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PubMed2006

Selective enrichment and detection of mycobacterial DNA in paucibacillary specimens.

A major challenge for tuberculosis control is mycobacterial detection in paucibacillary disease, particularly in pediatric, extrapulmonary and smear-negative pulmonary infections. We developed a simple and efficient DNA extraction and real-time quantitative PCR (qPCR) protocol for mycobacterial detection and quantification in paucibacillary specimens. The method was refined using an in vitro model mimicking blood specimens which are characterized by the presence of numerous qPCR inhibitors. Mycobacterial DNA detection in blood is of interest given the high sensitivity we previously reported using conventional PCR in blood of patients with tuberculosis lymphadenitis. Mechanical lysis of mycobacteria in the presence of an organic solvent provided the highest sensitivity. Mycobacterial DNA amplification was compromised when the human:bacterial genome ratio was at least 190:1. Separation of the specimen into bacterial- and host-rich fractions prior to DNA extraction improved mycobacterial DNA detection by 30%. Preliminary testing of our protocol in smear-negative, culture-positive specimens (gastric and lymph node aspirates, pleural and cerebrospinal fluid, and blood) confirmed the applicability of our technique to a range of paucibacillary specimens for the detection, quantification and speciation (M. tuberculosis versus M. avium) of mycobacteria, several weeks before culture results were available. Our protocol provides a novel, efficient and simple strategy to improve the performance of qPCR in paucibacillary specimens, including those with excess human DNA background. This tool is useful to study the pathophysiology of early pulmonary or occult tuberculosis, and for more rapid and accurate diagnosis in difficult to diagnose infections.

Benzothiazoles↗
PubMed2006

Detection and quantification of Erysiphe necator DNA in wine grapes and resultant must and juice.

Powdery mildew of grapevines is difficult to assess visually at the weighbridge, particularly in large consignments of machine-harvested fruit. To facilitate accurate methods for the detection and quantification of the disease in grape samples obtained from both the vineyard and winery, we developed a DNA probe for the pathogen Erysiphe necator. The E. necator-specific 450 bp DNA fragment pEnA1, targets highly repetitive sequences and was isolated from a partial genomic library. In screening for species specificity, clone pEnA1 was used in slot-blot hybridization and detected E. necator DNA from grapes and resultant must and juice, but not from clarified juice and wine. The detection threshold was approximately 50 pg of E. necator DNA per 100 ng total DNA of grape sample and was equivalent to 1-5% of a grape bunch visually affected by powdery mildew. Disease severity, expressed as the percentage of surface area of a bunch with powdery mildew, and E. necator DNA content were highly correlated, r2=0.955, P<0.001. The DNA-based hybridization assay has the potential to predict the severity of powdery mildew in grape samples from the vineyard and in must and juice samples at the winery. The DNA sequence of clone pEnA1 was used to design species-specific primers, the results maintaining the same specificity patterns observed in the initial hybridization assays. The PCR-based assay was sensitive enough to detect approximately 1 pg DNA, being equivalent to 1 conidium per sample. This is the first report to date of the detection of all known phenetic groups of E. necator DNA and of the quantification of DNA from grape samples at the winery. Accurate information on the extent of powdery mildew contamination of grape lots would enable wineries to make more informed decisions about the use of fruit and must.

Ascomycota↗
PubMed1999

Accuracy and sensitivity of DNA pooling with microsatellite repeats using capillary electrophoresis.

DNA pooling is a genetic screening method that combines DNA from many individuals in a single polymerase chain reaction (PCR) reaction to generate a representation of allele frequencies. The substantial saving in effort with DNA pooling over individual genotyping facilitates linkage disequilibrium scanning of the human genome using many thousands of genetic markers, and is applicable to mapping of complex diseases such as schizophrenia. However, the literature to date has not addressed several crucial technical aspects of DNA pooling. These include: DNA quantification; the choice of electrophoresis methods; sensitivity (the minimum reliably detectable difference between pools); and methods of dealing with 'plus-A' stutter. We have examined these points and make recommendations as to the best procedures to adopt as well as quantifying reproducibility and sensitivity. We conclude that, although allele frequencies derived from microsatellite pooling are distorted, differences of 5% or greater between pools can be reliably detected.

Alleles↗
PubMed2005

Development of a real-time RT-PCR assay for detection and quantitation of parainfluenza virus 3.

A TaqMan-based real-time RT-PCR assay was developed to detect and quantify human parainfluenza virus 3 (PIV3). Two sets of primer-probe pairs were designed based on the nucleotide (nt) sequence of the nucleocapsid (N) gene. The primer-probe pairs were derived from the 3' end of the N gene (set 1) and the 5' region of the gene (set 2), respectively. Using real-time RT-PCR, the sensitivity of set 1 was determined to be about 9 copies of PIV3 genome, while the sensitivity of set 2 was about 93 copies of PIV3 genome. Set 1 was chosen for subsequent experiments. This primer-probe pair detected PIV3, but not any of several other respiratory viruses, indicating that the assay is PIV3 specific. For clinical evaluation, the assay was employed to test 80 nasopharyngeal aspirates from children with respiratory symptoms. The results confirmed the presence of PIV3 in 12 specimens previously identified as positive by culture confirmation, and showed all of which contained more than 100 copies of PIV3 genome. In addition, the method also detected PIV3 genomes in specimens found negative by culture confirmation, indicating the value of this RT-PCR assay. These data thus demonstrate the application of the real-time RT-PCR assay for the detection and quantification of PIV3 in clinical specimens.

Child↗
PubMed2005

Quantitation of transgenic plant DNA in leachate water: real-time polymerase chain reaction analysis.

Roundup Ready (RR) genetically modified (GM) corn and soybean comprise a large portion of the annual planted acreage of GM crops. Plant growth and subsequent plant decomposition introduce the recombinant DNA (rDNA) into the soil environment, where its fate has not been completely researched. Little is known of the temporal and spatial distribution of plant-derived rDNA in the soil environment and in situ transport of plant DNA by leachate water has not been studied before. The objectives of this study were to determine whether sufficient quantities of plant rDNA were released by roots during growth and early decomposition to be detected in water collected after percolating through a soil profile and to determine the influence of temperature on DNA persistence in the leachate water. Individual plants of RR corn and RR soybean were grown in modified cylinders in a growth room, and the cylinders were flushed with rain water weekly. Immediately after collection, the leachate was subjected to DNA purification followed by rDNA quantification using real-time Polymerase Chain Reaction (PCR) analysis. To test the effects of temperature on plant DNA persistence in leachate water, water samples were spiked with known quantities of RR soybean or RR corn genomic DNA and DNA persistence was examined at 5, 15, and 25 degrees C. Differences in the amounts and temporal distributions of root-derived rDNA were observed between corn and soybean plants. The results suggest that rainfall events may distribute plant DNA throughout the soil and into leachate water. Half-lives of plant DNA in leachate water ranged from 1.2 to 26.7 h, and persistence was greater at colder temperatures (5 and 15 degrees C).

DNA, Plant↗
PubMed2002

Molecular studies of cerebrospinal fluid in human immunodeficiency virus type 1-associated opportunistic central nervous system diseases--an update.

Although the incidence of opportunistic central nervous system (CNS) diseases has markedly declined in developed countries following the advent of highly active antiretroviral therapies (HAARTs), they still represent a major diagnostic and therapeutic challenge over the world. The application of nucleic acid amplification techniques to the study of cerebrospinal fluid (CSF) has contributed substantially to their diagnosis. The detection of specific microbial genomes in the CSF is now the preferred test for some CNS opportunistic diseases, such as progressive multifocal leukoencephalopathy or cytomegalovirus encephalitis. More recent developments of these techniques are the quantitative amplification techniques and postamplification studies. Quantification of nucleic acids in CSF is an important aid both at the time of diagnosis, for the interpretation of positive findings, and during patient follow-up. Postamplification analyses can provide important information with regard to clinical patient management, e.g., detection of genotypic resistance to antimicrobial drugs, and in the attempt to elucidate disease epidemiology and pathogenesis.

AIDS Dementia Complex↗
PubMed2003

Past exposure to densely ionizing radiation leaves a unique permanent signature in the genome.

Speculation has long surrounded the question of whether past exposure to ionizing radiation leaves a unique permanent signature in the genome. Intrachromosomal rearrangements or deletions are produced much more efficiently by densely ionizing radiation than by chemical mutagens, x-rays, or endogenous aging processes. Until recently, such stable intrachromosomal aberrations have been very hard to detect, but a new chromosome band painting technique has made their detection practical. We report the detection and quantification of stable intrachromosomal aberrations in lymphocytes of healthy former nuclear-weapons workers who were exposed to plutonium many years ago. Even many years after occupational exposure, more than half the blood cells of the healthy plutonium workers contain large (>6 Mb) intrachromosomal rearrangements. The yield of these aberrations was highly correlated with plutonium dose to the bone marrow. The control groups contained very few such intrachromosomal aberrations. Quantification of this large-scale chromosomal damage in human populations exposed many years earlier will lead to new insights into the mechanisms and risks of cytogenetic damage.

Alpha Particles↗
PubMed2025

Bayesian estimation of allele-specific expression in the presence of phasing uncertainty.

MOTIVATION: Allele-specific expression (ASE) analyses aim to detect imbalanced expression of maternal versus paternal copies of an autosomal gene. Such allelic imbalance can result from a variety of cis-acting causes, including disruptive mutations within one copy of a gene that impact the stability of transcripts, as well as regulatory variants outside the gene that impact transcription initiation. Current methods for ASE estimation suffer from a number of shortcomings, such as relying on only one variant within a gene, assuming perfect phasing information across multiple variants within a gene, or failing to account for alignment biases and possible genotyping errors. RESULTS: We developed BEASTIE, a Bayesian hierarchical model designed for precise ASE quantification at the gene level, based on given genotypes and RNA-Seq data. BEASTIE addresses the complexities of allelic mapping bias, genotyping error, and phasing errors by incorporating empirical phasing error rates derived from Genome-in-a-Bottle individual NA12878. BEASTIE surpasses existing methods in accuracy, especially in scenarios with high phasing errors. This improvement is critical for identifying rare genetic variants often obscured by such errors. Through rigorous validation on simulated data and application to real data from the 1000 Genomes Project, we establish the robustness of BEASTIE. These findings underscore the value of BEASTIE in revealing patterns of ASE across gene sets and pathways. AVAILABILITY AND IMPLEMENTATION: The software is freely available from Github (https://github.com/x811zou/BEASTIE); and Zendo (DOI: 10.5281/zenodo.15062124).

Bayes Theorem↗
PubMed2026

Environmental antibiotic contamination and AMR: Integrating pathways, impacts, and artificial intelligence-driven mitigation.

The widespread contamination of the environment with antibiotic residues is a significant factor contributing to the global crisis of antimicrobial resistance (AMR). Antibiotics from various sources, such as effluents from municipal and hospital wastewater treatment plants, agricultural runoffs, discharges from pharmaceutical manufacturing and improper disposal of expired or unused medicines, create selective pressures in the spread of antibiotic resistance genes. These environmental reservoirs act as hotspots for horizontal gene transfer, facilitating the emergence of multidrug-resistant pathogens. Conventional detection methods including culture-based assays, chromatographic quantification, and molecular diagnostics, provide essential insights but are limited by low throughput, reduced sensitivity to new Antibiotic Resistance Genes, and challenges in real-time monitoring across complex environments. Recent advances, such as whole-genome sequencing, metagenomics, and biosensor-based detection, help to address these gaps by enabling more comprehensive surveillance of the resistome. Artificial intelligence further enhances these approaches by improving data interpretation and pattern recognition, thus complementing traditional and molecular methods rather than replacing them. This review examines the pathways of environmental antibiotic contamination, ecological and health impacts of AMR, and limitations of conventional detection methods. It aims to clarify how these pathways contribute to the AMR crisis, assess the effectiveness of existing surveillance techniques, and identify gaps in current research.

Anti-Bacterial Agents↗
PubMed2004

Evaluation of five diagnostic methods for the detection and quantification of Myxobolus cerebralis.

Diagnostic methods were used to identify and quantify Myxobolus cerebralis, a myxozoan parasite of salmonid fish. In this study, 7-week-old, pathogen-free rainbow trout (Oncorhynchus mykiss) were experimentally infected with M. cerebralis and at 7 months postinfection were evaluated with 5 diagnostic assays: 1) pepsin-trypsin digest (PTD) to detect and enumerate spores found in cranial cartilage, 2) 2 different histopathology grading scales that provide a numerical score for severity of microscopic lesions in the head, 3) a conventional single-round polymerase chain reaction (PCR), 4) a nested PCR assay, and 5) a newly developed quantitative real-time TaqMan PCR. There were no significant differences (P > 0.05) among the 5 diagnostic assays in distinguishing between experimentally infected and uninfected control fish. The 2 histopathology grading scales were highly correlated (P < 0.001) for assessment of microscopic lesion severity. Quantification of parasite levels in cranial tissues using PTD and real-time TaqMan PCR was significantly correlated r = 0.540 (P < 0.001). Lastly, 104 copies of the 18S rDNA gene are present in the M. cerebralis genome, a feature that makes this gene an excellent target for PCR-based diagnostic assays. Also, 2 copies of the insulin growth factor-I gene are found in the rainbow trout genome, whose detection can serve both as an internal quality control for amplifiable DNA and as a basis to quantify pathogen genome equivalents present in quantitative PCR assays.

Animals↗
PubMed1998

Quantification of proviral DNA load in human T-cell leukaemia virus type I infections.

A nested PCR was designed using primers from the pol and tax genes of human T-cell leukaemia virus type I (HTLV-I). The assay reliably detected a single copy of HTLV-I proviral genome in DNA from 1 x 10(5) Peripheral blood mononuclear cells (PBMCs). Using serial dilutions of sample DNA, the assay was applied prospectively to study proviral load in patients with HTLV-associated disease and carriers. The median proviral load expressed as number of copies/100 PBMCs was found to be 14.0 copies in patients with HAM and 1.55 copies in initially asymptomatic carriers. The assay was used to test for low proviral load in subjects who may have HTLV-I infection, and to monitor response to therapy.

Cell Line↗
PubMed1994

Quantitative trait loci underlying gene product variation: a novel perspective for analyzing regulation of genome expression.

A methodology to dissect the genetic architecture of quantitative variation of numerous gene products simultaneously is proposed. For each individual of a segregating progeny, proteins extracted from a given organ are separated using two-dimensional electrophoresis, and their amounts are estimated with a computer-assisted system for spot quantification. Provided a complete genetic map is available, statistical procedures allow determination of the number, effects and chromosomal locations of factors controlling the amounts of individual proteins. This approach was applied to anonymous proteins of etiolated coleoptiles of maize, in an F2 progeny between two distant lines. The genetic map included both restriction fragment length polymorphism and protein markers. Minimum estimates of one to five unlinked regulatory factors were found for 42 of the 72 proteins analyzed, with a large diversity of effects. Dominance and epistasis interactions were involved in the control of 38% and 14% of the 72 proteins, respectively. Such a methodology might help understanding the architecture of regulatory networks and the possible adaptive or phenotypic significance of the polymorphism of the genes involved.

Chromosome Mapping↗
PubMed2025

Absolute Quantification of Cellular and Cell-Free Mitochondrial DNA Copy Number from Human Blood and Urinary Samples Using Real Time Quantitative PCR.

Mitochondrial DNA copy number (mtDNA-CN) in human body fluids is widely used as a biomarker of mitochondrial dysfunction in common metabolic diseases. Here we describe protocols to measure cellular and/or cell free (cf)-mtDNA-CN in human peripheral blood and urine. Cellular mtDNA is located inside the mitochondria where it encodes key subunits of the respiratory complexes in mitochondria and is usually normalized with reference to the nuclear genome as the mitochondrial genome to nuclear genome ratio (Mt/N) in either whole blood, peripheral blood mononuclear cells (PBMCs), or whole urine. Cf -mtDNA is usually found outside of the mitochondria, often released following mitochondrial damage, can trigger inflammatory pathways, and is usually measured as mtDNA-CN per volume of the starting material. Here we describe how to (1) separate whole blood into PBMCs, plasma, and serum fractions and whole urine into urinary supernatant and pellet, (2) prepare DNA from each of these fractions, (3) prepare reference&#xa0;standards&#xa0;for absolute quantification, (4) carry out qPCR for either relative or absolute quantification from test samples, (5) analyze qPCR data, and (6) calculate the sample size to adequately power studies. The protocol presented here is suitable for high throughput use and can be modified to quantify mtDNA from other body fluids, human cells, and tissues.

Humans↗
PubMed2006

The role of epigenetic alterations in pancreatic cancer.

The past several years have witnessed an explosive increase in our knowledge about epigenetic features in human cancers. It has become apparent that pancreatic cancer is an epigenetic disease, as it is a genetic disease, characterized by widespread and profound alterations in DNA methylation. The introduction of genome-wide screening techniques has accelerated the discovery of a growing list of genes with abnormal methylation patterns in pancreatic cancer, and some of these epigenetic events play a role in the neoplastic process. The detection and quantification of DNA methylation alterations in pancreatic juice is likely a promising tool for the diagnosis of pancreatic cancer. The potential reversibility of epigenetic changes in genes involved in tumor progression makes them attractive therapeutic targets, but the efficacy of epigenetic therapies in pancreatic cancer, such as the use of DNA methylation inhibitors, remains undetermined. In this review, we briefly summarize recent research findings in the field of pancreatic cancer epigenetics and discuss their biological and clinical implications.

Cell Cycle Proteins↗
PubMed2026

Sitosterolemia: evolving strategies for earlier diagnosis.

PURPOSE OF REVIEW: Sitosterolemia is a rare autosomal recessive lipid disorder caused by biallelic pathogenic variants in ABCG5 or ABCG8 , resulting in excessive intestinal absorption and impaired biliary excretion of plant sterols. Although historically considered exceptionally rare, recent genetic studies suggest the disorder is substantially underdiagnosed, with marked phenotypic heterogeneity ranging from xanthomas and premature atherosclerosis to hematologic abnormalities, and frequently mimics familial hypercholesterolemia. This review summarizes recent advances in the clinical, biological, and genetic diagnosis of sitosterolemia, with a focus on strategies that may facilitate earlier detection. RECENT FINDINGS: Phytosterol quantification, particularly sitosterol, campesterol, and stigmasterol, remains indispensable for accurate diagnosis. Hematologic abnormalities, including hemolytic anemia, stomatocytosis, and macrothrombocytopenia, are increasingly recognized as valuable diagnostic clues complementing the biochemical approach. Expanded variant catalogs for ABCG5/ABCG8 and genome-wide association studies have revealed potentially polygenic contributions to phytosterol metabolism extending beyond these two genes. However, no specific guidelines have yet been established for cascade screening. SUMMARY: Earlier diagnosis requires integration of clinical, biochemical, hematologic, and genetic data. Plasma phytosterol measurement remains the diagnostic cornerstone. Improved disease awareness, broader access to sterol testing, and expanded genetic screening may reduce diagnostic delays and enable timely management, including ezetimibe and dietary phytosterol restriction.

Humans↗
PubMed1995

[Hepatitis C virus. Virological diagnosis].

Hepatitis C virus (HCV) has been discovered in 1989 and is probably the most common cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma. HCV is a single-stranded, positive-sense RNA virus, 9.4 kilobases in length. The genetic organisation and the properties of viral proteins have been characterized. At least 50 HCV genotypes or subtypes have been identified. Genotypes 1, 2 and 3 are the most commonly observed in patients from Europe and USA. Genotype 1 is more resistant to interferon treatment. The hypervariability of HCV is responsible, within a single patient, of the existence of a spectrum of very closely-related genomes reffered as quasispecies that may be a mechanism of escape from the immune response and may explain chronicity. Virological diagnosis of HCV infection is based on the detection of anti-HCV antibodies by ELISA. In some cases (acute hepatitis, problems in the interpretation of ELISA tests, or in immunosuppressed patients), it is necessary to search for HCV RNA using genomic amplification or amplification of hybridization. These technics can also be useful to predict the response to interferon, as it has been demonstrated that patients with low viremia are better responders than others. HCV RNA detection or quantification could also be useful to follow the efficiency of anti-viral drugs.

DNA Probes↗
PubMed2002

Quantification of radiation induced DNA double-strand breaks in human fibroblasts by PFGE: testing the applicability of random breakage models.

PURPOSE: To assess the applicability of methods of quantification of double-strand breaks (DSB) based on the random breakage paradigm, measuring yield and distribution of DSB induced by varying radiation quality. MATERIAL AND METHODS: 240 kVp X-rays and (238)Pu alpha-particles were used to induce DSB in AG01522B primary human fibroblasts. DNA molecular weight distributions were resolved by means of three pulsed-field gel-electrophoresis (PFGE) protocols, which, when combined together, allowed separation and quantification of double-stranded fragments between 5.7 Mbp and 12 kbp. Several analytical methods quantified the DSB yields. RESULTS: Data showed significant differences in the fragmentation patterns according to radiation quality. For both X-rays and alpha-particles, it was observed that the shape of the fragmentation profiles deviates from the prediction of a random breakage mechanism. This is in contrast to other studies where sparsely ionizing radiations appeared to distribute breaks uniformly throughout the genome. Deviations from random breakage were more evident after high linear energy transfer (LET) radiation, which showed an excess of breaks <1 Mbp and a deficit in the production of fragments >1 Mbp, a value that could be dose-dependent. CONCLUSIONS: Current methods of DNA fragmentation analysis after induction of DSB may lead to contradictory conclusions on both DSB yields and distributions. This study showed that the application of different DSB quantification methods, derived from random breakage or supported by its concepts, resulted in different radiation biological effectivenesses (RBE) for the induction of DSB, depending on how these methods were employed. To compare experimental results from different laboratories, care should be taken to provide as many details as possible about the application of methods of quantification of DNA damage. For all the methods used, total DSB yields resulted in RBE less than those for mutation induction or reproductive cell death, suggesting that total DSB yields only gave a limited indication of the severity of the inflicted damage. Production of correlated breaks on the chromatin loop structures by single particle-track traversals may explain the deviations observed between experimental data and the predictions of the random breakage paradigm.

Alpha Particles↗
PubMed2003

Genomic imprinting controls matrix attachment regions in the Igf2 gene.

Genomic imprinting at the Igf2/H19 locus originates from allele-specific DNA methylation, which modifies the affinity of some proteins for their target sequences. Here, we show that AT-rich DNA sequences located in the vicinity of previously characterized differentially methylated regions (DMRs) of the imprinted Igf2 gene are conserved between mouse and human. These sequences have all the characteristics of matrix attachment regions (MARs), which are known as versatile regulatory elements involved in chromatin structure and gene expression. Combining allele-specific nuclear matrix binding assays and real-time PCR quantification, we show that retention of two of these Igf2 MARs (MAR0 and MAR2) in the nuclear matrix fraction depends on the tissue and is specific to the paternal allele. Furthermore, on this allele, the Igf2 MAR2 is functionally linked to the neighboring DMR2 while, on the maternal allele, it is controlled by the imprinting-control region. Our work clearly demonstrates that genomic imprinting controls matrix attachment regions in the Igf2 gene.

Alleles↗