Regular ArticleQuantitative Polymerase Chain Reaction by Monitoring Enzymatic Activity of DNA Polymerase
Abstract
Nucleic acid amplification by polymerase chain reaction (PCR) is a very powerful technique in terms of sensitivity but is limited in terms of ability to perform accurate quantitation. While there is a theoretical correlation between copies of input target sequence and those of PCR product, the quantitative nature of this relationship is obscured by unpredictable variations in reaction conditions and by inhibitory and/or stimulatory substances which might be present in sample preparations, especially those derived from biological fluids. To reliably estimate copies of input DNA target from PCR product, we designed a combination of internal and external control systems coupled to DNA/RNA hybridization and enzymatic immunodetection techniques. The internal control system served to monitor amplification efficiency and to correct for the effects of inhibitors or stimuli on the efficiency of the DNA amplification. The assay is quantitative, nonisotopic, and can be widely applied to assessment of the quantity of DNA present in a wide range of preparations.
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Real-time PCR assays using internal controls for quantitation of HPV-16 and β-globin DNA in cervicovaginal lavages
2003, Journal of Virological MethodsHigh-risk human papillomavirus 16 (HPV-16) DNA viral load has been measured with real-time PCR assays by amplifying HPV-16 and a human gene. However, these assays have not used internal controls (ICs) to screen for the presence of inhibitors contained in samples. To quantitate HPV-16 DNA and cell content with real-time PCR, ICs for HPV-16 DNA and β-globin were synthesised and used to control for inhibition. The assays were sensitive and linear over 5 logs. Good reproducibility was achieved with inter-run coefficients of variation of 23% (102 HPV-16 copies), 12% (104 HPV-16 copies), 17% (274 β-globin DNA copies) and 7% (27 400 β-globin DNA copies). Samples containing 56 800 000, 306 000, 18 000, and 4070 HPV-16 copies/μg of cellular DNA were tested blindly and estimated to contain 48 800 000, 479 000, 20 300, and 6620 HPV-16 copies/μg of DNA (mean ratio of measured to expected viral load of 1.27±0.32). Inhibition of amplification of HPV-16 and β-globin ICs by six samples known to contain PCR inhibitors was variable: four inhibited both ICs while two inhibited only the HPV-16 IC. The use of internal controls with real-time PCR for HPV-16 quantitation allows to screen for the presence of inhibitors that do not affect equally primer-driven genomic amplification.
A flash-type bioluminescent immunoassay that is more sensitive than radioimaging: Quantitative detection of cytokine cDNA in activated and resting human cells
1998, Journal of Immunological MethodsBecause of its high sensitivity, bioluminescence (BL) is an excellent alternative to radioactive quantitation of cytokine RT-PCR-derived products. BL also allows detection of amplicons at cycle numbers not normally detectable using radioactivity. No direct comparisons between these two methods have been made. In this study, the sensitivities of BL using recombinant aequorin, a flash-type luminescent tag capable of detecting signal to attomolar (10−18 M) levels and radio imaging (RI) were directly compared. In addition, the application of BL for detecting cytokine message from biologic samples was examined. BL was 30- to 60-fold more sensitive than RI in detecting human IL-2 and CD3δ amplicons. This difference was particularly found during low cycle PCR, but was less at higher cycle numbers. The ability of BL to detect differences in cytokine message in stimulated and unstimulated human peripheral blood mononuclear cells was also evaluated. Using linear regression analysis, we observed up to 5,000-fold increases in RT-PCR amplified-mRNA in stimulated cells for IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10 and GM-CSF compared to unstimulated cells. Changes in CD3δ, TNFα or IL-12 were not observed or quantitated. We present a novel aequorin-based application of bioluminescent technology to directly quantitate RT-PCR amplicons and to investigate the induction of human cytokine expression. Significant advantages of this sensitive bioluminescent method compared with radioactive methods are its abilities to quantitate amplicons in a PCR cycle range where linear detection is most robust and to analyze products in an automated, open-architecture microtiter plate format.
How subtle differences in MHC class II affect the severity of experimental myasthenia gravis
1998, Clinical Immunology and ImmunopathologyMyasthenia gravis is an autoimmune disorder characterized by muscle weakness, due to an antibody-mediated deficit of acetylcholine receptors (AChRs) at neuromuscular junctions. We analyzed the factors that determine the severity of experimental myasthenia gravis (EAMG) induced by immunization withTorpedoAChR, in two congenic strains of mice—B6 mice, which are highly susceptible to EAMG; and bm12 mice, which are relatively resistant, and differ only in a change of three amino acids in MHC Class II. We prepared large numbers of AChR-specific T cell hybridomas from each strain and characterized their epitope specificities and T cell receptor (TCR) gene usage: Half the B6 hybridomas responded to a single AChR peptide (α 146–162), and their TCR genes encoded restricted Vα and Vβ chains and CDR3 motifs. bm12 hybridomas had different epitope specificities and different, less restricted TCR genes. APCs were able to present AChR or AChR-derived peptides virtually exclusively to hybridomas of their own strain. Levels of antibodies toTorpedoand autoantibodies to mouse AChR were higher in B6 mice, and were biased toward the IgG2b isotype. We conclude that the “better fit” of MHC II, peptide, and TCR in the B6 mice enhanced cognate interactions of APCs with T cells, and T cells with B cells, resulting in a more abundant and pathogenic AChR antibody response, and thus more severe EAMG.
Quantitation of RT-PCR amplified cytokine mRNA by aequorin-based bioluminescence immunoassay
1996, Journal of Immunological MethodsWe described here a bioluminescence-based immunoassay for the quantitation of RT-PCR amplified cytokine mRNA. This technique uses a standard RT-PCR procedure, with the following modifications. The forward primer in the PCR reaction is labeled with a 5′ biotin molecule. Following PCR, a digoxigenin-conjugated oligonucleotide probe is hybridized to the target biotin-labeled DNA template. The hybridized duplex is captured onto a streptavidin-coated microtiter plate. The bound product is quantitated by adding digoxigenin-specific antibodies conjugated with the photoprotein aequorin. the amount of specific DNA captured onto the plate is quantitated by triggering the bioluminescence reaction through the addition of calcium ions. This technique detected as low as 40 amol of amplified cytokine products, or 500 copies of templates when 27 PCR cycles were used. The high sensitivity of this technique enables the quantitation of target DNA during the exponential phase of the PCR reaction. The aequorin-bioluminescence assay is an alterative non-radioactive method for the quantitation of PCR products.
Quantification of polymerase chain reaction products: Enzyme immunoassay based systems for digoxigenin- and biotin-labelled products that quantify either total or specific amplicons
1996, Molecular and Cellular ProbesEnzyme immunoassays were developed for quantification of polymerase chain reaction (PCR) products, referred to as amplicons. Amplicons were dual labelled simultaneously by enzymatic incorporation of digoxigenin and biotin during PCR. For total amplicon quantification, Microfluor B polystyrene wells, compatible with chemiluminescent detection, were coated with streptavidin. Dual labelled amplicons were bound, treated with anti-digoxigenin antibody conjugated to alkaline phosphatase to complete the two-site sandwich immunoassay configuration, and detected by the chemiluminescence generated upon hydrolysis of a phosphate substituted dioxetane substrate, AMPPD. For specific amplicon quantification, the Microfluor B wells were coated with an unlabelled DNA probe complementary to the labelled amplicon target. Subsequent steps were performed as described above. This assay detects 2 pg of specifically amplified DNA. Chemiluminescent detection provides a linear range of four orders of magnitude for amplicon quantification. The non-radioactive labels are safe and stable. PCR as described here obviates the need for labelled primers and constitutes the initial report of concurrent dual non-radioactive labelling of DNA by a DNA polymerase.
Quantitative PCR for the measurement of circulating proviral load in HIV-infected individuals
1995, Clinical and Diagnostic VirologyBackground: PCR has been applied extensively as a tool for the detection of HIV. We have previously developed a semi-quantitative microtiter plate assay for the specific detection of HIV PCR products.
Objective: To develop and evaluate a novel quantitative assay for the measurement of circulating proviral load in HIV-infected individuals.
Study design: We evaluated 70 consecutive, unselected HIV-infected patients, divided into 3 groups, according to their CD4 cell count: greater than 500 cells/μl (10 subjects); 200–500 cells/μl (31 subjects); less than 200 cells/μl (29 subjects). Peripheral blood mononuclear cell lysates were amplified, and a portion of the product was added to streptavidin-coated wells with a biotinylated capture probe and a horseradish peroxidase-linked reporter probe, complementary to separate regions of the amplified product. Following incubation, readings were taken with an automated plate reader. Products were quantitated by interpolation into a standard curve of serial dilutions of an HIV-containing plasmid, included in each assay.
Results: HIV sequences were detected in all 70 clinical samples. Within each patient category, a wide range of proviral loads were observed. However, proviral load/106 CD4 cells was associated with disease progression when the patient groups were considered (up to 9.6% infected cells in subjects with CD4 cell counts below 200/μl).
Conclusions: This quantitative PCR assay will allow the measurement of proviral load in clinical samples. It may be useful in the managment of HIV-infected individuals and the evaluation of the efficacy of antiretroviral therapy.