluations of two assays: Oncotype DX(®) and MammaPrint. Studies of both tests provided evidence that their routine use was cost saving or cost-effective versus conventional approaches. Benefits were driven by optimal allocation of adjuvant chemotherapy and reduction in chemotherapy utilization. Findings were sensitive to variation in the frequency of chemotherapy prescription, chemotherapy costs, and patients' risk profiles. Evidence suggests that multigene assays are likely cost saving or cost-effective relative to current approaches to adjuvant therapy. They should benefit decision making in early-stage breast cancer in a variety of settings worldwide.

Development and Verification of an RNA Sequencing (RNA-Seq) Assay for the Detection of Gene Fusions in Tumors.

PubMed

Winters, Jennifer L; Davila, Jaime I; McDonald, Amber M; Nair, Asha A; Fadra, Numrah; Wehrs, Rebecca N; Thomas, Brittany C; Balcom, Jessica R; Jin, Long; Wu, Xianglin; Voss, Jesse S; Klee, Eric W; Oliver, Gavin R; Graham, Rondell P; Neff, Jadee L; Rumilla, Kandelaria M; Aypar, Umut; Kipp, Benjamin R; Jenkins, Robert B; Jen, Jin; Halling, Kevin C

2018-06-13

We assessed the performance characteristics of an RNA sequencing (RNA-Seq) assay designed to detect gene fusions in 571 genes to help manage patients with cancer. Polyadenylated RNA was converted to cDNA, which was then used to prepare next-generation sequencing libraries that were sequenced on an Illumina HiSeq 2500 instrument and analyzed with an in-house developed bioinformatic pipeline. The assay identified 38 of 41 gene fusions detected by another method, such as fluorescence in situ hybridization or RT-PCR, for a sensitivity of 93%. No false-positive gene fusions were identified in 15 normal tissue specimens and 10 tumor specimens that were negative for fusions by RNA sequencing or Mate Pair NGS (100% specificity). The assay also identified 22 fusions in 17 tumor specimens that had not been detected by other methods. Eighteen of the 22 fusions had not previously been described. Good intra-assay and interassay reproducibility was observed with complete concordance for the presence or absence of gene fusions in replicates. The analytical sensitivity of the assay was tested by diluting RNA isolated from gene fusion-positive cases with fusion-negative RNA. Gene fusions were generally detectable down to 12.5% dilutions for most fusions and as little as 3% for some fusions. This assay can help identify fusions in patients with cancer; these patients may in turn benefit from both US Food and Drug Administration-approved and investigational targeted therapies. Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

78 FR 9060 - Request for Nominations for Voting Members on Public Advisory Panels or Committees

Federal Register 2010, 2011, 2012, 2013, 2014

2013-02-07

... diagnostic assays, e.g., hepatologists; molecular biologists. Molecular and Clinical 2 June 1, 2013. Genetics.... Individuals with training in inborn errors of metabolism, biochemical and/or molecular genetics, population genetics, epidemiology and related statistical training, and clinical molecular genetics testing (e.g...

77 FR 26294 - Government-Owned Inventions; Availability for Licensing

Federal Register 2010, 2011, 2012, 2013, 2014

2012-05-03

... ELISA-like assay entirely to the molecular level, complex macroscopic or microfluidic washing and... for ELISA assays Modify or destroy target molecules, while detecting them Detect genetic diseases in...

SWATH Mass Spectrometry Performance Using Extended Peptide MS/MS Assay Libraries.

PubMed

Wu, Jemma X; Song, Xiaomin; Pascovici, Dana; Zaw, Thiri; Care, Natasha; Krisp, Christoph; Molloy, Mark P

2016-07-01

The use of data-independent acquisition methods such as SWATH for mass spectrometry based proteomics is usually performed with peptide MS/MS assay libraries which enable identification and quantitation of peptide peak areas. Reference assay libraries can be generated locally through information dependent acquisition, or obtained from community data repositories for commonly studied organisms. However, there have been no studies performed to systematically evaluate how locally generated or repository-based assay libraries affect SWATH performance for proteomic studies. To undertake this analysis, we developed a software workflow, SwathXtend, which generates extended peptide assay libraries by integration with a local seed library and delivers statistical analysis of SWATH-quantitative comparisons. We designed test samples using peptides from a yeast extract spiked into peptides from human K562 cell lysates at three different ratios to simulate protein abundance change comparisons. SWATH-MS performance was assessed using local and external assay libraries of varying complexities and proteome compositions. These experiments demonstrated that local seed libraries integrated with external assay libraries achieve better performance than local assay libraries alone, in terms of the number of identified peptides and proteins and the specificity to detect differentially abundant proteins. Our findings show that the performance of extended assay libraries is influenced by the MS/MS feature similarity of the seed and external libraries, while statistical analysis using multiple testing corrections increases the statistical rigor needed when searching against large extended assay libraries. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of fatty acid amide hydrolase activity by a fluorescence-based assay.

PubMed

Dato, Florian M; Maaßen, Andreas; Goldfuß, Bernd; Pietsch, Markus

2018-04-01

Fatty acid amide hydrolase (FAAH) is involved in many human diseases, particularly cancer, pain and inflammation as well as neurological, metabolic and cardiovascular disorders. Therefore, FAAH is an attractive target for the development of low-molecular-weight inhibitors as therapeutics, which requires robust assays that can be used for high-throughput screening (HTS) of compound libraries. Here, we report the development of a fluorometric assay based on FAAH's ability to effectively hydrolyze medium-chain fatty acid amides, introducing N-decanoyl-substituted 5-amino-2-methoxypyridine (D-MAP) as new amide substrate. D-MAP is cleaved by FAAH with an 8-fold larger specificity constant than the previously reported octanoyl-analog Oc-MAP (V max /K m of 1.09 and 0.134 mL min -1 mg -1 , respectively), with both MAP derivatives possessing superior substrate properties and much increased aqueous solubility compared to the respective p-nitroaniline compounds D-pNA and Oc-pNA. The new assay with D-MAP as substrate is highly sensitive using a lower enzyme concentration (1 μg mL -1 ) than literature-reported fluorimetric FAAH assays. In addition, D-MAP was validated in comparison to the substrate Oc-MAP for the characterization of FAAH inhibitors by means of the reference compounds URB597 and TC-F2 and was shown to be highly suitable for HTS in both kinetic and endpoint assays (Z' factors of 0.81 and 0.78, respectively). Copyright © 2018 Elsevier Inc. All rights reserved.

An Integrated Approach to Rapid Diagnosis of Tuberculosis and Multidrug Resistance Using Liquid Culture and Molecular Methods in Russia

PubMed Central

Balabanova, Yanina; Drobniewski, Francis; Nikolayevskyy, Vladyslav; Kruuner, Annika; Malomanova, Nadezhda; Simak, Tatyana; Ilyina, Nailya; Zakharova, Svetlana; Lebedeva, Natalya; Alexander, Heather L.; O'Brien, Rick; Sohn, Hojoon; Shakhmistova, Anastasia; Fedorin, Ivan

2009-01-01

Objective To analyse the feasibility, cost and performance of rapid tuberculosis (TB) molecular and culture systems, in a high multidrug-resistant TB (MDR TB) middle-income region (Samara, Russia) and provide evidence for WHO policy change. Methods Performance and cost evaluation was conducted to compare the BACTEC™ MGIT™ 960 system for culture and drug susceptibility testing (DST) and molecular systems for TB diagnosis, resistance to isoniazid and rifampin, and MDR TB identification compared to conventional Lowenstein-Jensen culture assays. Findings 698 consecutive patients (2487 sputum samples) with risk factors for drug-resistant tuberculosis were recruited. Overall M. tuberculosis complex culture positivity rates were 31.6% (787/2487) in MGIT and 27.1% (675/2487) in LJ (90.5% and 83.2% for smear-positive specimens). In total, 809 cultures of M. tuberculosis complex were isolated by any method. Median time to detection was 14 days for MGIT and 36 days for LJ (10 and 33 days for smear positive specimens) and indirect DST in MGIT took 9 days compared to 21 days on LJ. There was good concordance between DST on LJ and MGIT (96.8% for rifampin and 95.6% for isoniazid). Both molecular hybridization assay results correlated well with MGIT DST results, although molecular assays generally yielded higher rates of resistance (by approximately 3% for both isoniazid and rifampin). Conclusion With effective planning and logistics, the MGIT 960 and molecular based methodologies can be successfully introduced into a reference laboratory setting in a middle incidence country. High rates of MDR TB in the Russian Federation make the introduction of such assays particularly useful. PMID:19774085

Specific detection and identification of [Actinobacillus] muris by PCR using primers targeting the 16S-23S rRNA internal transcribed spacer regions.

PubMed

Benga, Laurentiu; Benten, W Peter M; Engelhardt, Eva; Gougoula, Christina; Sager, Martin

2013-08-01

[Actinobacillus] muris represents along with [Pasteurella] pneumotropica the most prevalent Pasteurellaceae species isolated from the laboratory mouse. Despite the biological and economic importance of Pasteurellaceae in relation to experimental animals, no molecular based methods for the identification of [A.] muris are available. The aim of the present investigation was to develop a PCR method allowing detection and identification of [A.] muris. In this assay, a Pasteurellaceae common forward primer based on a conserved region of the 16S rRNA gene was used in conjunction with two different reverse primers specific for [A.] muris, targeting the 16S-23S internal transcribed spacer sequences. The specificity of the assay was tested against 78 reference and clinical isolates of Pasteurellaceae, including 37 strains of [A.] muris. In addition, eight other mice associated bacterial species which could pose a diagnostic problem were included. The assay showed 100% sensitivity and 97.95% specificity. Identification of the clinical isolates was validated by ITS profiling and when necessary by 16S rRNA sequencing. This multiplex PCR represents the first molecular tool able to detect [A.] muris and may become a reliable alternative to the present diagnostic methods. Copyright © 2013 Elsevier B.V. All rights reserved.

Development and Validation of a Real-Time PCR Assay for Rapid Detection of Two-Spotted Spider Mite, Tetranychus urticae (Acari: Tetranychidae)

PubMed Central

Li, Dongmei; Fan, Qing-Hai; Waite, David W.; Gunawardana, Disna; George, Sherly; Kumarasinghe, Lalith

2015-01-01

Spider mites of the genus Tetranychus are difficult to identify due to their limited diagnostic characters. Many of them are morphologically similar and males are needed for species-level identification. Tetranychus urticae is a common interception and non-regulated pest at New Zealand’s borders, however, most of the intercepted specimens are females and the identification was left at Tetranychus sp. Consequently, the shipments need to be fumigated. DNA sequencing and PCR-restriction fragment length polymorphism (PCR-RFLP) protocols could be used to facilitate the accurate identification. However, in the context of border security practiced in New Zealand, insect identifications are required to be provided within four hours of receiving the samples; thus, those molecular methods are not sufficient to meet this requirement. Therefore, a real-time PCR TaqMan assay was developed for identification of T. urticae by amplification of a 142 bp Internal Transcribed Spacer (ITS) 1 sequence. The developed assay is rapid, detects all life stages of T. urticae within three hours, and does not react with closely related species. Plasmid DNA containing ITS1 sequence of T. uritcae was serially diluted and used as standards in the real-time PCR assay. The quantification cycle (Cq) value of the assay depicted a strong linear relationship with T. urticae DNA content, with a regression coefficient of 0.99 and efficiency of 98%. The detection limit was estimated to be ten copies of the T. urticae target region. The assay was validated against a range of T. urticae specimens from various countries and hosts in a blind panel test. Therefore the application of the assay at New Zealand will reduce the unnecessary fumigation and be beneficial to both the importers and exporters. It is expected that the implementation of this real-time PCR assay would have wide applications in diagnostic and research agencies worldwide. PMID:26147599

Editor's Highlight: Development of an In vitro Assay Measuring Uterine-Specific Estrogenic Responses for Use in Chemical Safety Assessment.

PubMed

Miller, Michelle M; Alyea, Rebecca A; LeSommer, Caroline; Doheny, Daniel L; Rowley, Sean M; Childs, Kristin M; Balbuena, Pergentino; Ross, Susan M; Dong, Jian; Sun, Bin; Andersen, Melvin A; Clewell, Rebecca A

2016-11-01

A toxicity pathway approach was taken to develop an in vitro assay using human uterine epithelial adenocarcinoma (Ishikawa) cells as a replacement for measuring an in vivo uterotrophic response to estrogens. The Ishikawa cell was determined to be fit for the purpose of recapitulating in vivo uterine response by verifying fidelity of the biological pathway components and the dose-response predictions to women of child-bearing age. Expression of the suite of estrogen receptors that control uterine proliferation (ERα66, ERα46, ERα36, ERβ, G-protein coupled estrogen receptor (GPER)) were confirmed across passages and treatment conditions. Phenotypic responses to ethinyl estradiol (EE) from transcriptional activation of ER-mediated genes, to ALP enzyme induction and cellular proliferation occurred at concentrations consistent with estrogenic activity in adult women (low picomolar). To confirm utility of this model to predict concentration-response for uterine proliferation with xenobiotics, we tested the concentration-response for compounds with known uterine estrogenic activity in humans and compared the results to assays from the ToxCast and Tox21 suite of estrogen assays. The Ishikawa proliferation assay was consistent with in vivo responses and was a more sensitive measure of uterine response. Because this assay was constructed by first mapping the key molecular events for cellular response, and then ensuring that the assay incorporated these events, the resulting cellular assay should be a reliable tool for identifying estrogenic compounds and may provide improved quantitation of chemical concentration response for in vitro-based safety assessments. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology.

Development of an updated PCR assay for detection of African swine fever virus.

PubMed

Luo, Yuzi; Atim, Stella A; Shao, Lina; Ayebazibwe, Chrisostom; Sun, Yuan; Liu, Yan; Ji, Shengwei; Meng, Xing-Yu; Li, Su; Li, Yongfeng; Masembe, Charles; Ståhl, Karl; Widén, Frederik; Liu, Lihong; Qiu, Hua-Ji

2017-01-01

Due to the current unavailability of vaccines or treatments for African swine fever (ASF), which is caused by African swine fever virus (ASFV), rapid and reliable detection of the virus is essential for timely implementation of emergency control measures and differentiation of ASF from other swine diseases with similar clinical presentations. Here, an improved PCR assay was developed and evaluated for sensitive and universal detection of ASFV. Primers specific for ASFV were designed based on the highly conserved region of the vp72 gene sequences of all ASFV strains available in GenBank, and the PCR assay was established and compared with two OIE-validated PCR tests. The analytic detection limit of the PCR assay was 60 DNA copies per reaction. No amplification signal was observed for several other porcine viruses. The novel PCR assay was more sensitive than two OIE-validated PCR assays when testing 14 strains of ASFV representing four genotypes (I, V, VIII and IX) from diverse geographical areas. A total of 62 clinical swine blood samples collected from Uganda were examined by the novel PCR, giving a high agreement (59/62) with a superior sensitive universal probe library-based real-time PCR. Eight out of 62 samples tested positive, and three samples with higher Ct values (39.15, 38.39 and 37.41) in the real-time PCR were negative for ASFV in the novel PCR. In contrast, one (with a Ct value of 29.75 by the real-time PCR) and two (with Ct values of 29.75 and 33.12) ASFV-positive samples were not identified by the two OIE-validated PCR assays, respectively. Taken together, these data show that the novel PCR assay is specific, sensitive, and applicable for molecular diagnosis and surveillance of ASF.

A new LAMP-based assay for the molecular diagnosis of toxoplasmosis: comparison with a proficient PCR assay.

PubMed

Varlet-Marie, Emmanuelle; Sterkers, Yvon; Perrotte, Marina; Bastien, Patrick

2018-05-01

Toxoplasmosis is generally a benign infection caused by the protozoan parasite Toxoplasma gondii but can have severe consequences in fetuses of mothers infected during pregnancy (congenital toxoplasmosis) and immunocompromised individuals. PCR-based diagnostic tests have become crucial for its diagnosis. However, this molecular diagnosis essentially relies upon laboratory-developed methods and suffers from a lack of standardization, leading to great variation in methods and performance among laboratories. With the need for accreditation of clinical microbiological laboratories, the use of commercial PCR kits has become an attractive alternative; but thorough evaluation of newly commercialized kits by proficient groups is necessary before any recommendation can be made to parasitology laboratories by health authorities or learned societies. Here, we compared the performance of an original commercial method, the Iam TOXO Q-LAMP (DiaSorin®), using Loop-mediated isothermal amplification (LAMP) technology, with our reference laboratory-developed method using real-time PCR. The kit was first tested using amniotic fluid (AF) and plasma samples (either negative or spiked with live T. gondii tachyzoites at different concentrations (from 7 to 10 5  tachyzoites/mL)). It was then assessed using a cohort of 11 AF, five placental and 32 blood clinical samples preserved at -20 °C. For the processing of placental/blood samples, a pretreatment step was used, which did not strictly follow the manufacturer's recommendations. The practical ease of use and compliance with good laboratory practices were also evaluated. Although the LAMP assay was less sensitive than the laboratory-developed method at very low parasite concentrations (0.1 T. gondii genome equivalents/mL), the two methods yielded identical results qualitatively and, in some instances, quantitatively, particularly for AF samples. Copyright © 2018. Published by Elsevier Ltd.

The hypothalamic-pituitary-thyroid (HPT) axis in fish and its role in fish development and reproduction.

PubMed

Blanton, Michael L; Specker, Jennifer L

2007-01-01

Bony fishes represent the largest vertebrate class and are a very diverse animal group. This chapter provides a thorough review of the available scientific literature on the thyroid system in these important vertebrate animals. The molecular components of the hypothalamic-pituitary-thyroid (HPT) axis in this group correspond closely to those of mammals. The thyroid tissue in the fishes is organized as diffuse follicles, with a few exceptions, rather than as an encapsulated gland as is found in most other vertebrate species. The features of this diffuse tissue in fishes are reviewed with an emphasis on feedback relationships within the HPT axis, the molecular biology of the thyroid system in fishes, and comparisons versus the thyroid systems of other vertebrate taxa. A review of the role of thyroid hormone in fish development and reproduction is included. Available information about the HPT axis in fishes is quite detailed for some species and rather limited or absent in others. This review focuses on species that have been intensively studied for their value as laboratory models in assays to investigate disruption in normal function of the thyroid system. In addition, in vitro and in vivo assay methods for screening chemicals for their potential to interfere with the thyroid system are reviewed. It is concluded that there are currently no in vitro or in vivo assays in fish species that are sufficiently developed to warrant recommendation for use to efficiently screen chemicals for thyroid disruption. Methods are available that can be used to measure thyroid hormones, although our ability to interpret the causes and implications of potential alterations in T4 or T3 levels in fishes is nonetheless limited without further research.