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At least 685 records · Page 38Linked to original sources
PubMed2001

Detection and quantification of infectious hypodermal and hematopoietic necrosis virus in penaeid shrimp by real-time PCR.

A real-time PCR method using a fluorogenic 5' nuclease assay and a PE Applied Biosystems GeneAmp 5700 sequence detector was developed to detect infectious hypodermal and hematopoietic necrosis virus (IHHNV) in penaeid shrimp. A pair of PCR primers to amplify an 81 bp DNA fragment and a fluorogenic probe (TaqMan probe) were selected from ORF1 (open reading frame 1) of the IHHNV genome. The primers and TaqMan probe used in this assay were shown to be specific for IHHNV and did not react with either hepatopancreatic parvovirus (HPV), white-spot syndrome virus (WSSV), or shrimp DNA. A plasmid, pIHHNV-P4, containing the target IHHNV sequence was constructed and used as a positive control. The concentration of pIHHNV-P4 was determined through spectrophotometric analysis and the plasmid was used for quantitative studies. This real-time PCR assay had a detection limit of 10 copies and a log-linear range up to 5 x 10(7) copies of IHHNV DNA. The assay was then used to quantify IHHNV in infected shrimp collected from 5 locations: Hawaii, Panama, Mexico, Guam, and the Philippines. The quantitative analysis showed that wild-caught, large juvenile Penaeus stylirostris collected from the Gulf of California (Mexico) in 1996 were naturally infected with IHHNV and contained up to 10(9) copies of IHHNV microg(-1) of DNA. Similar quantities of IHHNV were detected in hatchery-raised, small juvenile P. stylirostris collected from Guam in 1995 and in farm-raised, post-larval P. monodon from the Philippines in 1996. Laboratory-infected P. stylirostris contained approximately 10(8) copies of IHHNV 31 d after being fed with IHHNV-infected shrimp tissue. In contrast, individuals of Super Shrimp, a line of P. stylirostris selected for IHHNV resistance, showed no signs of infection 32 d after ingesting IHHNV-infected shrimp tissue. Laboratory-infected P. vannamei also contained approximately 10(8) copies of IHHNV 30 d after being fed infected shrimp tissue. A time-course study of IHHNV replication in juvenile P. vannamei showed that the doubling time in the exponential growth phase was approximately 22 h.

Animals↗
PubMed1998

Phylogenetic information and experimental design in molecular systematics.

Despite the widespread perception that evolutionary inference from molecular sequences is a statistical problem, there has been very little attention paid to questions of experimental design. Previous consideration of this topic has led to little more than an empirical folklore regarding the choice of suitable genes for analysis, and to dispute over the best choice of taxa for inclusion in data sets. I introduce what I believe are new methods that permit the quantification of phylogenetic information in a sequence alignment. The methods use likelihood calculations based on Markov-process models of nucleotide substitution allied with phylogenetic trees, and allow a general approach to optimal experimental design. Two examples are given, illustrating realistic problems in experimental design in molecular phylogenetics and suggesting more general conclusions about the choice of genomic regions, sequence lengths and taxa for evolutionary studies.

Animals↗
PubMed2005

Regulation of type 1 fimbriae synthesis and biofilm formation by the transcriptional regulator LrhA of Escherichia coli.

Type 1 fimbriae of Escherichia coli facilitate attachment to the host mucosa and promote biofilm formation on abiotic surfaces. The transcriptional regulator LrhA, which is known as a repressor of flagellar, motility and chemotaxis genes, regulates biofilm formation and expression of type 1 fimbriae. Whole-genome expression profiling revealed that inactivation of lrhA results in an increased expression of structural components of type 1 fimbriae. In vitro, LrhA bound to the promoter regions of the two fim recombinases (FimB and FimE) that catalyse the inversion of the fimA promoter, and to the invertible element itself. Translational lacZ fusions with these genes and quantification of fimE transcript levels by real-time PCR showed that LrhA influences type 1 fimbrial phase variation, primarily via activation of FimE, which is required for the ON-to-OFF transition of the fim switch. Enhanced type 1 fimbrial expression as a result of lrhA disruption was confirmed by mannose-sensitive agglutination of yeast cells. Biofilm formation was stimulated by lrhA inactivation and completely suppressed upon LrhA overproduction. The effects of LrhA on biofilm formation were exerted via the changed levels of surface molecules, most probably both flagella and type 1 fimbriae. Together, the data show a role for LrhA as a repressor of type 1 fimbrial expression, and thus as a regulator of the initial stages of biofilm development and, presumably, bacterial adherence to epithelial host cells also.

Animals↗
PubMed2025

AmpSeqR: an R package for amplicon deep sequencing data analysis.

Amplicon sequencing (AmpSeq) is a methodology that targets specific genomic regions of interest for polymerase chain reaction (PCR) amplification so that they can be sequenced to a high depth of coverage. Amplicons are typically chosen to be highly polymorphic, usually with several highly informative, high frequency single nucleotide polymorphisms (SNPs) segregating in an amplicon of 100-200 base pair (bp). This allows high sensitivity detection and quantification of the frequency of each sequence within each sample making it suitable for applications such as low frequency somatic mosaicism detection or minor clone detection in mixed samples. AmpSeq is being increasingly applied to both biological and medical studies, in applications such as cancer, infectious diseases and brain mosaicism studies. Current bioinformatics pipelines for AmpSeq data processing lack downstream analysis, have difficulty distinguishing between true sequences and PCR sequencing errors and artifacts, and often require bioinformatic expertise. We present a new R package: AmpSeqR, designed for the processing of deep short-read amplicon sequencing data, with a focus on infectious diseases. The pipeline integrates several existing R packages combining them with newly developed functions to perform optimal filtering of reads to remove noise and improve the accuracy of the detected sequences data, permitting detection of very low frequency clones in mixed samples. The package provides useful functions including data pre-processing, amplicon sequence variants (ASVs) estimation, data post-processing, data visualization, and automatically generates a comprehensive Rmarkdown report that contains all essential results facilitating easy inclusion into reports and publications. AmpSeqR is publicly available at https://github.com/bahlolab/AmpSeqR.

High-Throughput Nucleotide Sequencing↗
PubMed2006

Performance characteristics of two real-time PCR assays for the quantification of Epstein-Barr virus DNA.

We compared the performance of a laboratory-developed 5'-nuclease real-time polymerase chain reaction assay and a commercial assay (LightCycler, Roche Diagnostics, Indianapolis, IN) for quantification of Epstein-Barr virus (EBV) DNA. Using standards provided by the manufacturer, the LightCycler assay was linear from 100 to 1 million copies per reaction. Based on dilution of a plasmid containing the amplicon, the laboratory-developed assay was linear from 22 to 45 million copies per reaction. Both assays detected 0.5 copies of genomic EBV DNA per reaction; both showed good reproducibility with coefficients of variation from 0.3% to 2.4% for the LightCycler and 1.8% to 5.1% for the laboratory-developed assay. For 31 whole blood specimens with measurable values in both assays, the viral load values obtained with the LightCycler averaged 2.3-fold higher than those obtained with the laboratory-developed assay. Testing 30 matched whole blood and plasma samples in the laboratory-developed assay showed whole blood viral load values averaged 10-fold higher than those for plasma. The LightCycler and laboratory-developed assays are sensitive and reproducible with broad linear ranges. Further clinical evaluation is needed to determine the viral load cutoff that is predictive of posttransplantation lymphoproliferative disorders.

DNA, Viral↗
PubMed2006

The complex of ethidium bromide with genomic DNA: structure analysis by polarized Raman spectroscopy.

Structural properties of the complex formed between genomic DNA and the intercalating drug ethidium bromide (EtBr) have been determined by use of a Raman microscope equipped with near-infrared laser excitation. The polarized spectra, which were obtained from oriented fibers of the EtBr:DNA complex, are interpreted in terms of the relative orientations of the phenanthridinium ring of EtBr and bases of DNA. Quantification of structure parameters of EtBr and DNA in the complex were assessed using Raman tensors obtained from polarized Raman analyses of oriented specimens of EtBr (single crystal) and DNA (hydrated fiber). We find that the phenanthridinium plane is tilted by 35+/-5 degrees from the plane perpendicular to the fiber (DNA helix) axis. Assuming coplanarity of the phenanthridinium ring and its immediate base neighbors at the intercalation site, such bases would have a tilt angle closer to that of A-DNA (20 degrees) than to that of B-DNA (6 degrees). The average base tilt in stretches of DNA between intercalation sites remains that of B-DNA.

Binding Sites↗
PubMed1996

Quantification of human herpesvirus 6 in immunocompetent persons and post-mortem tissues from AIDS patients by PCR.

A quantitative competitive PCR assay for human herpesvirus 6 (HHV-6) was developed. Firstly, viral burden was determined in the blood of 25 healthy persons. Using 1 microgram of DNA, the prevalence of HHV-6 was 36% (9/25). Eight persons had viral loads of < or = 32 HHV-6 genomes/microgram DNA. The viral burden in the ninth individual was 1.2 x 10(6) HHV-6 genome copies/microgram DNA, which remained constant over a period of 10 months. This demonstrates the persistence of a high HHV-6 load in the absence of apparent disease. Secondly, HHV-6 burden was determined in 100 post-mortem tissues from seven AIDS patients and three controls. For all tissues combined, there was a statistically significant higher median viral load in AIDS patients (56 copies/microgram DNA, range 0-43321) compared to controls (10 copies/microgram DNA, range 0-423) (P = 0.04). The precision and reproducibility of this assay will allow hypotheses concerning the pathogenic potential of HHV-6 to be tested quantitatively.

AIDS-Related Opportunistic Infections↗
PubMed2005

Role of mRNA stability during genome-wide adaptation of Lactococcus lactis to carbon starvation.

The stability of mRNA was investigated for the first time at the genomic scale during carbon starvation adaptation of Lactococcus lactis IL1403. In exponential phase, mRNA half-lives were correlated positively to open reading frame length. A polypurine sequence, AGGAG, was identified as a putative 5'-stabilizer and inverted repeated sequences as a 3'-destabilizer. These original findings suggested that multiple pathways of mRNA degradation should coexist: internal cleavage, endonuclease cleavage initiated at the 5'-end, and exonuclease attack at the 3'-end. During carbon starvation adaptation, mRNA stability globally increased, but specific mechanisms allowing a wide range of stabilization factors between genes and differential kinetic evolution were involved. A formal method allowing the quantification of the relative influences of transcription and degradation on the mRNA pool control was developed and applied in L. lactis. Gene expression was mostly controlled by altered transcription prior to carbon source exhaustion, while the influence of mRNA stability increased during the starvation phase. This study highlighted that stability modulation in response to adverse growth conditions can govern gene regulation to the same extent as transcription in bacteria.

Carbon↗
PubMed2026

An end-to-end computational framework for "Record-seq" transcriptional recording data.

MOTIVATION: Record-seq captures cumulative transcriptional activity over time in engineered Escherichia coli by integrating cellular RNA-derived spacer sequences into clustered regularly interspaced short palindromic repeats (CRISPR) arrays, which are read out by sequencing. Unlike the approximately uniform transcript sampling of RNA-seq, Record-seq records biological signal as spacers sampled by the CRISPR spacer acquisition machinery. Consequently, standard RNA-seq analysis strategies are not directly applicable, limiting sensitivity and interpretability. Our previous pipeline addressed these challenges only partially, retained inherited RNA-seq assumptions, and had limited algorithmic efficiency. RESULTS: Here, we present an end-to-end computational framework for Record-seq data. To address the primary computational bottleneck of spacer sequence extraction, we implemented a wavefront alignment approach for efficient quasi-local pattern matching, achieving an approximately 30-fold speedup. We introduce transcription unit-based feature counting as an alternative to gene-body quantification to better represent prokaryotic transcription and increase statistical power by capturing signal from untranslated regions, which are spacer acquisition hotspots. For downstream analyses, we incorporate multiple normalization strategies and a nonparametric differential expression testing framework designed for sparse datasets. Further, we analyze spacer acquisition patterns and train sequence-based neural models that predict acquisition propensity from genomic sequence and annotations, providing a framework for assessing whether acquisition rules generalize as Record-seq is extended to new microbial hosts. AVAILABILITY AND IMPLEMENTATION: The primary analysis workflow, the recoRdseq package, acquisition modeling repository, and relevant data are all linked at https://github.com/plattlab/Record-seq-Framework. Acquisition models and training data are on Zenodo at https://doi.org/10.5281/zenodo.18891434.

Escherichia coli↗
PubMed2001

Competitive polymerase chain reaction as a method to detect the amplification of bcr-abl gene of chronic myeloid leukemia.

BACKGROUND AND OBJECTIVES: The chimeric product of the bcr-abl rearranged gene is critical in the pathogenesis of chronic myeloid leukemia (CML), yet its role in the progression of the disease remains unclear. There is some evidence that increased bcr-abl expression levels, possibly due to gene amplification, precede the clonal evolution of CML hematopoietic progenitors toward a fully transformed phenotype and might be involved in their resistance to interferon-alpha or tyrosine kinase inhibitors. DESIGN AND METHODS: To quantify the bcr-abl gene both at the genomic and at the transcriptional levels we developed a competitive polymerase chain reaction (PCR) strategy. The competitive PCR technique is based upon the co-amplification of the sample template (target) together with increasing amounts of a DNA fragment (competitor) sharing with the target the primer recognition sites, but differing in size. We constructed a competitor for the quantification of both b2a2 and b3a2 alternative splicing forms of the bcr-abl chimera and established the accuracy and reproducibility of our competitive strategy in a clone of the murine 32DG hematopoietic cell line (32D LG7), which bears a stable integration of a single copy of p210 bcr-abl fusion gene. We utilized this technique to follow, over a period of 200 days, the fusion gene copy numbers and transcription rates in several p210 bcr-abl-transduced 32D cell clones, an experimental condition mimicking the evolution of CML myeloid progenitors in vivo. RESULTS: Our results are consistent with p210 bcr-abl overexpression but not gene amplification associated with their clonal evolution. Increased p210 bcr-abl transcription rate is associated with the abrogation of radiation-induced apoptotic cell death, suggesting a role for the chimeric gene expression level in cell life expectancy after a genotoxic insult. INTERPRETATION AND CONCLUSIONS: We conclude that the assessment of gene amplification and expression might serve to improve prognostic classification and follow-up of CML patients.

Alternative Splicing↗
PubMed1992

Detection and quantification of latently infected B lymphocytes in Epstein-Barr virus-seropositive, healthy individuals by polymerase chain reaction.

We designed a highly sensitive and specific polymerase chain reaction assay for the detection of Epstein-Barr virus (EBV)-related sequences in B-cell DNA of EBV-seropositive healthy individuals. By using this assay, we were able to amplify at least 10 copies of a plasmid containing the BamHI-W region, which is repeated up to 11 times within the EBV genome, in the presence of 1 microgram of EBV-negative DNA, indicating that one virus genome was detectable from 150,000 cells. In 15 of 16 tested individuals, EBV-related sequences were found frequently when the DNA from 10(6) B lymphocytes was examined and 1 microgram of DNA was used in each polymerase chain reaction. When the results of amplifying the diluted plasmid were used as a semiquantitative standard, the number of EBV genomes detected could be estimated to range between 50 and less than 1 from 10(6) B lymphocytes. The results of our study will provide the basis for further investigations of the characteristics of the latent carrier state in healthy EBV-seropositive individuals, such as the determination of the number of virus copies per cell and expression of antigens.

Antibodies, Viral↗
PubMed1999

Hypermutation targets a green fluorescent protein-encoding transgene in the presence of immunoglobulin enhancers.

Hypermutation introduces point mutations into the gene segments encoding immunoglobulin variable regions at a rate that is a million-fold higher than the spontaneous mutation rate in most of the genome. Because Ig enhancers are required to target hypermutation, transcription appears to play a critical role for the hypermutation mechanism. We have developed a novel system for detecting mutations that enables us to determine the influence of expression levels on the mutability of a transgene. This system utilizes a green fluorescent protein receptor gene and the powerful enumeration and quantification properties of flow cytometry. We have tested this system with several constructs bearing Ig enhancers in cell lines with active and inactive hypermutation systems.

Cell Line↗
PubMed1992

Detection of Plasmodium falciparum DNA in a microtitration plate-based hybridization test.

A rapid DNA-test, depending on the affinity based hybrid collection principle, was developed for the detection of Plasmodium falciparum DNA from clinical specimens. In this method, hybridization takes place in solution and the hybrids are collected onto a solid phase for measurement. Two probes are used, one labelled with an affinity tag (biotin) and the other with a detectable label (32P). In the present test a single oligonucleotide complementary to a 21-base pair sequence which is highly repeated in the parasite genome served both as capture and detector probe. The test is a 2-h hybridization performed in streptavidin coated microtitration plate wells, onto which the labelled hybrids simultaneously bind. The sensitivity of the assay with a crude erythrocyte lysate specimen was 1.6 x 10(9) repeat units corresponding to about 160 parasites in one microliter blood. The results allowed quantification of the repeat sequences and thus estimation of the degree of parasitemia in clinical specimens.

Animals↗
PubMed1997

What technique should be used for routine detection and quantification of HBV DNA in clinical samples?

Detection of hepatitis B virus (HBV) DNA in serum allows monitoring of HBV replication and assessing responses to antiviral treatment. HBV DNA quantification measures virus replication and can be used as a prognosis indicator of liver disease and an index of response to antiviral drugs. The aim of this study was to compare the performances of three HBV DNA detection and/or quantification techniques for assessing HBV replication. Three hundred unselected sera with a request for HBV DNA detection and quantification were tested with a molecular hybridisation technique without amplification (Digene Hybrid-Capture, Murex Diagnostics Ltd), a signal amplification assay based on branched DNA technology (Quantiplex HBV DNA, Chiron diagnostics), and an 'in-house' qualitative, non quantitative target amplification assay based on the polymerase chain reaction (PCR) with primers located in the S gene of the HBV genome. Hybrid-capture and branched DNA gave concordant results in 278 cases (93%). In the 128 samples positive by both assays, DNA titres in pg/ml were related significantly (r = 0.70, P < 0.0001). but branched DNA titres increased more rapidly than hybrid-capture titres when the amount of HBV DNA in the sample increased. Twenty-two sera (7%) were negative by hybrid-capture, but positive in branched DNA (detection rate gain: 15%). In these 22 patients, DNA titres were low, HBsAg was present in all instances and alanine aminotransferase activity was elevated in 18 patients (82%); HBeAg was present in seven patients (32%) and anti-HBe antibodies in 18 patients (82%); liver biopsy, undertaken in 18 patients, revealed chronic active hepatitis in all instances, associated with cirrhosis in eight cases. Qualitative, non-quantitative HBV DNA PCR was positive in 75 (50%) of the 150 hybrid-capture-negative, branched DNA-negative samples, including a significant proportion of patients without evidence of ongoing HBV-related liver disease. The results show that in general, the branched DNA assay detects HBV DNA in more patients than hybrid-capture and that this improved detection rate is relevant clinically and genome equivalents/ml are preferred to pg/ml to quantify HBV DNA in clinical specimens and finally qualitative, non-quantitative polymerase chain reaction can detect HBV DNA in patients without evidence of active HBV-related liver disease. This study emphasizes the need for more sensitive, university standardised quantitative HBV DNA assays and for the definition of clinically relevant cutoffs with these assays.

DNA, Viral↗
PubMed1996

Increasing DNA extraction yield from saliva stains with a modified Chelex method.

Recovery, preservation and analysis of body fluid stains is an important aspect of forensic science. PCR-based typing of DNA extracted from recovered stains is often a crucial method to identify a perpetrator or exclude an innocent suspect. This paper reports an improved method of extracting genomic DNA from saliva stains deposited on human skin in simulated bite mark situations. Results of organic (phenol-chloroform) extraction and Chelex extraction were compared to a modified Chelex method developed by the authors. Modifications include pre-extraction preparation with proteinase K and incubations at 56 degrees C and 100 degrees C plus microconcentration of the solution. Quantification results using the classical Chelex extraction method showed that 31.9 +/- 4.22% of the deposited DNA was recovered, but using the modified Chelex extraction method DNA recovery was increased to 47.7 +/- 6.90%. The quantity and quality of extracted DNA was shown to be adequate for PCR-based typing at two STR loci.

Bites and Stings↗
PubMed2004

[Genomic medicine. Polymorphisms and microarray applications].

This update shows new concepts related to the significance of DNA variations among individuals, as well as to their detection by using a new technology. The sequencing of the human genome is only the beginning of what will enable us to understand genetic diversity. The unit of DNA variability is the polymorphism of a single nucleotide (SNP). At present, studies on SNPs are restricted to basic research but the large number of papers on this subject makes feasible their entrance into clinical practice. We illustrate here the use of SNPs as molecular markers in ethnical genotyping, gene expression in some diseases and as potential targets in pharmacological response, and also introduce the technology of arrays. Microarrays experiments allow the quantification and comparison of gene expression on a large scale, at the same time, by using special chips and array designs. Conventional methods provide data from up to 20 genes, while a single microarray may provide information about thousands of them simultaneously, leading to a more rapid and accurate genotyping. Biotechnology improvements will facilitate our knowledge of each gene sequence, the frequency and exact location of SNPs and their influence on cellular behavior. Although experimental efficiency and validity of results from microarrays are still controversial, the knowledge and characterization of a patient's genetic profile will lead, undoubtedly, to advances in prevention, diagnosis, prognosis and treatment of human diseases.

Genetics, Medical↗
PubMed2001

Quantification of tumor cell injury in vitro and in vivo using expression of green fluorescent protein under the control of the GADD153 promoter.

The GADD153 gene is strongly transcriptionally activated by many types of cellular injury and the magnitude of the change in GADD153 expression is proportional to the extent of damage. We developed a novel reporter system in which a chimeric gene containing the GADD153 promoter linked to the coding region of an enhanced green fluorescent protein (EGFP) gene was stably integrated into the genome of a clone of UMSCC10b head and neck carcinoma cells. Activation of the exogenous GADD153 promoter was quantified using flow cytometric measurement of EGFP expression following drug exposure. The exogenous GADD153 promoter in this clone was activated by N-methl-N'-nitro-N-nitrosoguanidine (MNNG) in a concentration-dependent manner with kinetics that closely paralleled perturbation of cell cycle phase distribution. EGFP expression was strongly activated by a variety of genotoxic agents including DNA cross-linking and methylating agents, oxygen free radicals, DNA intercalator, UV and gamma-radiation and hypoxia. When grown as a xenograft in nude mice, the stably transfected clone also demonstrated dose-dependent EGFP expression when measured 4 days after cisplatin treatment. The reporter system accurately categorized the relative potency of adducts produced by 6 related platinum-containing drugs. In conclusion, this reporter system can facilitate in vitro and in vivo screening for agents capable of producing cytotoxicity via a wide variety of different mechanisms, and can be utilized to investigate the relative potency of structurally related DNA adducts.

Animals↗
PubMed2006

A stem-loop-mediated reverse transcription real-time PCR for the selective detection and quantification of the replicative strand of an RNA virus.

A stem-loop-based method to quantify the replicative strand of a model system, dengue virus, with high specificity and sensitivity is described. The high specificity of this approach is achieved at two levels: the use of a reverse transcription primer folded into a stem-loop structure with optimal energetics and the use of specific PCR primers to the loop structure. This approach has exceptional specificity to the replicative RNA as compared with the genomic sequence (>10(5)-fold difference), with a detection sensitivity of 10 copies. The high correlation to the biological "gold standard" plaque assay, used to quantify infectious virus, renders this method a useful quantitative tool that can replace the time-consuming, labor-intensive, and low-throughput plaque-based assays. The method has been extended to the detection of replicative strands of other RNA viruses (West Nile virus and human respiratory syncytial virus) with similar results. This real-time PCR method is reliable, simple to perform, and easily adaptable to different targets. The ability to detect and rapidly quantify replicating viruses is an important step in the elucidation of pathogenesis and is also useful for the evaluation of drugs designed to inhibit viral replication.

Animals↗