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Results for “Isothermal amplification”

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At least 145 records · Page 8Linked to original sources

PubMed1994

Identification of Campylobacter jejuni, Campylobacter coli and campylobacter lari by the nucleic acid amplification system NASBAR.

NASBAR, an isothermal amplification technique for nucleic acids, was evaluated for the specific identification of Campylobacter jejuni, Camp. coli and Camp. lari. A set of primers and a probe were chosen from the 16S rRNA sequence of Campylobacter. The probe was hybridized in solution with the amplified nucleic acids of 12 Campylobacter species and nine other Gram-negative bacteria. The probe was shown to hybridize specifically to the amplified single-stranded RNA of Camp. jejuni, Camp. coli and Camp. lari in an enzyme-linked gel assay (ELGA). In a Camp. jejuni model system the combination of NASBAR and ELGA was able to detect ca 1000 rRNA molecules. The presence of an excess of Gram-negative bacteria did not influence the sensitivity of detection. A number of 6 cfu of Camp. jejuni, present in a total count of 4 x 10(6) cfu of Gram-negative bacteria, resulted in a positive hybridization signal.

Base Sequence↗

PubMed2003

Whole genome analysis of genetic alterations in small DNA samples using hyperbranched strand displacement amplification and array-CGH.

Structural genetic alterations in cancer often involve gene loss or gene amplification. With the advent of microarray approaches for the analysis of the genome, as exemplified by array-CGH (Comparative Genomic Hybridization), scanning for gene-dosage alterations is limited only by issues of DNA microarray density. However, samples of interest to the pathologist often comprise small clusters of just a few hundred cells, which do not provide sufficient DNA for array-CGH analysis. We sought to develop a simple method that would permit amplification of the whole genome without the use of thermocycling or ligation of DNA adaptors, because such a method would lend itself to the automated processing of a large number of tissue samples. We describe a method that permits the isothermal amplification of genomic DNA with high fidelity and limited sequence representation bias. The method is based on strand displacement reactions that propagate by a hyperbranching mechanism, and generate hundreds, or even thousands, of copies of the genome in a few hours. Using whole genome isothermal amplification, in combination with comparative genomic hybridization on cDNA microarrays, we demonstrate the ability to detect gene losses in yeast and gene dosage imbalances in human breast tumor cell lines. Although sequence representation bias in the amplified DNA presents potential problems for CGH analysis, these problems have been overcome by using amplified DNA in both control and tester samples. Gene-dosage alterations of threefold or more can be observed with high reproducibility with as few as 1000 cells of starting material.

Breast Neoplasms↗

PubMed2004

[Principle of LAMP method--a simple and rapid gene amplification method].

So far nucleic acid test (NAT) has been employed in various fields, including infectious disease diagnoses. However, due to its complicated procedures and relatively high cost, it has not been widely utilized in many actual diagnostic applications. We have therefore developed a simple and rapid gene amplification technology, Loop-mediated Isothermal Amplification (LAMP) method, which has shown prominent results of surpassing the performance of the conventional gene amplification methods. LAMP method acquires three main features: (1) all reaction can be carried out under isothermal conditions; (2) the amplification efficiency is extremely high and tremendous amount of amplification products can be obtained; and (3) the reaction is highly specific. Furthermore, developed from the standard LAMP method, a rapid LAMP method, by adding in the loop primers, can reduce the amplification time from the previous 1 hour to less than 30 minutes. Enormous amount of white precipitate of magnesium pyrophosphate is produced as a by-product of the amplification, therefore, direct visual detection is possible without using any reaction indicators and detection equipments. We believe LAMP technology, with the integration of these features, can rightly apply to clinical genetic testing, food and environmental analysis, as well as NAT in different fields.

Chemical Precipitation↗

PubMed2006

Detection and identification of IHN and ISA viruses by isothermal DNA amplification in microcapillary tubes.

Unique base sequences derived from RNA of both infectious hematopoietic necrosis virus (IHNV) and infectious salmon anemia virus (ISAV) were detected and identified using a combination of surface-associated molecular padlock DNA probes (MPPs) and rolling circle amplification (RCA) in microcapillary tubes. DNA oligonucleotides with base sequences identical to RNA obtained from IHNV or ISAV were recognized by MPPs. Circularized MPPs were then captured on the inner surfaces of glass microcapillary tubes by immobilized DNA oligonucleotide primers. Extension of the immobilized primers by isothermal RCA produced DNA concatamers, which were labeled with fluorescent SYBR Green II nucleic acid stain, and measured by microfluorimetry. Molecular padlock probes, combined with this method of surface-associated isothermal RCA, exhibited high selectivity without the need for thermal cycling. This method is applicable to the design of low-power field sensors capable of multiplex detection of viral, bacterial, and protozoan pathogens within localized regions of microcapillary tubes.

Biosensing Techniques↗

PubMed1997

Machines for automated evolution experiments in vitro based on the serial-transfer concept.

Two machine setups for automated evolution experiments in vitro are described. Both machines enable the monitoring of growing populations of RNA or DNA molecules in real time using high-sensitivity glass fiber laser fluorimeters and an automated sample handling facility for volumes in the microliter range. Growth conditions are kept constant by means of the serial-transfer technique, that is, the successive transfer of a small fraction of a growing population into a fresh solution containing no individuals prior to the transfer. The serial transfer technique was modified to work with large populations and constant growth conditions. In the single-channel evolution machine isothermal amplification reactions (Qbeta-system, 3SR, NASBA, SDA) are monitored successively in single test tubes. This machine is particularly well suited for the investigation of optimal adaptation to altered environmental conditions, as is experimentally demonstrated in the evolution of an RNA quasi-species using ribonuclease A as the selection pressure. The new variant of RNA appeared very rapidly (within approximately 80 generations) without stable intermediates, and it was selected by steadily increasing the RNaseA concentration during the serial-transfer experiment. The other machine, which is described in the second part of this article, is a consequent extension of the single-channel machine, and was designed to allow the multichannel detection of up to 960 samples simultaneously. Thus, high-throughput screening can be applied to evolution experiments. In addition to monitoring isothermal amplification reactions, it is also possible to follow PCR amplifications through thin plastic foils. Initial experiments have demonstrated the suitability of the apparatus for uniformly processing samples and for performing thermocycling.

Base Sequence↗

PubMed2004

Isothermal whole genome amplification from single and small numbers of cells: a new era for preimplantation genetic diagnosis of inherited disease.

Preimplantation genetic diagnosis (PGD) of single gene defects following assisted conception typically involves removal of single cells from preimplantation embryos and analysis using highly sensitive PCR amplification methods taking stringent precautions to prevent contamination from foreign or previously amplified DNA. Recently, whole genome amplification has been achieved from small quantities of genomic DNA by isothermal amplification with bacteriophage 29 DNA polymerase- and exonuclease-resistant random hexamer primers. Here we report that isothermal whole genome amplification from single and small numbers of lymphocytes and blastomeres isolated from cleavage stage embryos yielded microgram quantities of amplified DNA, and allowed analysis of 20 different loci, including the DeltaF508 deletion causing cystic fibrosis and polymorphic repeat sequences used in DNA fingerprinting. As with analysis by PCR-based methods, some preferential amplification or allele drop-out at heterozygous loci was detected with single cells. With 2-5 cells, amplification was more consistent and with 10 or 20 cells results were indistinguishable from genomic DNA. The use of isothermal whole genome amplification as a universal first step marks a new era for PGD since, unlike previous PCR-based methods, sufficient DNA is amplified for diagnosis of any known single gene defect by standard methods and conditions.

Cystic Fibrosis Transmembrane Conductance Regulato↗

PubMed1996

Typing of Mycoplasma pneumoniae by nucleic acid sequence-based amplification, NASBA.

Nucleic acid sequence-based amplification, NASBA, is an isothermal amplification technique for nucleic acids and was used for typing a collection of 24 Mycoplasma pneumoniae strains. A set of primers was chosen from the 16S rRNA sequence alignment of Mycoplasma species. The nucleotide sequences of the (-)RNA amplicons were determined for M. pneumoniae strains M15/83 (type 1) and FH (type 2), and revealed a one-point difference at the 16S rRNA level between the two types. Based on this result, two type-specific probes were constructed. The probes were hybridized in solution with the amplified nucleic acids of 24 M. pneumoniae strains in an enzyme-linked gel assay (ELGA). The results obtained by NASBA-based typing are in agreement with the classification of the 24 M. pneumoniae strains into two types by other typing methods, confirming the reliability of this technique.

Bacterial Typing Techniques↗

PubMed1995

Detection of HIV-1 infection in vitro using NASBA: an isothermal RNA amplification technique.

Establishment of a sensitive infection assay for HIV-1 is essential for successful screening of antiviral agents and neutralizing antibodies. In this report, an infection assay is described which measures the expression of viral genomic RNA and spliced mRNA intermediates in infected cells by an amplification-based technique called NASBA. The extreme sensitivity of this method permits the detection of viral RNA in peripheral blood mononuclear cells (PBMC) within 48 h of infection by a low dose of virus. Similarly, spliced HIV-1 mRNA could be detected within 24 h of infection of CEM cells by HIV-1IIIB. This NASBA-based infection assay was shown to titer the neutralization of the HIV-1IIIB isolate by serum from an infected human and by a monoclonal antibody to gp120. Furthermore, the inhibitory effects of azidothymidine (AZT) and soluble CD4 on HIV-1IIIB infection were quantitated by this assay. The early detection of virus by NASBA minimizes the contribution of secondary infection, thereby permitting more accurate evaluation of antiviral agents and neutralizing antibodies. This assay may be useful for the study of infection of phenotypically distinct HIV-1 isolates, which differ in terms of their replication kinetics.

Antibodies, Viral↗

PubMed2005

Increased precision of microbial RNA quantification using NASBA with an internal control.

Detection and quantification of low abundance target RNA has wide utility in the fields of clinical diagnostics, environmental monitoring, gene expression analysis, and biodefense. Nucleic acid based sequence amplification (NASBA) is an isothermal amplification method that provides the sensitivity needed for these applications. However, the requirement for three separate enzymes in NASBA often results in a greater variability between replicate samples than that seen in PCR-based assays. To overcome this problem, we have adapted the bioMérieux Nuclisens Basic Kit and Nuclisens EasyQ Analyzer along with the introduction of a synthetic internal control RNA (IC-RNA) for quantification of potentially any RNA sequence. Using the rbcL gene from the Florida red tide organism Karenia brevis as our target, we describe a simple method to accurately quantify the native target by computing the ratio of the time to positivity (TTP) values for both the wild-type and IC-RNA, and plotting this ratio against the starting number of target molecules or cells. By utilizing this simple method, we have significantly increased our accuracy and precision of prediction over the standard TTP calculations.

Animals↗

PubMed2001

Detection of rare DNA targets by isothermal ramification amplification.

We described previously a novel DNA amplification technique, termed ramification amplification (RAM) (Zhang et al., Gene 211 (1998) 277). This method was designed to utilize a circular probe (C-probe) that is covalently linked by a DNA ligase when it hybridizes to a target. Then, a DNA polymerase extends the bound forward primer along the C-probe and continuously displaces a downstream strand, generating a multimeric single-stranded DNA (ssDNA), analogous to in vivo 'rolling circle' replication of bacteriophage. This multimeric ssDNA then serves as a template for multiple reverse primers to hybridize, extend, and displace downstream DNA, generating a large ramified (branching) DNA complex, and resulting in an exponential amplification. Previously, we were able to achieve a significant amplification using phi29 DNA polymerase that has a high processivity and strong displacement activity. However, due to the intrinsic limitations of the polymerase, we only achieved a sensitivity of 10,000 target molecules, which is insufficient for most practical uses. Therefore, we tested several DNA polymerases and found that exo(-) Bst DNA polymerase meets the requirement for high sensitivity. By further improving the assay condition and format, we are able to detect fewer than ten targets in 1 h and to apply successfully this method for detection of Epstein-Barr virus in human lymphoma specimens.

Blotting, Southern↗

PubMed1998

[A method of isothermal gene amplification].

A gene amplification technique applied to the detection of HIV and which can be implemented without programmable thermocyclers is described. The reaction products are high molecular weight strands of DNA that can be detected using simple methods, even without electrophoresis. This is a promising technique for in situ amplification experiments. Our findings show that this amplification method is compatible with the temperature levels encountered in living cells. This technology could thus be the basis for a new class of drugs intended for treatment by gene therapy of certain diseases, including malaria and AIDS.

Base Sequence↗

PubMed2006

Multiple displacement amplification to create a long-lasting source of DNA for genetic studies.

In many situations there may not be sufficient DNA collected from patient or population cohorts to meet the requirements of genome-wide analysis of SNPs, genomic copy number polymorphisms, or acquired copy number alternations. When the amount of available DNA for genotype analysis is limited, high performance whole-genome amplification (WGA) represents a new development in genetic analysis. It is especially useful for analysis of DNA extracted from stored histology slides, tissue samples, buccal swabs, or blood stains collected on filter paper. The multiple displacement amplification (MDA) method, which relies on isothermal amplification using the DNA polymerase of the bacteriophage phi29, is a recently developed technique for high performance WGA. This review addresses new trends in the technical performance of MDA and its applications to genetic analyses. The main challenge of WGA methods is to obtain balanced and faithful replication of all chromosomal regions without the loss of or preferential amplification of any genomic loci or allele. In multiple comparisons to other WGA methods, MDA appears to be most reliable for genotyping, with the most favorable call rates, best genomic coverage, and lowest amplification bias.

DNA↗

PubMed2005

Optimization and design of oligonucleotide setup for strand displacement amplification.

Several advantages of strand displacement amplification (SDA) as an all-purpose DNA amplification reaction are due to it isothermal mechanism. The major problem of isothermal amplification mechanism is the accumulation of non-predictable byproduct especially for longer incubation time and low concentrations of initial template DNA. New theoretical strategies to tackle the difficulties regarding the specificity of the reaction are experimentally verified. Besides improving the reaction conditions, the stringency of primer hybridization can be distinctly improved by computer based sequence prediction algorithms based on the thermodynamic stability of DNA hybrid a described by the partition function of the hybridization reaction. An alternative SDA mechanism, with sequences developed by this means is also investigated.

Algorithms↗

PubMed2003

[Rapid and simple detection of Legionella species by LAMP, a mew DNA amplification method].

Legionella is a common cause of community-acquired respiratory tract infections and occasionally causes nosocomial pneumonia. Rapid and accurate detection of legionellae is important for diagnosis and treatment of patients. In order to detect legionellae, a new DNA amplification method was designed and evaluated. Loop-mediated isothermal amplification (LAMP) method amplifies DNA with high specificity, sensitivity, and rapidity under isothermal conditions at 65 degrees C. This method employs a DNA polymerase with strand displacement activity and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. The primers targeting 16S rRNA gene were designed in order to detect a wide range of Legionella species. We could specifically detect Legionella species including Legionella pneumophila, Legionella anisa, Legionella bozemanii, Legionella dumoffii, Legionella erythra, Legionella feeleii, Legionella gormanii, Legionella longbeachae, Legionella micdadei, Legionella oakridgensis, and Legionella sainthelensi. The detection limit of the assay was 6 cfu per test of L. pneumophila strain. Furthermore, all of the positive LAMP results could be obtained within 50 minutes. The LAMP method was able to detect a wide range of Legionella species with high specificity, sensitivity, rapidity, and a simple procedure.

Legionella↗

PubMed2001

Rolling circle amplification: a new approach to increase sensitivity for immunohistochemistry and flow cytometry.

Immunohistochemistry is a method that can provide complementary diagnostic and prognostic information to morphological observations and soluble assays. Sensitivity, specificity, or requirements for arduous sample preparation or signal amplification procedures often limit the application of this approach to routine clinical specimens. Rolling circle amplification (RCA) generates a localized signal via an isothermal amplification of an oligonucleotide circle. The application of this approach to immunohistochemistry could extend the utility of these methods to include a more complete set of immunological and molecular probes. RCA-mediated signal amplification was successfully applied to the sensitive and specific detection of a variety of cell surface antigens (CD3, CD20, and epithelial membrane antigen) and intracellular molecules (vimentin and prostate-specific antigen) within a variety of routinely fixed specimens, as well as samples prepared for flow cytometry. RCA technology, which has an intrinsically wide dynamic range, is a robust and simple procedure that can provide a universal platform for the localization of a wide variety of molecules as a function of either antigenicity or nucleic acid sequence. The use of RCA in this way could enhance the use of markers of current interest as well as permit the integration of emerging information from genomics and proteomics into cell- and tissue-based analyses.

Antibodies, Monoclonal↗

PubMed2005

In situ detection of specific DNA double strand breaks using rolling circle amplification.

We have developed a method to localize DNA double strand breaks (DSBs) in situ in cultured mammalian cells. Adenoviruses encoding Saccharomyces cerevisiae HO endonuclease and its cleavage site were used to induce site-specific DSBs. Rolling circle amplification (RCA), a sensitive method that allows the detection of single molecular event by rapid isothermal amplification, was used to localize the broken ends in situ. Punctate RCA signals were only seen in the cells that had been infected with both adenoviruses encoding HO endonuclease and HO cleavage site, but not in the cells mock-infected or infected with the site or endonuclease virus only. With use of a chemical crosslinker, in situ RCA and immunofluorescence (IF) can be performed simultaneously on the same sample. This methodology provides a novel approach for investigation of DNA recombination, DNA repair, and checkpoint controls in mammalian cells.

Adenoviridae↗

PubMed2006

[The laboratory diagnosis of influenza viruses].

The methods of the pathogen diagnosis for confirm the infection of the influenza virus, there are the viral isolation and the method of detected the viral antigen. In the each method of inspection, the time of sample extraction, the method of extraction and preservation must be suited to fully improve the sensitivity and specificity. The viral isolation is necessary and indispensable to analyze the antigen of the epidemic viruses and it is the high level of sensitivity. The several methods of detecting the antigen of the gene amplification include the reverse transcription polymerase chain reaction (RT-PCR) method, the loop-mediated isothermal amplification (LAMP) method, the real-time PCR method, and the nucleic acid sequence-based amplification (NASBA) method, and these methods are excellent promptly. As for these detection methods, being chosen according to the purpose is desirable.

Antigens, Viral↗

PubMed1997

Evaluation of the Gen-Probe Chlamydia trachomatis transcription-mediated amplification assay with urine specimens from women.

We evaluated the Gen-Probe Chlamydia trachomatis transcription-mediated amplification (TMA) assay with urine specimens for the detection of C. trachomatis infections in women. The novel test, based on the isothermal amplification of chlamydial RNA, was compared with the Roche Amplicor PCR with urine and cell culture with endocervical specimens. First-catch urine and endocervical swab specimens were collected from a total of 561 patients, of whom 70 (12.3%) were confirmed to have chlamydial infection. The diagnostic sensitivity and specificity of TMA with urine were 91.4 and 99.6%, respectively, and those of Amplicor PCR were 97.1 and 99.8%, respectively. By repeated analysis of the specimens with discrepant results, the sensitivity of TMA could be increased to 99%, indicating that some methodological improvements in the assay are still to be expected. The sensitivity of PCR could be increased to 100% by the elimination of DNA polymerase inhibitors in a repeated analysis. The sensitivity and specificity of cell culture with cervical specimens were 85.7 and 100%, respectively. The results indicate that TMA with urine specimens from women is a sensitive and specific assay for the detection of C. trachomatis, providing a new noninvasive technique for the screening of chlamydial infections in women.

Bacteriological Techniques↗