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At least 253 records · Page 14Linked to original sources

PubMed2004

Multiparametric duplex real-time nucleic acid sequence-based amplification assay for mRNA profiling.

Nucleic acid sequence-based amplification (NASBA) is a sensitive isothermal transcription-based amplification method. We have developed real-time NASBA assays to detect mRNA coding for the estrogen receptor alpha (ESR1) and the progesterone receptor (PGR) in breast tumors by means of duplex reactions using cyclophilin B (PPIB) as the normalizing gene. Both the ESR1/PPIB and PGR/PPIB duplex NASBA assays are highly sensitive, specific, and reproducible. Quantification is determined using external standard calibration curves and the ratio between the number of target and housekeeping gene mRNA copies. Amplification of the target gene in the duplex NASBA assay was disrupted when this latter was mixed with a large amount of the housekeeping PPIB gene, suggesting that it is preferable for the normalizing gene chosen to have an expression level comparable to the target gene. Sensitivity and robustness of the duplex NASBA assays were assessed in breast cancer cell lines. Such a rapid and easy-to-use multiparametric duplex real-time NASBA assay could also advantageously be set up for other mRNA profiling applications.

Biological Assay↗

PubMed2004

DNA nucleic acid sequence-based amplification-based genotyping for polymorphism analysis.

Nucleic acid sequence-based amplification (NASBA) is a sensitive isothermal transcription-based amplification method known to be a suitable tool for RNA research. We demonstrate that NASBA technology can be applied to single nucleotide polymorphism (SNP) analysis using human genomic DNA as a template. Combination of DNA NASBA with multiplex hybridization of specific molecular beacons makes it possible to unambiguously discriminate the presence of the SNP of interest. This protocol is easy-to-use, robust, and makes it possible to rapidly detect single nucleotide substitutions in clinical or cell line DNA sequences using a large range of DNA input. Such a real-time genotyping DNA NASBA assay can find broad application in clinical diagnostics.

Base Sequence↗

PubMed2003

Homogeneous real-time detection of single-nucleotide polymorphisms by strand displacement amplification on the BD ProbeTec ET system.

BACKGROUND: The BD ProbeTec ET System is based on isothermal strand displacement amplification (SDA) of target nucleic acid coupled with homogeneous real-time detection using fluorescent probes. We have developed a novel, rapid method using this platform that incorporates a universal detection format for identification of single-nucleotide polymorphisms (SNPs) and other genotypic variations. METHOD: The system uses a common pair of fluorescent Detector Probes in conjunction with unlabeled allele-specific Adapter Primers and a universal buffer chemistry to permit analysis of multiple SNP loci under generic assay conditions. We used Detector Probes labeled with different dyes to facilitate differentiation of two alternative alleles in a single reaction with no postamplification manipulation. We analyzed six SNPs within the human beta(2)-adrenergic receptor (beta(2)AR) gene, using whole blood, buccal swabs, and urine samples, and compared results with those obtained by DNA sequencing. RESULTS: Unprocessed whole blood was successfully genotyped with as little as 0.1-1 micro L of sample per reaction. All six beta(2)AR assays were able to accommodate >/==" BORDER="0">20 micro L of unprocessed whole blood. For the 14 individuals tested, genotypes determined with the six beta(2)AR assays agreed with DNA sequencing results. CONCLUSION: SDA-based allelic differentiation on the BD ProbeTec ET System can detect SNPs rapidly, using whole blood, buccal swabs, or urine.

Alleles↗

PubMed2002

Use of signal-mediated amplification of RNA technology (SMART) to detect marine cyanophage DNA.

Here, we describe the application of an isothermal nucleic acid amplification assay, signal-mediated amplification of RNA technology (SMART), to detect DNA extracted from marine cyanophages known to infect unicellular cyanobacteria from the genus Synechococcus. The SMART assay is based on the target-dependent production of multiple copies of an RNA signal, which is measured by an enzyme-linked oligosorbent assay. SMART was able to detect both synthetic oligonucleotide targets and genomic cyanophage DNA using probes designed against the portal vertex gene (g20). Specific signals were obtained for each cyanophage strain (S-PM2 and S-BnMI). Nonspecific genomic DNA did not produce false signals or inhibit the detection of a specific target. In addition, we found that extensive purification of target DNA may not be required since signals were obtained from crude cyanophage lysates. This is the first report of the SMART assay being used to discriminate between two similar target sequences.

Bacteriophages↗

PubMed2002

A method to detect major serotypes of foot-and-mouth disease virus.

Nucleic acid sequence-based amplification (NASBA) is an isothermal technique that allows the rapid amplification of specific regions of nucleic acid obtained from a diverse range of sources. It is especially suitable for amplifying RNA sequences. A rapid and specific NASBA technique was developed, allowing the detection of foot-and-mouth disease virus genetic material in a range of sample material, including preserved skin biopsy material from infected animals, vaccines prepared from denatured cell-free material, and cell-free antigen-based detection kits. A single pair of DNA oligonucleotide primers was able to amplify examples of all major FMD virus subtypes. The amplified viral RNA was detected by electrochemiluminescence. The method was at least as sensitive as existing cell-free antigen detection methods.

Animals↗

PubMed1995

Development of NASBA, a nucleic acid amplification system, for identification of Listeria monocytogenes and comparison to ELISA and a modified FDA method.

NASBA, an isothermal nucleic acid amplification system was used for identification of Listeria monocytogenes. A primer set and a species-specific probe were selected from the 16S rRNA sequence. The probe was shown to hybridize specifically to the amplified single-stranded RNA of L. monocytogenes. No hybridization occurred with amplification product of L. seeligeri, L. innocua, L. ivanovii, and L. welshimeri. Detection sensitivity for the NASBA assay was determined at 10(6) cfu/ml. The possibility of using the NASBA assay for detection of L. monocytogenes in foods after a 2-day enrichment procedure was explored. NASBA was compared to a modified FDA method and ELISA for detection of L. monocytogenes artificially inoculated (1-100 cfu/25 g) in eight food products. False-negative results were obtained with the modified FDA method (6.75%). NASBA and ELISA were shown in this study to detect the pathogen with equal efficiency (no false negative or false positive results). Both methods allowed detection of less than 10 cfu/25 g within 3 days but ELISA can only be used for diagnosis of Listeria spp. while the NASBA procedure permitted specific identification of the human pathogen L. monocytogenes.

Bacteriological Techniques↗

PubMedDate not supplied

Nucleic acid amplification mediated microbial identification.

Nucleic acid amplification techniques have and are revolutionising the way research, academia, medicine, environmental and quality control issues are dealt with. In particular, the polymerase chain reaction, (PCR), originally mentioned in 1985 has greatly modified the ways in which scientists and clinicians go about nucleic acid sequence analysis and manipulation and has great implications for molecular biology in general and diagnostics in particular. This report attempts to review the use of PCR and isothermal nucleic acid amplification techniques in the rapid identification of microorganisms. Key methodologies have been noted, their strengths and weaknesses commented upon and comparisons have been drawn between isothermal and thermally cycled nucleic amplification techniques in rapid microbial identification.

Bacteria↗

PubMed2003

Rapid and sensitive detection of avian influenza virus subtype H7 using NASBA.

Nucleic acid sequence-based amplification with electrochemiluminescent detection (NASBA/ECL) is an isothermal technique allowing rapid amplification and detection of specific regions of nucleic acid from a diverse range of sources. It is especially suitable for amplifying RNA. A NASBA/ECL technique has been developed allowing the detection of RNA from avian influenza virus subtype H7 derived from allantoic fluid harvested from inoculated chick embryos and from cell cultures. Degenerate amplification primers and amplicon capture probes were designed enabling the detection of low and highly pathogenic avian influenza of the H7 subtype from the Eurasian and North American lineages and the Australian sub-lineage. The NASBA/ECL technique is specific for subtype H7 and does not cross-react with other influenza subtypes or with viruses containing haemagglutinin-like genes. The assay is 10- to 100-fold more sensitive than a commercially available antigen capture immunoassay system. The NASBA/ECL assay could be used in high throughput poultry screening programmes.

Animals↗

PubMed2006

DNA detection using recombination proteins.

DNA amplification is essential to most nucleic acid testing strategies, but established techniques require sophisticated equipment or complex experimental procedures, and their uptake outside specialised laboratories has been limited. Our novel approach, recombinase polymerase amplification (RPA), couples isothermal recombinase-driven primer targeting of template material with strand-displacement DNA synthesis. It achieves exponential amplification with no need for pretreatment of sample DNA. Reactions are sensitive, specific, and rapid and operate at constant low temperature. We have also developed a probe-based detection system. Key aspects of the combined RPA amplification/detection process are illustrated by a test for the pathogen methicillin-resistant Staphylococcus aureus. The technology proves to be sensitive to fewer than ten copies of genomic DNA. Furthermore, products can be detected in a simple sandwich assay, thereby establishing an instrument-free DNA testing system. This unique combination of properties is a significant advance in the development of portable and widely accessible nucleic acid-based tests.

Bacillus subtilis↗

PubMed2004

Whole genome amplification of DNA from laser capture-microdissected tissue for high-throughput single nucleotide polymorphism and short tandem repeat genotyping.

Genome-wide screening of genetic alterations between normal and cancer cells, as well as among subgroups of tumors, is important for establishing molecular mechanism and classification of cancer. Gene silencing through loss of heterozygosity is widely observed in cancer cells and detectable by analyzing allelic loss of single nucleotide polymorphism and/or short tandem repeat markers. To use minute quantities of DNA that are available through laser capture microdissection (LCM) of cancer cells, a whole genome amplification method that maintains locus and allele balance is essential. We have successfully used a ø29 polymerase-based isothermal whole genome amplification method to amplify LCM DNA using a proteinase K lysis procedure coupled with a pooling strategy. Through single nucleotide polymorphism and short tandem repeat genotype analysis we demonstrate that using pooled DNA from two or three separate amplification reactions significantly reduces any allele bias introduced during amplification. This strategy is especially effective when using small quantities of source DNA. Although a convenient alkaline lysis DNA extraction procedure provided satisfactory results from using 1500 to 3000 LCM cells, proteinase K digestion was superior for lower cell numbers. Accurate genotyping is achieved with as few as 100 cells when both proteinase K extraction and pooling are applied.

Female↗

PubMed1993

Cloning of RNA molecules in vitro.

A method for RNA amplification in an immobilized medium is described. The medium contains a complete set of nucleotide substrates and purified Q beta replicase, an enzyme capable of exponentially amplifying RNAs under isothermal conditions. RNA amplification in the immobilized medium results in the formation of separate 'colonies', each comprising the progeny of a single RNA molecule (a clone). The colonies were visualized by staining with ethidium bromide, by utilizing radioactive substrates, and by hybridization with sequence-specific labeled probes. The number and identity of the RNA colonies corresponded to that of the RNAs seeded. When a mixture of different RNA species was seeded, these species were found in different colonies. Possible implementations of this technique include a search for recombinant RNAs, very sensitive nucleic acid diagnostics, and gene cloning in vitro.

Cloning, Molecular↗

PubMed1998

Rapid detection of genetic mutations using the chemiluminescent hybridization protection assay (HPA): overview and comparison with other methods.

The detection of genetic mutations is of paramount importance for the study, diagnosis, and treatment of human genetic disease. Methods of detection generally fall into one of two categories: those to scan for unknown mutations and those to detect known mutations. This review focuses on methods for the detection of known mutations. The hybridization protection assay (HPA) is described in detail. The HPA method utilizes short oligonucleotide probes covalently labeled with a highly chemiluminescent acridinium ester (AE). The assay format is completely homogeneous, requiring no physical separation steps, and can rapidly and sensitively detect all single-base mismatches as well as multiple mismatches, insertions, deletions, and genetic translocations. When very low copy number targets are assayed, HPA is coupled with transcription-mediated amplification (TMA), an isothermal method that amplifies DNA or RNA targets. Other methods that are described for the detection of known mutations include hybridization with sequence-specific oligonucleotides, hybridization to oligonucleotide arrays, allele-specific amplification, ligase-mediated detection, primer extension, and restriction fragment analysis. The advantages and limitations of each of these methods are discussed. Methods to scan for unknown mutations are briefly described.

Base Pair Mismatch↗

PubMed2004

Genomic profiling by DNA amplification of laser capture microdissected tissues and array CGH.

Comparative genomic hybridization by means of BAC microarrays (array CGH) allows high-resolution profiling of copy-number aberrations in tumor DNA. However, specific genetic lesions associated with small but clinically relevant tumor areas may pass undetected due to intra-tumor heterogeneity and/or the presence of contaminating normal cells. Here, we show that the combination of laser capture microdissection, phi29 DNA polymerase-mediated isothermal genomic DNA amplification, and array CGH allows genomic profiling of very limited numbers of cells. Moreover, by means of simple statistical models, we were able to bypass the exclusion of amplification distortions and variability prone areas, and to detect tumor-specific chromosomal gains and losses. We applied this new combined experimental and analytical approach to the genomic profiling of colorectal adenomatous polyps and demonstrated our ability to accurately detect single copy gains and losses affecting either whole chromosomes or small genomic regions from as little as 2 ng of DNA or 1000 microdissected cells.

Adenomatous Polyps↗

PubMed1997

Localization of dopamine D1A receptor protein and messenger ribonucleic acid in rat adrenal cortex.

Pharmacological, physiological, and autoradiographic studies have suggested the presence of dopamine receptors in the adrenal gland. Dopaminergic ligands have been shown to modulate adrenocortical aldosterone biosynthesis and secretion as well as adrenomedullary catecholamine production and release. Using a combination of light microscopic immunochemistry and in situ amplification and hybridization, the present study sought to determine the site-specific expression of the recently cloned D1A receptor subtype in rat adrenal gland. Light microscopic immunohistochemistry was conducted using polyclonal antisera raised to the putative rat D1A receptor. Immunoreactive product was detected using an avidin-biotin immunoperoxidase method. D1A receptor messenger RNA (mRNA) was detected using a transcription-based isothermal in situ amplification and hybridization approach using receptor-specific mRNA oligonucleotide probes. The amplified product was localized using an alkaline phosphatase 4-nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate technique. This combined experimental approach, using both receptor subtype-selective antibodies and oligonucleotide probes, allows for the site-specific localization of the D1A receptor subtype, which would otherwise not be possible with the pharmacological methods currently available. The D1A receptor protein and mRNA were expressed solely in the zona glomerulosa of the rat adrenal gland, with no signal evident in any of the other cortical layers or in the medulla. Such a distribution raises the possibility that the D1A receptor subtype could modulate, at least in part, some of the known effects of dopamine on aldosterone secretion.

Adrenal Cortex↗

PubMed1996

Expression of the subtype 1A dopamine receptor in the rat heart.

The subtype 1A dopamine receptor (D1A) has recently been detected in the rat kidney. In the present study using light microscopic immunohistochemistry, electron microscopic immunocytochemistry, and in situ amplification of mRNA, we demonstrate the D1A receptor in Sprague-Dawley and Wistar Kyoto rat hearts. For immunohistochemistry and immunocytochemistry, anti-peptide polyclonal antibodies were directed toward amino acid sequences of the third extracellular and intracellular domains of the native receptor. Selectivity was validated by recognition of the D1A receptor expressed in stably transfected LTK- cells. D1A receptor mRNA was detected with a novel transcription-based isothermal in situ amplification system as well as with reverse transcription-polymerase chain reaction. D1A receptor protein was distributed throughout the atrium and ventricular myocardium. Preimmune and preabsorption controls were negative. Electron microscopic immunocytochemistry using the protein A gold method demonstrated the D1A receptor along the cellular membranes of coronary smooth muscle cells and ventricular myocytes and in the myosin thick filaments and M-lines. D1A receptor mRNA was present in coronary vessels and myocardium in amplified but not in unamplified sections. Western blot analysis showed specific D1A bands in transfected LTK- cells and the atrium but not in nontransfected LTK- cells and the ventricle. The selective D1-like receptor agonist SKF38393 stimulated adenylyl cyclase in ventricular myocardial plasma membranes in a dose-related fashion, and the response was abolished by the selective D1-like receptor antagonist SCH23390. These results demonstrate that the D1A receptor gene and protein are expressed in normal rat heart. The physiological and pathophysiological roles and predominant cell signaling mechanism or mechanisms of this receptor remain to be determined.

Animals↗

PubMed1999

Amplification of padlock probes for DNA diagnostics by cascade rolling circle amplification or the polymerase chain reaction.

CONTEXT: Padlock probes are highly specific reagents for DNA diagnostics that can discriminate gene sequences with single base mutations. When the 3' and 5' terminal regions of the oligonucleotide probes are juxtaposed on a target DNA sequence, they can be circularized by enzymatic ligation and become topologically locked to the target. However, to be useful in solution-based diagnostics, the sensitivity of padlock probes must be markedly enhanced. OBJECTIVE: To describe two methods for geometric amplification of circularized padlock probes. DESIGN: Cascade rolling circle amplification is an isothermal system that uses generic primers and a DNA polymerase with strong strand displacement activity to amplify circularized probes by a mechanism combining rolling circle replication and strand displacement synthesis. One of the primers was designed as an energy transfer-labeled primer, which generates a fluorescence signal only when incorporated into the amplified product, enabling a direct means of detection. RESULTS: Using pUC19 as a model target to circularize an 89-base probe, a 10 billion-fold amplification was achieved with Bst DNA polymerase (large fragment) within 1 hour starting with as few as 10 probe molecules. The polymerase chain reaction was also used to amplify ligated padlock probes in a rare target detection system. In mixing experiments containing both normal and mutant p53 or c-Ki-ras2 gene target sequences, mutant targets were easily detected in the presence of a 500-fold excess of normal target copies. CONCLUSION: These results indicate that padlock probes can be amplified to the high levels required for solution-based DNA diagnostics.

Base Sequence↗

PubMed2026

A CRISPR-Based Rapid Detection Assay for Crayfish Plague (Aphanomyces astaci) From Environmental Samples.

Crayfish plague, caused by Aphanomyces astaci (Aa), is an infectious disease invasive in Europe, where its rapid spread has resulted in sharp declines of native crayfish species. Monitoring currently relies on a highly sensitive, but costly and time-consuming qPCR approach. Here, we designed a simplified, rapid and cost-efficient molecular assay for on-site detection of Aa. The novel rapid assay employs a combination of isothermal recombinase polymerase amplification and CRISPR-Cas12a-based detection that can be coupled with fluorescence or lateral flow visualisation. We demonstrate that the novel assay can detect A. astaci from tissue and environmental DNA with higher sensitivity than the available qPCR assay and readily distinguishes Aa from its non-pathogenic sister taxon A. fennicus. We tested two genomic marker sites for Aa that discriminate closely related oomycetes and incorporate field-deployable lateral flow and fluorescence readouts. Our work will make crayfish plague monitoring broadly accessible to practitioners and non-academic stakeholders as a tool to curb further Aa-driven loss of Europe's imperilled freshwater crustaceans and strengthen preparedness against future incursions of the pathogen in other regions.

Cas12a↗

PubMed2005

Rapid detection of Mycobacterium tuberculosis in respiratory samples by transcription-reverse transcription concerted reaction with an automated system.

The aim of this study was to evaluate the performance of the transcription-reverse transcription concerted (TRC) method for the detection of Mycobacterium tuberculosis complex (MTC) 16S rRNA in clinical respiratory samples for the diagnosis of pulmonary tuberculosis. TRC is a novel method that enables the rapid and the completely homogeneous real-time monitoring of isothermal sequence RNA amplification without any postamplification procedure. The detection limit of the TRC method for MTC was one organism per 100 mul of sputum. The specificity of the method was confirmed by the absence of positive signals for sputum containing 10(6) M. avium or M. kansasii organisms per 100 microl. A total of 201 respiratory samples from patients diagnosed with or suspected of having tuberculosis were tested. Of the 72 MTC culture-positive samples, the TRC method was positive for 52 (sensitivity, 72.2%), whereas the Roche COBAS AMPLICOR PCR was positive for 58 (sensitivity, 80.6%). Both the TRC method and the COBAS AMPLICOR PCR showed no positive identification for any of the 129 culture-negative samples. The percent agreement between the two methods was 95% (191 of 201 samples). The high sensitivity and specificity together with shorter detection time (within 1 h) of the TRC method allow it to be proposed as a useful method for the rapid detection of MTC in respiratory samples.

Bacteriological Techniques↗