ABSTRACT
Recent advances in nucleic acid amplification techniques have allowed for quantitation of viral nucleic acid levels in clinical specimens. The most prevalent testing is carried out for HIV viral load. Strand displacement amplification (SDA) is an isothermal DNA amplification system utilizing a restriction enzyme and a DNA polymerase with strand displacement properties. SDA was adapted for quantitative RNA amplification (QRT-SDA) of an HIV gag sequence by including AMV reverse transcriptase, a quantitative control sequence, and 32P-labeled detector oligonucleotides for the HIV and the control sequences. We have also improved the amplification efficiency by including the single-strand binding protein from gene 32 of T4 bacteriophage (T4gp32) to enhance strand displacement replication. In a preliminary analytical demonstration of the technique, RT-SDA was quantitative to within twofold over a range of 500-500,000 transcripts that were generated from a plasmid bearing an HIV gag sequence. QRT-SDA potentially represents a convenient alternative for viral load testing in a clinical setting.
Subject(s)
Nucleic Acid Amplification Techniques , Nucleic Acids/analysis , Base Sequence , DNA Primers , Gene Products, gag/genetics , HIV/genetics , HIV/isolation & purification , Nucleic Acids/genetics , RNA-Directed DNA Polymerase/chemistry , Viral LoadABSTRACT
A total of 294 clinical respiratory specimens, including 75 with culture-positive results, were tested for the presence of Mycobacterium tuberculosis by strand displacement amplification (SDA) of DNA. A region of the IS6110 insertion element and an internal control sequence were amplified and then detected by a chemiluminescence assay. Receiver operator-characteristic curves were used to evaluate three methods for declaring specimens positive for M. tuberculosis. By the preferred method, SDA chemiluminescence results were converted to theoretical numbers of M. tuberculosis organisms. A positive threshold (PT) value, above which 95% of the SDA results were judged to be M. tuberculosis positive (sensitivity = 95%), was found to be 2.4 M. tuberculosis organisms per SDA reaction. The analogous PT value for 95% sensitivity on smear-positive specimens was 3.6 M. tuberculosis organisms per reaction. The PT of 2.4 M. tuberculosis organisms per reaction detected 100% of culture-positive, smear-positive specimens (sensitivity = 100%), while 95% sensitivity was achieved with a PT of 15.5 M. tuberculosis organisms per reaction. Specificities, which were calculated with respect to culture- and smear-negative specimens, ranged from 96% at a PT of 15.5 M. tuberculosis organisms to 84% at a PT of 2.4 M. tuberculosis organisms per reaction. The M. tuberculosis-negative specimens were also segregated according to whether the patients received antituberculosis chemotherapy. SDA specificity ranged from 90% (PT = 2.4 M. tuberculosis organisms) to 98% (PT = 15.5 M. tuberculosis organisms) for the M. tuberculosis-negative specimens from patients who had not received chemotherapy. SDA specificity in the M. tuberculosis-negative specimens from patients who received chemotherapy was lower (85 to 94%). This study represents the first large-scale demonstration of M. tuberculosis detection in clinical sputum specimens by isothermal DNA amplification with SDA.
Subject(s)
Bacteriological Techniques , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/isolation & purification , Nucleic Acid Amplification Techniques , Tuberculosis, Pulmonary/diagnosis , Tuberculosis, Pulmonary/microbiology , Bacteriological Techniques/standards , Bacteriological Techniques/statistics & numerical data , False Positive Reactions , Humans , Luminescent Measurements , ROC Curve , Reference Standards , Sensitivity and Specificity , Sputum/microbiologyABSTRACT
Modern automated drug-screening can generate hundreds of inhibitor leads from diverse chemical sources in a short period of time. Traditional methods of inhibitor analysis are resource intensive and limit the number of inhibitors that can be analyzed for their mechanism of inhibition. This paper presents methods we have developed for rapid estimation of both potency and mechanism of potential inhibitor leads for a biochemically complex screening target (protein kinase C) using commercially available computer programs for statistical experimental design. Our findings indicate that, with careful choice of factor levels, statistical experimental design clearly identifies the various interactions of the assay components with inhibitors. Suitably plotted, the data can be used to examine the competitive nature of the inhibitor and can provide estimates of IC50 and Michaelis constants useful for planning further kinetic work. The techniques used are amenable to automation and should be useful for identifying inhibitors that may have only marginal potency, but exhibit desirable mechanistic profiles suitable for structural analoging efforts.
Subject(s)
Antifungal Agents/metabolism , Azepines , Computer Simulation , Enzyme Inhibitors/metabolism , Hydroxybenzoates , Models, Statistical , Protein Kinase C/antagonists & inhibitors , Drug Design , Factor Analysis, Statistical , Kinetics , Pyrrolidines/metabolism , Research DesignABSTRACT
Strand displacement amplification, a new isothermal in vitro DNA amplification technique, was used to amplify target DNA contained within the IS6110 insertion element of the species within the Mycobacterium complex (Mycobacterium tuberculosis, M. bovis, M. bovis-BCG, M. africanum and M. microti). The target nucleic acid sequence is present in approximately ten, two, one, five and five copies in M. tuberculosis, M. bovis, M. bovis-BCG, M. africanum and M. microti, respectively. Amplified products were detected using a non-isotopic microtitre plate assay employing a biotinylated oligodeoxynucleotide probe and an alkaline phosphatase conjugated oligodeoxynucleotide probe. Lumiphos 530 was the chemiluminescent substrate for alkaline phosphatase. The combination of the strand displacement amplification method with this sensitive and rapid (less than 2 h) detection system resulted in the specific detection of as few as 1-25 initial IS6110 targets in the five Mycobacterium complex species based on signal/noise criteria. Negative results were obtained with eight other Mycobacterium species as well as with 32 non-Mycobacterium species.
Subject(s)
Gene Amplification , Mycobacterium tuberculosis/genetics , Mycobacterium/genetics , Oligonucleotide Probes , Tuberculosis/diagnosis , Base Sequence , Biotin , DNA Polymerase I , DNA Transposable Elements , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , False Positive Reactions , Humans , Luminescent Measurements , Molecular Sequence Data , Mycobacterium/isolation & purification , Mycobacterium bovis/genetics , Mycobacterium bovis/isolation & purification , Mycobacterium tuberculosis/isolation & purification , Nucleic Acid Hybridization , Sensitivity and SpecificityABSTRACT
The contributions of several components to the variance in lodoxamide delivery from lodoxamide tromethamine metered-dose aerosol containers have been estimated. Two aerosol lots, manufactured with mean diameters of 2.3 and 7.2 micron, exhibited approximately equal variances. The variance was apportioned to the following components: container-to-container, 27%; mouthpiece-to-mouthpiece 18%; valve-to-valve, 11%; assay, 6%. The largest single contribution to the variance (38%) is attributed to unassignable variations which include within-container variations in the dose; quality improvement efforts should concentrate on this area. Little effort should be expended to minimize the assay, valve delivery, or mouthpiece variation as their contribution to lodoxamide dose variation is small. Likewise, the bulk drug particle size did not contribute appreciably to within-lot dose variation.