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At least 631 records · Page 35Linked to original sources

PubMed2003

Electrocatalytic detection of pathogenic DNA sequences and antibiotic resistance markers.

The detection of specific DNA sequences using electrochemical readout would permit the rapid and inexpensive detection and identification of bacterial pathogens. A new assay developed for this purpose is described that harnesses a sensitive electrocatalytic process to monitor DNA hybridization. Two sequences belonging to the pathogenic microbe Helicobacter pylori are used to demonstrate the versatility and specificity of the assay: one that codes for an unique H. pylori protein and one that represents a small portion of the 23S rRNA from this organism. Both sequences can be monitored into the nanomolar concentration range. Target sequences introduced to the electrode surface as synthetic oligonucleotides, PCR products, and RNA transcripts are all detected with high specificity. In addition to reporting the presence of pathogen-related sequences, this assay can accurately resolve single-base changes in target sequences. An A2143C substitution within the H. pylori rRNA that confers antibiotic resistance significantly attenuates hybridization to an immobilized probe corresponding to the WT sequence. The single-base mismatch introduced by this mutation slows the kinetics of hybridization and permits discrimination of the two sequences when short hybridization times are employed. The remarkable sensitivity of this label-free assay to small sequence changes may provide the basis of a new method for the detection and genotyping of infectious bacteria using electrochemical methods.

Bacterial Proteins↗

PubMed2026

Artificial intelligence in molecular diagnostics for pandemic preparedness.

INTRODUCTION: Molecular diagnostics focusing on the detection and analysis of nucleic acids are indispensable tools for early pathogen identification, transmission monitoring, and genomic surveillance during pandemics. Recent technological advances have broadened the diagnostic landscape, incorporating PCR-based methods, isothermal amplification, high-CRISPR-based amplification detection, and sequencing. Despite their diagnostic potential, widespread implementation remains limited by high validation costs, time and logistical constraints, the need for specialized professional knowledge, and a lack of adaptability in resource-limited settings. Artificial intelligence (AI) is increasingly recognized as a promising but challenging approach, offering tools that streamline assay development, automate data interpretation, and optimize real-time diagnostic performance. AREAS COVERED: This review introduces recently published AI tools with potential to enhance the in-silico design validation process of oligonucleotides for molecular assays. These cover tools for initial assay design and optimization to validation and continuous assay updates. The limitations, including concerns regarding data accuracy, the lack of transparency in data processing ('black box' models), and unresolved licensing and regulatory issues, are highlighted for each tool and as expert opinion. EXPERT OPINION: Collectively, these challenges currently confine most AI-based approaches to research settings and prevent their routine implementation in clinical molecular diagnostics. Their widespread adoption depends on addressing remaining technical, regulatory, and practical challenges.

Humans↗

PubMed2003

Development of generic continuous-flow enzyme immunoassay system for analysis of aminoglycosides in serum.

A simple generic continuous-flow enzyme immunoassay (CFEIA) for analysis of aminoglycosides in serum has been successfully developed. The developed assay employed a specific monoclonal antibody and beta-galactosidase (beta-GAL) enzyme as label. The assay involves an off-line competitive binding reaction between the analyte and free labelled analyte for the binding sites of the antibody. After equilibrium is reached, the sample was injected into the flow system. The bound antibody complexes with the analyte and the labelled analyte were trapped in a protein G column, while the unbound free labelled analyte was eluted and detected colorimetrically down-stream, after reaction with chlorophenolic red-beta-D-galactopyranoside as a substrate for the beta-GAL enzyme. The concentration of the analyte in a sample was quantified by its ability to inhibit the binding of the analyte-enzyme conjugate to the antibody, and the signal was directly proportional to the concentration of the analyte in the original sample. The optimum conditions for the developed CFEIA were investigated and applied to the analysis of tobramycin, as a representative example of the aminoglycosides, in serum samples. The detection limit of the assay was 0.06 microgml(-1). The assay showed good precision; the coefficients of variation were 2.49-4.33 and 3.30-6.82% for intra- and inter-assay precision, respectively. Serum matrix constituents and the endogenous compounds did not interfere with the assay. Analytical recovery of spiked tobramycin, in the concentration range between 0.5 and 8.0 microgml(-1), was 101.55+/-3.14. The assay results correlated well with those obtained by high-performance liquid chromatography (r=0.991). All the obtained results strongly demonstrate that the developed CFEIA is a suitable method for a rapid and reliable analysis of aminoglycosides in serum.

Aminoglycosides↗

PubMed2002

Multiplex assay for detection of strain-specific antibodies against the two variable regions of the G protein of respiratory syncytial virus.

The role of strain differences in respiratory syncytial virus (RSV) disease has not been clearly defined. To investigate the possibility that strain differences contribute to susceptibility to repeat infections, we developed assays to detect antibodies to the two variable regions of the RSV G protein by cloning and expressing the internal variable region at amino acids (aa) 60 to 172 (g1) and the carboxy-terminal variable region at aa 193 to the carboxy terminus (g2) from different genotypes of RSV. The purified proteins were covalently linked to beads with different proportions of red and orange fluorescent dyes and reacted against serum specimens. Antibody reacting against the differently colored beads, and thus against different G polypeptides, was detected by use of flow cytometry and the Luminex system. This assay system detected group- and, to some extent, genotype-specific responses to RSV infection and can be used to investigate the role of strain differences in RSV disease.

Animals↗

PubMed1998

Development of a radiolabeled ATP assay for carboxylic acid:CoA ligases and its use in the characterization of the xenobiotic carboxylic acid:CoA ligases of bovine liver mitochondria.

A radiolabeled ATP assay was developed for measuring carboxylic acid:CoA ligase activity. The assay was designed to measure the formation of [gamma-33P]pyrophosphate from [gamma-33P]ATP in the course of the reaction. The assay was linear with protein concentration, and rates as low as 1 pmol/min were measurable. Rates determined with this assay were in agreement with rates determined with [14C]carboxylic acids. The assay was used to characterize the substrate specificity of the XL-I, XL-II, and XL-III ligases from bovine liver mitochondria. Forty carboxylic acids were tested for activity. The enzymes differed in their substrate specificities with XL-I and XL-II being the most similar and XL-III having the broadest specificity. This study has uncovered 19 new carboxylic acids that are substrates for these enzymes.

Adenosine Triphosphate↗

PubMed1998

A model for ATM heterozygote identification in a large population: four founder-effect ATM mutations identify most of Costa Rican patients with ataxia telangiectasia.

Ataxia telangiectasia (A-T) is an autosomal recessive disorder with a broad range of clinical manifestations and a frequency of 1:40,000-100,000 live births. Epidemiological studies have suggested that A-T heterozygotes are at an elevated risk of breast cancer. ATM mutations occur worldwide over the entire ATM gene, making it difficult to identify heterozygotes in large populations. However, some founder-effect mutations are specific for certain populations. Here, we present four mutations in Costa Rican A-T patients that accounted for 86-93% of 41 patients studied in two batches. We have developed assays for rapid detection of these four mutations which can be used diagnostically. They will also enable the Costa Rican population to be used as a model for analyzing the role of ATM heterozygosity in cancer development and other disorders.

Ataxia Telangiectasia↗

PubMed2000

Development of an immunomagnetic capture reverse transcription-PCR assay for the detection of Norwalk virus.

Norwalk virus (NV) is the prototype human virus of the family Caliciviridae. A rapid immunomagnetic capture/reverse transcription-(IMC/RT-)PCR assay was developed for the detection of NV. Immunomagnetic capture (IMC) utilizes paramagnetic beads coupled to a virus-specific antibody and allows separation of virus from contaminating materials and virus concentration in a single step. The detection limit of the developed assay was approximately 250-750 genomic equivalents/ml of 10% stool suspension. The detection limit of the assay was not altered by the presence of excess hepatitis A virus (HAV), although non-specific binding of HAV to the paramagnetic beads was observed. A panel of 100 stools from experimental human infections was screened for NV using a previously described heat release method, an antigen ELISA, or IMC/RT-PCR. NV was detected in 65/100 of these samples by IMC/RT-PCR compared to only 38/99 by heat release and 31/95 by antigen detection ELISA. All samples that were negative by IMC were also negative by both heat release and antigen ELISA. The number of samples in which RT-PCR was inhibited was greatly reduced by the use of IMC/RT-PCR compared to the heat release method (1/100 and 16/95 samples inhibited, respectively). The ability of IMC to concentrate virus (> or =2000-fold greater than heat release) and effectively remove inhibitory substances gives this assay distinct advantages over both the heat release and antigen ELISAs.

Caliciviridae Infections↗

PubMed1987

Activation of a cryptic 5' splice site in the upstream exon of the phage T4 td transcript: exon context, missplicing, and mRNA deletion in a fidelity mutant.

A collection of 100 td mutants defective in phage T4 thymidylate synthase (TS) production was screened for splicing impairments. Splicing-defective mutants were identified by a rapid assay developed to detect imbalances in the td protein products (TS, the exon ligation product, and NH2TS, encoded by the pre-mRNA). Thirteen selected mutants, confirmed to be splicing defective by an RNA-oligodeoxynucleotide hybridization assay, were all shown to be inhibited in the first step of the group I splicing pathway, cleavage at the 5' splice site. Of these, only one, SC99, appeared to be a specificity mutant. Whereas the 12 other mutants had sequence changes within the functionally important 5' and 3' domains of the intron, SC99 was shown to be an exon mutant. The G----A change at residue -3 of the upstream exon of SC99 resulted in loss of normal 5' splice site recognition. Furthermore, activation of a remote cryptic splice site at residue -29 of the upstream exon and missplicing of mRNA that is deleted for 29 nucleotides of the 5' exon are characteristic for this mutant. These results underscore the role of exon sequences in guiding the fidelity of the splicing reaction and they raise provocative questions about the alignment of introns within exon contexts that are consistent with accurate splicing and synthesis of an intact gene product.

Base Sequence↗

PubMed2001

Heminested multiplex reverse transcription-PCR for detection and differentiation of Norwalk-like virus genogroups 1 and 2 in fecal samples.

The present study describes a heminested multiplex reverse transcription (RT)-PCR assay which enables simultaneous detection and differentiation of Norwalk-like virus (NLV) genogroups from clinical fecal samples without the need to perform sequencing or hybridization. The assay developed was able to detect concentrations of fewer than 100 viral particles per 5 microl of clarified fecal extract and could differentiate the two genogroups with a specificity of 100%. Although the multiplex RT-PCR assay failed to detect NLV in about 3% of the fecal samples which were NLV positive by electron microscopy (EM), the assay was approximately six times more sensitive than EM for NLV detection.

Caliciviridae↗

PubMed1990

Immunoaffinity purification and immunoassay determination of human erythrocyte acylphosphatase.

Specific anti-human erythrocyte acylphosphatase antibodies were raised in rabbits, purified by affinity chromatography, and used to develop an enzyme purification procedure based on an immunoaffinity chromatography step. This procedure permitted the rapid purification of the enzyme, with a high final yield and with a specific activity very similar to that found for the enzyme purified by the standard procedure. The noncompetitive enzyme-linked immunoadsorbent assay developed with the affinity-purified antibodies was very specific and sensitive in that a positive reaction could be detected in the presence of antigen amounts of as little as 0.01 ng/ml. By this assay the enzyme content was determined in normal cells, tissues, and organs as well as in blood samples from hemopathy-affected patients. This test could possibly have clinical applications.

Acid Anhydride Hydrolases↗

PubMed1996

Caprine beta-mannosidase: sequencing and characterization of the cDNA and identification of the molecular defect of caprine beta-mannosidosis.

The complete sequence of the caprine beta-mannosidase cDNA coding region has been determined, and a mutation that is associated with caprine beta-mannosidosis has been identified. Reverse transcriptase-polymerase chain reactions were performed using primers based on bovine and, later, goat cDNA sequences to produce an overlapping series of amplicons covering the entire coding region. The composite cDNA codes for an 879-amino-acid peptide that has four potential N-glycosylation sites. Comparison of the caprine and bovine cDNAs reveals that 96.3% of the nucleotides and 95.2% of the deduced amino acids are identical. A single-base deletion at position 1398 of the coding sequence was identified in the cDNA isolated from a goat affected with beta-mannosidosis. This deletion results in a shift in the reading frame and a premature termination of translation, yielding a deduced peptide of 481 amino acids. An assay, developed to determine the presence or absence of this mutation, confirmed that animals affected with beta-mannosidosis were homozygous for the mutation and that obligate carriers in a caprine beta-mannosidosis colony were heterozygous. This assay accurately distinguished between mutation carrier and noncarrier goats and was used for prenatal diagnosis using DNA collected from fetal fluids. The assay also confirmed chimerism in a goat with an atypically mild beta-mannosidosis phenotype. Thus, this application enables assessment of the efficacy of engraftment of hematopoietic stem cells after prenatal transfer from donor sources.

Amino Acid Sequence↗

PubMed2001

Simian immunodeficiency virus replicates to high levels in naturally infected African green monkeys without inducing immunologic or neurologic disease.

African green monkeys can maintain long-term persistent infection with simian immunodeficiency viruses (SIVagm) without developing AIDS and thus provide an important model for understanding mechanisms of natural host resistance to disease. This study assessed the levels and anatomic distribution of SIVagm in healthy, naturally infected monkeys. Quantitative competitive reverse transcriptase PCR assays developed to measure SIVagm from two African green monkey subspecies demonstrated high levels of SIV RNA in plasma (>6 x 10(6) RNA copies/ml) in sabaeus and vervet monkeys. Infectious virus was readily recovered from plasma and peripheral blood mononuclear cells and shown to be highly cytopathic in human cell lines and macrophages. SIVagm DNA levels were highest in the gastrointestinal tract, suggesting that the gut is a major site for SIVagm replication in vivo. Appreciable levels of virus were also found within the brain parenchyma and the cerebrospinal fluid (CSF), with lower levels detected in peripheral blood cells and lymph nodes. Virus isolates from the CSF and brain parenchyma readily infected macrophages in culture, whereas lymph node isolates were more restricted to growth in human T-cell lines. Comparison of env V2-C4 sequences showed extensive amino acid diversity between SIVagm recovered from the central nervous system and that recovered from lymphoid tissues. Homology between brain and CSF viruses, macrophage tropism, and active replication suggest compartmentalization in the central nervous system without associated neuropathology in naturally infected monkeys. These studies provide evidence that the nonpathogenic nature of SIVagm in the natural host can be attributed neither to more effective host control over viral replication nor to differences in the tissue and cell tropism from those for human immunodeficiency virus type 1-infected humans or SIV-infected macaques.

Amino Acid Sequence↗

PubMed1994

Isolation, characterization and functional activity of human marrow stromal progenitors in hemopoiesis.

In conclusion, monoclonal antibody STRO-1 has proven to be an extremely valuable reagent for the identification, isolation and functional characterization of human bone marrow stromal cell precursors. We were able to isolate CFU-F free of contaminating hemopoietic progenitors and are beginning to construct a detailed picture of their cell surface phenotype and have developed assays to examine the conditions required for their growth and differentiation. In the future, we hope to determine whether multipotential stromal stem cells exist in human bone marrow and to establish culture conditions that selectively promote self-renewal or development along any of a number of stromal cell lineages. Such cells would be an ideal target for gene therapy and may provide a means of treating disorders of the hemopoietic system with a suspected stromal etiology, such as aplastic anemia. We have recently succeeded in generating bone cells from STRO-1+ marrow cells (Gronthos et al, in preparation) and thus marrow stromal cell precursors may also find application in the treatment of other disorders such as those of the osteogenic system. For the present, these must remain tantalizing possibilities.

Adult↗

PubMed1994

Direct colorimetric determination of solid-supported functional groups and ligands using bicinchoninic acid.

We describe new colorimetric methods for the direct determination of total solid-supported sulfydryl, aldehydo, hydrazido, and N-hydroxysuccinimido carboxylate groups as well as of immobilized cysteine, tyrosine, and thyroxin using only the commercially available bicinchoninic acid/copper protein assay reagent. The method is based on the ability of these groups to reduce Cu2+ to Cu+, which forms a chelate complex with bicinchoninic acid absorbing at 562 nm. Each assay requires only one incubation step of the solids with the reagent for 1 h at 60 degrees C. The quantitation of the different groups is finally carried out through standard curves of appropriate substances. Using the assays developed we determined the amount of the above-mentioned functional groups and ligands onto several commercially available solid supports. The values obtained were in agreement with those provided by relative literature methods and/or by the manufacturers. The assays were found to be accurate, precise (interassay CV less than 3%), and very sensitive, allowing the determination of nmol quantities of functional groups per assay tube.

Aldehydes↗

PubMed2005

In vitro estrogenicity of polybrominated flame retardants.

Estrogenicity of five brominated flame retardants (BFRs), namely BDE-47, BDE-99, BDE-205, PBB-153 and technical Firemaster BP-6, were assessed by in vitro assays developed to detect chemicals with estrogenic properties. Recombinant yeast cells containing a human estrogen receptor gene failed to give any response to the chemicals tested. However, the positive control compound, estradiol-17beta, showed that the yeast cell assays had worked properly. The freshly separated fish hepatocyte assay based on the synthesis and secretion of vitellogenin from the isolated liver cells produced a clear dose-response curve in the presence of all tested flame retardants except Firemaster BP-6. The toxicity of the BFRs was detected by determining the cell ethoxyresorufin-O-deethylase activity (EROD). The BFRs tested induced hepatic EROD activity at low test concentrations, but started to inhibit activity at higher concentrations. The decreased detoxification capacity of the hepatocytes resulted in a decrease in the vitellogenin production of the cells. The capability of in vitro assays to detect estrogenic properties of chemicals seems to vary. Thus, further work is needed to understand the mechanisms responsible for these reactions.

Analysis of Variance↗

PubMed2006

Elaboration of silica colloid/polymer hybrid support for oligonucleotide synthesis.

One way to increase the sensitivity of DNA diagnostic assays developed on microarrays is to improve the solid phase that allows the extraction of the target from a biological sample, before detection. Two parameters are influencing the performances of this capture step: (i) the specific surface area being offered for the capture and (ii) the number and the accessibility of oligonucleotide probes immobilized on the surface. In this context, we have developed an attractive approach which fulfills these two points. Our strategy was to elaborate a new material of high specific surface area, suitable to serve as support for both solid-phase oligonucleotide synthesis and in vitro diagnostic assay. This material has consisted of aggregates of colloidal amino-silica nanoparticles covalently linked by poly(ethylene oxide) (PEO) arms. The aggregation of amino-silica particles in the presence of reactive bis-isocyanate PEO was achieved in a controlled manner. The aggregate size and structure were examined by microscopy. The specific surface area of this material was measured by nitrogen adsorption technique. The composition of aggregate was studied by thermogravimetry and X-ray photoelectron spectroscopy. Then, this material has been successfully used as support for oligonucleotide synthesis of high yield and purity. The resulting system will be further evaluated in a diagnostic assay on a microarray.

Adsorption↗

PubMed1997

Evaluation of polymerase chain reaction for detection of Mycoplasma meleagridis infection in turkeys.

Different methods were compared for the detection of the turkey pathogen Mycoplasma meleagridis in a turkey field flock before and after antibiotic (oxytetracycline) treatment. They included culture, serology (detection of antibodies by ELISA) and a polymerase chain reaction (PCR) based assay developed by Boyle et al. [Boyle, J.S., Good, R., Morrow, C.J., 1995. Detection of the turkey pathogens Mycoplasma meleagridis and M. iowae by amplification of genes coding for rRNA. J. Clin. Microbiol. 33, 1335-1338]. Culture and PCR assay with tracheal swab samples were much more sensitive than ELISA with serum samples. Percentages of infected birds detected by culture or PCR for samples collected prior to antibiotic treatment were almost identical but the percentage of positive samples detected after antibiotic treatment was much higher with the PCR test.

Animals↗

PubMed2006

Multiplex real-time reverse transcription-PCR assay for determination of hepatitis C virus genotypes.

A variety of methods have been used to determine hepatitis C virus (HCV) genotypes. Because therapeutic decisions for chronic HCV-related hepatitis are made on the basis of genotype, it is important that genotype be accurately determined by clinical laboratories. Existing methods are often subjective, inaccurate, manual, time-consuming, and contamination prone. We therefore evaluated real-time reverse transcription-PCR (RT-PCR) reagents that have recently become commercially available (Abbott HCV Genotype ASR). The assay developed by our laboratory starts with purified RNA and can be performed in 4 to 5 h. An initial evaluation of 479 samples was done with a restriction fragment length polymorphism (RFLP) method and the RT-PCR assay, and discrepant samples were sequenced. An additional 1,200 samples were then tested, and data from all assays were used to evaluate the efficiency and specificity of each genotype-specific reaction. Good correlation between results by the two methods was seen. Discrepant samples included those indeterminate by the RT-PCR assay (n = 110) and a subset that were incorrectly called 2a by the RFLP method (n = 75). The real-time RT-PCR assay performed well with genotype 1, 2, and 3 samples. Inadequate numbers of samples were available to evaluate fully genotypes 4, 5, and 6. Analysis of each primer-probe set demonstrated that weak cross-reactive amplifications were common but usually did not interfere with the genotype determination. However, in about 1% of samples, two or more genotypes amplified at roughly equivalent amounts. Further studies are necessary to determine whether these mixed-genotype samples are true mixtures or a reflection of occasional cross-reactive amplifications.

Cross Reactions↗