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Real-time polymerase chain reaction

Real-time polymerase chain reaction (qPCR), also known as quantitative PCR, is a molecular biology technique that amplifies specific DNA sequences while simultaneously monitoring the progress of the reaction in real time through fluorescence detection.^[1] This method allows for the precise quantification of the initial amount of target DNA in a sample by measuring the cycle threshold (Ct), the point at which fluorescence exceeds a baseline threshold during the exponential amplification phase.^[2] Unlike conventional endpoint PCR, which requires post-amplification analysis such as gel electrophoresis, qPCR eliminates these steps by integrating detection directly into the amplification process, enabling both qualitative and quantitative results in a single reaction.^[1] The foundation of qPCR lies in the polymerase chain reaction (PCR), invented by Kary Mullis in 1985 as a method to exponentially amplify specific DNA segments using repeated cycles of denaturation, annealing, and extension.^[3] Mullis's innovation, which earned him the Nobel Prize in Chemistry in 1993, relied on thermostable DNA polymerase from Thermus aquaticus (Taq polymerase) to automate the process without manual enzyme replenishment after each cycle.^[4] Real-time monitoring was introduced in 1992 by Russell Higuchi and colleagues at Roche Molecular Systems, who demonstrated simultaneous amplification and detection using ethidium bromide, a DNA-intercalating dye that fluoresces upon binding to double-stranded DNA products.^[5] This breakthrough was expanded in 1993 with kinetic PCR analysis, employing a video camera to track fluorescence changes across multiple reactions, establishing the quantitative framework for modern qPCR.^[6] At its core, qPCR involves a thermal cycler that alternates temperatures to facilitate DNA denaturation at approximately 95°C, primer annealing at 50–60°C, and extension at 72°C using Taq polymerase, which synthesizes new DNA strands.^[1] Detection occurs via fluorescent reporters: either non-specific intercalating dyes like SYBR Green, which bind all double-stranded DNA and increase fluorescence proportionally to amplicon accumulation, or sequence-specific probes such as TaqMan, where a fluorophore is released from a quencher upon polymerase-mediated cleavage during extension.^[7] The resulting amplification curves, plotted as fluorescence versus cycle number, follow an exponential phase followed by a plateau, with the Ct value inversely correlating to the starting template quantity for absolute or relative quantification.^[2] qPCR has become indispensable in diagnostics, research, and forensics due to its high sensitivity—capable of detecting as few as 1–10 target copies—and specificity, which minimizes false positives through probe-based validation.^[1] Key applications include viral load measurement (e.g., for HIV or SARS-CoV-2), gene expression profiling via reverse transcription qPCR (RT-qPCR) for mRNA analysis, microbial identification, genotyping, and copy number variation studies in cancer research.^[2] Its closed-tube format reduces carryover contamination, while real-time data acquisition shortens turnaround times to under two hours, making it a cornerstone for rapid outbreak response and personalized medicine.^[1] Despite requiring specialized instrumentation and optimized primers, qPCR's reliability has revolutionized nucleic acid analysis across biomedical fields.^[7]

Introduction

Definition and Overview

Real-time polymerase chain reaction (real-time PCR), also known as quantitative PCR (qPCR), is a laboratory technique that amplifies specific DNA sequences while simultaneously monitoring the progress of the reaction in real time through fluorescence signals that are proportional to the amount of amplified product (amplicon).^[8] This method enables the sensitive and specific detection and quantification of nucleic acids, making it essential for applications such as gene expression analysis, pathogen detection, and genotyping.^[1] Introduced as a refinement of the standard polymerase chain reaction (PCR), real-time PCR was first demonstrated by Higuchi et al. in 1992, who coupled ethidium bromide fluorescence with video imaging to observe amplification kinetics without opening the reaction vessel.^[9] Unlike traditional PCR, which relies on end-point analysis after amplification has reached a plateau phase where quantification is unreliable due to limiting reagents and enzyme inefficiencies, real-time PCR measures DNA accumulation during the exponential phase of the reaction.^[10] This allows for accurate determination of the initial template concentration, as the fluorescence signal correlates linearly with amplicon quantity before plateau effects introduce bias.^[1] By avoiding post-amplification processing steps like gel electrophoresis, real-time PCR reduces contamination risks and provides faster, more reproducible results.^[8] The basic workflow of real-time PCR involves thermal cycling to drive DNA amplification, coupled with fluorescence detection at the end of each cycle. The process includes three main steps: denaturation at approximately 95°C to separate DNA strands, annealing at 50–65°C for primers to bind to target sequences, and extension at 72°C where a thermostable DNA polymerase synthesizes new strands using deoxynucleotide triphosphates.^[1] Fluorescent reporters, such as dyes or probes, are incorporated or activated proportionally to the amplicon amount, generating a signal that is measured by an optical detection system integrated into the thermal cycler.^[8] Quantification in real-time PCR is primarily based on the threshold cycle (Ct), defined as the fractional cycle number at which the fluorescence signal exceeds a predetermined threshold above background noise, indicating the onset of exponential amplification.^[1] The Ct value inversely correlates with the initial amount of target DNA: lower Ct values signify higher starting concentrations, as amplification reaches detectability earlier.^[10] This metric enables relative or absolute quantification when calibrated against standards, with typical efficiency assuming a twofold increase in product per cycle during the exponential phase.^[1]

Historical Development

The development of real-time polymerase chain reaction (PCR), also known as quantitative PCR (qPCR), emerged from efforts to overcome the limitations of traditional end-point PCR, which relied on post-amplification analysis and suffered from issues like plateau effects and imprecise quantification. These foundational ideas set the stage for direct observation during amplification cycles. A pivotal advancement occurred in 1992 when Russell Higuchi and colleagues at Cetus introduced the first real-time monitoring system, using ethidium bromide fluorescence to track double-stranded DNA accumulation via a video camera setup, enabling kinetic analysis of PCR reactions without post-run processing. This was followed in 1993 by Lee et al. at Cetus (later acquired by Roche), who developed the 5' nuclease assay with fluorogenic probes—now known as TaqMan technology—allowing sequence-specific detection and quantification during amplification through reporter dye release. These innovations transformed PCR into a tool for precise, real-time measurement of nucleic acid levels. Commercialization accelerated adoption in the mid-1990s, with Applied Biosystems launching the ABI PRISM 7700 Sequence Detection System in 1996, the first integrated instrument combining thermal cycling with fluorescence detection for high-throughput qPCR. During the 2000s, standardization efforts addressed variability in qPCR reporting; notably, Stephen Bustin led the development of the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines in 2009, establishing protocols for experimental design, validation, and data reporting to enhance reproducibility and reliability.^[11] The COVID-19 pandemic from 2020 onward dramatically scaled real-time PCR applications, with qPCR becoming the gold standard for SARS-CoV-2 detection due to its sensitivity and specificity in diagnosing viral RNA. This surge drove innovations in portable devices, such as the 2022 Columbia Engineering-Rover Diagnostics platform delivering RT-PCR results in 23 minutes, facilitating point-of-care testing in resource-limited settings.^[12] By 2025, global deployment of such compact systems had expanded access, with the overall real-time PCR market projected to grow at a compound annual growth rate (CAGR) of 7.2% from 2025 to 2033 (as of 2024 estimates).^[13]

Principles of Operation

DNA Amplification Mechanism

The DNA amplification mechanism in real-time polymerase chain reaction (PCR) relies on the core components of the reaction mixture, which include the DNA template serving as the starting genetic material, forward and reverse primers that define the target sequence boundaries, a thermostable DNA polymerase such as Taq polymerase for catalyzing DNA synthesis, deoxynucleotide triphosphates (dNTPs) as building blocks for new strands, and an appropriate buffer to maintain optimal pH and ionic conditions.^[1] These elements enable the in vitro replication of specific DNA segments through repeated cycles of enzymatic activity. The process unfolds through three primary thermal cycling phases: denaturation, annealing, and extension. During denaturation, the reaction mixture is heated to 94–98°C for 15–30 seconds, causing the double-stranded DNA template to separate into single strands by disrupting hydrogen bonds.^[1] In the annealing phase, the temperature is lowered to 50–65°C for 20–40 seconds, allowing the primers to hybridize specifically to their complementary sequences on the single-stranded DNA templates.^[1] The extension phase follows at 72°C, where the Taq polymerase extends the primers by incorporating dNTPs, synthesizing new DNA strands at a rate of approximately 30 seconds per kilobase pair.^[1] Under ideal conditions, each thermal cycle results in exponential amplification, theoretically doubling the number of target DNA molecules per cycle and yielding up to 10^9 copies from a single starting molecule after 30–40 cycles, assuming 100% efficiency with no limiting factors such as reagent depletion.^[1] This exponential growth is the foundation of PCR's sensitivity, enabling detection of low-abundance targets. In real-time PCR, the amplification mechanism is adapted to a closed-tube format, which minimizes contamination risks by eliminating the need to open the reaction vessel between cycles, while fluorescence measurement is integrated during or immediately after the extension phase to monitor product accumulation continuously.^[5]

Real-Time Fluorescence Detection

In real-time polymerase chain reaction (qPCR), fluorescence detection operates on the principle that the fluorescent signal intensifies in proportion to the accumulation of amplified DNA products during each thermal cycle. As double-stranded DNA amplicons form through the enzymatic replication process, they bind or activate fluorescent reporters, generating emissions that are captured immediately after the extension step. These emissions are detected by sensitive photodetectors, such as photomultiplier tubes (PMTs) or charge-coupled devices (CCDs), integrated into the qPCR instrument's optical system, enabling continuous monitoring without interrupting the reaction. This real-time readout allows for the quantification of starting template amounts based on the kinetics of signal increase, as first demonstrated in early kinetic PCR assays using ethidium bromide fluorescence.^[14]^[15]^[16]^[17] The dynamics of the fluorescence signal follow a characteristic sigmoidal pattern across PCR cycles. Initially, during the baseline phase (typically cycles 1–10), fluorescence remains low and stable, representing background noise from unbound dyes, probes, or instrument autofluorescence, with no detectable amplification. In the subsequent exponential phase (cycles 10–25, depending on template abundance), the signal rises steeply as amplicons double with each cycle, producing a linear increase in fluorescence on a logarithmic scale that correlates directly with template quantity. This phase transitions into a plateau (cycles >25), where the signal levels off due to reagent exhaustion, product inhibition, or enzyme denaturation, limiting further amplification despite the continued cycling. Accurate threshold setting above the baseline during the exponential phase is crucial for determining the cycle threshold (Ct) value, which inversely reflects initial target concentration.^[18]^[19]^[20] To mitigate variations in optical path length, reaction volume, or instrument-specific artifacts that could skew fluorescence readings across wells, normalization is achieved using passive reference dyes such as ROX (carboxy-X-rhodamine). ROX is a non-amplification-dependent fluorophore that emits a stable, constant signal throughout the reaction, serving as an internal standard for correcting well-to-well differences in signal intensity. By dividing the reporter fluorescence by the ROX signal, normalized data enhance precision and reproducibility, particularly in multi-well formats. This approach is widely recommended by instrument manufacturers and is essential for high-throughput applications.^[21]^[22]^[23] qPCR fluorescence detection offers exceptional sensitivity, with typical limits of detection ranging from 1 to 10 target molecules per reaction volume, allowing the identification of rare nucleic acid sequences in complex samples. This capability stems from the low background noise and high signal-to-noise ratios of modern optical systems. The method also provides a broad dynamic range of approximately $10^2 to $10^9-fold, enabling accurate quantification from low-copy templates (e.g., single-digit molecules) to high-abundance targets (e.g., $10^9 copies), though optimal performance requires assay optimization to maintain linearity across this span. Such parameters make real-time fluorescence detection indispensable for applications demanding precise absolute or relative quantification.^[24]^[25]^[26]^[27]

Detection Methods

Intercalating Dye-Based Detection

Intercalating dyes represent a non-specific method for detecting DNA amplification in real-time polymerase chain reaction (qPCR), where these fluorescent molecules bind to double-stranded DNA (dsDNA) produced during each cycle, allowing real-time monitoring of product accumulation through increased fluorescence.^[28] SYBR Green I, the most commonly used intercalating dye, binds to the minor groove of dsDNA and exhibits negligible fluorescence in solution or when bound to single-stranded DNA (ssDNA); upon excitation at approximately 497 nm, its fluorescence increases dramatically—up to 1000-fold—specifically upon intercalation into dsDNA, correlating directly with the amount of amplified product.^[29] This non-sequence-specific binding enables detection of any dsDNA, making the method broadly applicable without the need for custom probes.^[28] Other common intercalating dyes include EvaGreen, which similarly binds dsDNA with high affinity and shows enhanced fluorescence upon binding, as well as YO-PRO-1 and SYTO dyes, each offering variations in spectral properties and stability for qPCR applications.^[28] These dyes preferentially interact with dsDNA over ssDNA due to their intercalative nature, with binding affinity increasing substantially in the presence of double helices, though exact fold enhancements can vary by dye chemistry.^[28]^[30] The primary advantages of intercalating dye-based detection lie in its cost-effectiveness and simplicity, as it requires no probe design or synthesis, facilitates high sensitivity for low-copy targets, and eliminates post-amplification processing steps, thereby reducing contamination risks in routine workflows.^[28] However, a key limitation is its lack of specificity, as the dyes bind indiscriminately to all dsDNA, including non-target amplicons, primer dimers, or off-target products, which can lead to overestimation of target quantity and necessitate confirmatory techniques like melting curve analysis to verify product identity.^[28] Despite this, the method remains ideal for initial screening of unknown targets, gene expression monitoring, or applications where cost outweighs the need for absolute specificity, such as pathogen detection or genotyping assays.^[28]

Sequence-Specific Probe-Based Detection

Sequence-specific probe-based detection in real-time polymerase chain reaction (PCR) utilizes oligonucleotide probes designed to hybridize to a defined target sequence within the amplicon, allowing for precise fluorescence monitoring during amplification cycles. These probes incorporate fluorogenic moieties that emit detectable signals upon target binding or enzymatic modification, offering greater sequence discrimination than non-specific intercalating dyes. This approach minimizes background noise from primer-dimers or off-target products, enhancing assay reliability for targeted quantification.^[8] Hydrolysis probes, commonly known as TaqMan probes, represent a primary type of sequence-specific detection system. These probes are linear oligonucleotides labeled with a fluorescent reporter dye at the 5' terminus, such as 6-carboxyfluorescein (FAM), and a non-fluorescent quencher at the 3' end, such as 6-carboxytetramethylrhodamine (TAMRA). In the intact probe, the quencher suppresses reporter fluorescence through proximity-based energy transfer. During the PCR extension phase, the probe anneals to the target sequence between the forward and reverse primers; the 5' to 3' exonuclease activity of the thermostable DNA polymerase (e.g., Taq polymerase) then hydrolyzes the bound probe, releasing the reporter dye from the quencher and generating a proportional increase in fluorescence with each cycle of product accumulation. This mechanism enables real-time detection without post-amplification processing and was originally developed by exploiting the exonuclease properties of Thermus aquaticus DNA polymerase.^[8] Förster resonance energy transfer (FRET)-based probes, such as Molecular Beacons, provide an alternative non-hydrolytic detection strategy. These probes adopt a stem-loop (hairpin) conformation in solution, positioning a 5' reporter fluorophore (e.g., FAM) and a 3' quencher (e.g., DABCYL) in close proximity to quench fluorescence via FRET. Upon hybridization to the complementary target sequence during the annealing step, the probe unfolds, spatially separating the fluorophore from the quencher and restoring fluorescence emission. This reversible binding allows for dynamic monitoring of amplicon formation without probe degradation, though signal intensity depends on hybridization kinetics and temperature control. Molecular Beacons were introduced as homogeneous probes for nucleic acid detection in 1996.^[31] Several variants extend the utility of sequence-specific probes. Scorpion probes integrate a primer sequence with an adjacent probe element in a single hairpin-structured oligonucleotide, featuring a 5' fluorophore and 3' quencher. After primer extension incorporates the probe into the amplicon, a stem-loop structure forms intramolecularly, bringing the fluorophore and quencher apart to produce fluorescence in a unimolecular manner, which accelerates signal generation and supports multiplexing. This design was reported in 1999 for efficient PCR product detection. Hybridization probes, in contrast, employ two non-overlapping, adjacent oligonucleotides: one labeled with a donor fluorophore (e.g., fluorescein) at the 3' end and the other with an acceptor (e.g., LightCycler Red) at the 5' end. When both probes bind to the target amplicon, FRET occurs between the dyes, yielding acceptor emission only upon specific dual hybridization, without requiring nuclease activity. This FRET-mediated approach builds on early demonstrations of nonradiative energy transfer for nucleic acid hybridization detection. The specificity of sequence-specific probes is particularly advantageous for applications requiring discrimination of subtle sequence variations, such as single nucleotide polymorphisms (SNPs). By designing probes to span the polymorphic site, even a single base mismatch destabilizes hybridization, preventing fluorescence signal in mismatched cases and enabling allele-specific detection with high accuracy. This targeted binding reduces non-specific amplification artifacts, providing cleaner signals than intercalating dye methods, which bind any double-stranded DNA indiscriminately.^[32]^[8]

Instrumentation and Procedure

Key Components of Real-Time PCR Systems

Real-time PCR systems rely on specialized instrumentation to enable precise thermal cycling and simultaneous fluorescence detection during nucleic acid amplification. The core hardware component is the thermal cycler, which uses Peltier-based heating and cooling elements to achieve rapid and accurate temperature transitions essential for the denaturation, annealing, and extension phases of PCR. These cyclers maintain temperature uniformity and precision within ±0.1°C across the reaction block, ensuring reproducible results even in multi-well formats.^[33] Integrated optical modules in modern thermal cyclers facilitate real-time monitoring by positioning the detection system directly above or below the sample block, allowing fluorescence measurements without interrupting the cycling process.^[2] The detection system in real-time PCR instruments comprises excitation sources, emission filters, and sensitive detectors to quantify fluorescence signals proportional to amplified DNA. Excitation is typically provided by light-emitting diodes (LEDs) or lasers, which emit narrow-bandwidth light tailored to specific fluorophores, such as FAM or SYBR Green, enabling excitation at wavelengths around 450–600 nm.^[34] Emission filters selectively pass fluorescence signals while blocking excitation light, supporting multiplex assays with up to five or six color channels in advanced systems for simultaneous detection of multiple targets. Detectors, including charge-coupled devices (CCDs) or photomultiplier tubes (PMTs), capture these signals with high sensitivity, often achieving dynamic ranges exceeding six orders of magnitude to detect low-abundance templates.^[35] Consumables form the disposable foundation of real-time PCR workflows, optimized for optical clarity and thermal efficiency to minimize signal interference and evaporation. Standard formats include 96-well or 384-well optical plates made from polypropylene, designed for 10–50 µL reaction volumes and compatible with automated liquid handlers. These plates are sealed with optically transparent adhesive films or caps to prevent cross-contamination and ensure uniform light transmission during detection. Master mix kits, pre-formulated with thermostable DNA polymerase, dNTPs, buffers, and sometimes fluorescent dyes or probes, simplify setup and standardize reaction conditions across experiments.^[36]^[37]^[38] As of 2025, advancements in real-time PCR systems emphasize portability and integration for point-of-care applications, particularly through microfluidic chips that miniaturize reactions into channels for reduced reagent use and faster cycling times. These devices often incorporate on-chip sample preparation modules, such as lysis and purification steps, powered by compact Peltier elements and battery-operated optics, enabling deployment in field settings like clinics or remote diagnostics without laboratory infrastructure. Examples include compact systems using PCB-based disposable chips with open-platform CMOS cameras for fluorescence detection, supporting pathogen assays with limits of detection comparable to benchtop systems.^[39]

Preparation

The preparation phase of a real-time PCR experiment begins with nucleic acid template extraction, where genomic DNA or RNA is isolated from samples using methods such as silica-based columns or magnetic beads to yield high-purity templates suitable for amplification.^[40] Typical input amounts range from 1 to 100 ng of DNA or RNA per reaction to ensure sensitive detection without inhibition.^[41] Next, primers and probes are designed with specific parameters: primers should have a melting temperature (Tm) of 58-60°C, a GC content of 40-60%, and generate amplicons of 75-150 base pairs to optimize efficiency and specificity in real-time detection.^[42] For probe-based assays, hydrolysis probes like TaqMan are positioned to span the amplicon junction, with a Tm 8-10°C higher than the primers.^[43] Master mix assembly follows, typically using a commercial 2X PCR master mix containing Taq DNA polymerase, dNTPs, MgCl₂, and buffer to minimize pipetting errors and ensure consistency across reactions.^[44] For a standard 20-25 µL reaction volume, add 10-12.5 µL of 2X master mix, 0.2-1 µM forward and reverse primers, 0.1-0.25 µM probe (if applicable), and nuclease-free water to volume, excluding the template which is added later.^[45] Calculations should account for the total number of reactions plus 10% extra volume to compensate for pipetting variability.^[46]

Setup

Reactions are pipetted into optical-grade multiwell plates (e.g., 96-well or 384-well formats) compatible with the real-time instrument to allow fluorescence transmission, with each well receiving 1-5 µL of template solution added last to avoid contamination.^[40] Essential controls include no-template controls (NTCs) to detect contamination, positive controls with known template quantities for validation, and negative controls like reverse-transcription-minus (RT-minus) for RNA assays to identify genomic DNA carryover.^[47] Plates are sealed with optical adhesive film, briefly centrifuged at 200-500 × g to eliminate air bubbles, and loaded into the instrument.^[48] Instrument programming involves setting the thermal cycling profile: an initial denaturation at 95°C for 2-10 minutes to activate the hot-start polymerase, followed by 40-45 cycles of denaturation at 95°C for 15 seconds and annealing/extension at 55-60°C for 30-60 seconds, with data acquisition during the annealing step for fluorescence measurement.^[41] Ramp rates are typically programmed at 1-2°C per second to balance speed and specificity, and a final hold at 4-10°C for post-run stability.^[49]

Execution

The experiment is initiated by placing the plate in the real-time PCR instrument, which performs automated thermal cycling while monitoring fluorescence at each cycle to track amplification in real time.^[40] Detection occurs via intercalating dyes or sequence-specific probes, with the instrument's optics exciting and detecting emitted light proportional to amplified product.^[25] The run duration is approximately 1-2 hours, depending on cycle number and ramp rates, after which the plate is removed and stored at 4°C if needed.^[41]

Quality Checks

All reactions should be performed in at least technical triplicates to assess reproducibility and reduce variability.^[11] Efficiency validation follows MIQE guidelines, requiring amplification efficiencies of 90-110% determined from standard curves, along with checks for Ct variability less than 0.5 cycles across replicates.^[50] Additional quality metrics include baseline subtraction in software and confirmation of no amplification in NTCs to ensure assay reliability.^[51]

Data Analysis

Amplification Curve Interpretation

The amplification curve in real-time PCR represents the accumulation of fluorescent signal over successive cycles, typically plotted with cycle number on the x-axis and fluorescence intensity (often on a logarithmic scale for the y-axis) to visualize the sigmoidal progression of product formation.^[52] This curve is fundamental for quantifying initial template concentration, as it reflects the kinetics of DNA amplification monitored in real time.^[53] The curve consists of three distinct phases. The baseline phase occurs in the early cycles, usually cycles 1–10 or 0–15, where fluorescence remains low and relatively constant, dominated by background noise from the detection system rather than amplified product.^[53] During this period, template concentration is minimal, and any signal adjustment, such as baseline correction, subtracts this background to normalize subsequent data.^[52] The exponential phase follows, appearing linear on a logarithmic scale, where amplification efficiency is maximal (ideally approaching 100%) as reagents remain in excess, leading to a doubling of product each cycle.^[8] Finally, the plateau phase emerges in later cycles (typically after 30–40), where the signal flattens due to reagent depletion, enzyme inactivation, or product saturation, halting further exponential growth.^[52] To extract quantitative data, a threshold line is established above the baseline, often automatically by software or manually adjusted, typically at 10 times the standard deviation of baseline fluorescence to ensure it intersects the exponential phase reliably.^[54] The cycle threshold (Ct), also termed quantification cycle (Cq), is defined as the fractional cycle number where the amplification curve first crosses this threshold, providing a direct measure inversely proportional to the starting template amount—lower Ct values indicate higher initial concentrations.^[55] Baseline correction is crucial prior to threshold setting, as it adjusts for instrument noise or optical artifacts by subtracting the average fluorescence from early cycles (e.g., 3–15), ensuring accurate Ct determination.^[53] For relative quantification, Ct values are normalized using a reference gene or housekeeping gene assumed to have stable expression across samples. The difference is calculated as ΔCt = Ct_target - Ct_reference, which accounts for variations in sample input, reverse transcription efficiency, or reaction conditions, enabling comparison of target gene levels between experimental and control groups.^[56] This ΔCt forms the basis for methods like the 2^{-ΔΔCt} approach, where further differencing against a calibrator sample yields fold-change estimates.^[56] Amplification curves can reveal artifacts that compromise data quality. Inhibitors, such as humic acids or immunoglobulins from sample carryover, reduce polymerase activity or quench fluorescence, resulting in delayed Ct values, flattened exponential slopes, or incomplete amplification without reaching plateau.^[57] Conversely, contamination from extraneous DNA or primer dimers causes premature signal rise, yielding erroneously low Ct values and early exponential onset, often mimicking high-template positives.^[25] These issues underscore the need for rigorous controls, such as no-template reactions, to validate curve integrity.^[52]

Melting Curve Analysis

Melting curve analysis is a post-amplification technique in real-time PCR that evaluates the specificity and purity of amplified products by monitoring their thermal dissociation. Following the completion of PCR cycles, the reaction mixture is subjected to a gradual temperature increase from approximately 60°C to 95°C at a rate of 0.1 to 0.5°C per second, during which fluorescence is continuously measured to track the release of intercalating dyes from double-stranded DNA as it denatures into single strands.^[35]^[58] The resulting melting curve plots fluorescence intensity against temperature, revealing the melting temperature (Tm)—the point of maximum DNA dissociation—as the peak in the negative derivative plot (-dF/dT versus temperature). A single, sharp peak in this derivative plot indicates a homogeneous amplicon with high specificity, while multiple peaks suggest the presence of non-specific products or primer artifacts. The Tm value is primarily determined by the amplicon's length, GC content, and sequence, allowing differentiation of products with Tm differences as small as 2°C.^[58] This analysis is particularly valuable for validating reactions using intercalating dyes like SYBR Green, where it distinguishes primer dimers—short, non-specific artifacts with low Tm values around 70°C—from target amplicons exhibiting higher Tm values, typically around 85°C. By identifying such contaminants without requiring gel electrophoresis, melting curve analysis enhances the reliability of quantitative results and reduces false positives in downstream applications.^[58]^[59] High-resolution melting (HRM) represents an advanced iteration of this technique, employing precision instrumentation to detect minute Tm shifts of 0.1°C or less, enabling applications such as single nucleotide polymorphism (SNP) genotyping directly from PCR products. In HRM, saturating DNA-binding dyes like LCGreen are used during amplification, followed by high-density fluorescence sampling during the melt phase, which generates normalized and difference curves for variant calling without post-PCR processing. This method has been demonstrated to accurately genotype both heterozygous and homozygous SNPs in amplicons up to 544 bp, offering a rapid, closed-tube alternative to probe-based assays.^[60]

Quantitative Modeling and Efficiency

Quantitative modeling in real-time PCR relies on mathematical frameworks to determine amplification efficiency and quantify target nucleic acid copy numbers, ensuring assay reliability and reproducibility. Efficiency (E), expressed as the fold increase in target copies per cycle, is calculated from the slope of a standard curve generated by plotting threshold cycle (Ct) values against the logarithm of known template concentrations from serial dilutions. The formula is E = 10^{-1/\text{slope}}, where an ideal slope of -3.32 corresponds to 100% efficiency (E = 2), and slopes between -3.1 and -3.6 indicate 90-110% efficiency, validating optimal primer and reaction conditions.^[50] Absolute quantification estimates the initial copy number of the target using the efficiency-derived standard curve. Serial dilutions of a reference standard with known copy numbers (e.g., purified plasmid DNA) produce the curve, allowing interpolation of unknown samples via the equation \text{Copy number} = K \times E^{-\text{Ct}}, where K is the calibration factor determined from the standard's known quantity and Ct. This approach provides precise absolute values, such as copies per microliter, essential for applications requiring exact pathogen loads or viral titers.^[61] Relative quantification compares target gene expression across samples without standards, using the Livak-Schmittgen method to compute fold changes normalized to housekeeping genes. The formula \text{Fold change} = 2^{-\Delta\Delta\text{Ct}} assumes near-100% efficiency, where \Delta\text{Ct} = \text{Ct}_{\text{target}} - \text{Ct}_{\text{reference}} and \Delta\Delta\text{Ct} = \Delta\text{Ct}_{\text{sample}} - \Delta\text{Ct}_{\text{control}}, yielding normalized ratios like upregulated or downregulated expression levels. This method simplifies analysis for gene expression studies by accounting for input variations through endogenous controls.^[56] To validate these models, statistical analysis incorporates technical and biological replicates to compute confidence intervals, typically at 95%, reflecting variability in Ct values. Error sources, such as pipetting variance (contributing up to 1-2% coefficient of variation in replicates), are minimized through multiple runs, with broader intervals indicating potential inhibition or inconsistency; software tools often derive these from standard deviations of triplicate Ct measurements.

Applications

Gene Expression Analysis

Real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a cornerstone technique for quantifying messenger RNA (mRNA) levels to assess gene expression in eukaryotic cells, enabling precise measurement of transcript abundance across diverse biological contexts.^[62] The process begins with the conversion of RNA to complementary DNA (cDNA) using reverse transcriptase enzymes, typically employing random hexamers as primers to initiate synthesis from the entire RNA population, ensuring comprehensive coverage without bias toward specific sequences. This step is critical as it transforms unstable RNA into stable cDNA suitable for subsequent PCR amplification and real-time detection. RT-qPCR protocols are implemented in either one-step or two-step formats, each with distinct advantages for gene expression studies. In the one-step approach, reverse transcription and qPCR amplification occur in a single tube using a combined reverse transcriptase and DNA polymerase, minimizing handling to reduce contamination risk and enabling high-throughput analysis of multiple targets from limited RNA samples. Conversely, the two-step method separates cDNA synthesis from qPCR, allowing greater flexibility for optimizing reverse transcription conditions, archiving cDNA for repeated use, and multiplexing different gene targets in downstream assays, though it involves more pipetting steps that can introduce variability. Relative quantification of gene expression, often using methods like the 2^(-ΔΔCt) approach, compares target mRNA levels to a calibrator sample after normalization, providing fold-change estimates that reveal regulatory dynamics.^[56] Accurate interpretation of RT-qPCR data requires normalization to endogenous reference genes to account for variations in RNA input, reverse transcription efficiency, and sample quality. Common reference genes include glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin (ACTB), selected for their presumed stable expression across conditions, though validation is essential as their stability can vary by tissue or perturbation.^[63] Algorithms such as geNorm, which calculates pairwise variation to identify the optimal set of multiple reference genes via geometric averaging, and NormFinder, which estimates expression stability by modeling inter- and intra-group variances, facilitate rigorous selection of the most reliable normalizers for a given experimental design. In applications, RT-qPCR excels in profiling differential gene expression in cancer research, where it quantifies upregulated oncogenes or downregulated tumor suppressors in tumor versus normal tissues, aiding biomarker discovery and therapeutic response monitoring.^[64] For developmental biology, time-course studies leverage RT-qPCR to track dynamic mRNA changes during embryogenesis or differentiation, revealing temporal regulation of key pathways such as those governing cell fate decisions in model organisms like Xenopus.^[65] However, challenges persist, including RNA instability due to ubiquitous RNases, which necessitates immediate extraction and storage protocols, and variable reverse transcription efficiency, typically ranging from 70% to 90% depending on RNA quality and enzyme performance, potentially biasing quantification toward abundant transcripts.^[66] Adherence to guidelines like MIQE ensures reproducibility by mandating reporting of efficiency metrics and validation steps.

Pathogen Detection and Diagnostics

Real-time polymerase chain reaction (PCR) plays a pivotal role in pathogen detection by enabling the rapid identification and quantification of microbial nucleic acids in clinical samples, facilitating timely diagnosis of infectious diseases. This technique targets specific genetic sequences from bacteria and viruses, offering high sensitivity and specificity that surpass traditional culture methods, particularly for fastidious or non-culturable organisms.^[67] In clinical settings, it supports both qualitative detection for initial diagnosis and quantitative analysis for monitoring disease progression and treatment efficacy.^[68] For bacterial pathogens, real-time PCR is widely used to detect Mycobacterium tuberculosis in respiratory samples, where it identifies the IS6110 insertion sequence or other conserved regions, achieving sensitivities of 85-95% in smear-positive cases.^[69] In viral diagnostics, it quantifies human immunodeficiency virus (HIV) type 1 RNA in plasma to measure viral load, with assays like the Abbott RealTime HIV-1 detecting as few as 40 copies per milliliter and providing linear quantification up to 10 million copies per milliliter for antiretroviral therapy monitoring.^[68] Multiplex real-time PCR formats allow simultaneous detection of multiple pathogens in co-infection scenarios, such as respiratory panels targeting bacteria like Streptococcus pneumoniae alongside viruses like influenza, reducing diagnostic turnaround time to under 2 hours while maintaining specificity above 95%.^[70] A prominent application emerged during the COVID-19 pandemic, where reverse transcription real-time PCR (RT-PCR) assays targeted the envelope (E) and nucleocapsid (N) genes of SARS-CoV-2 in upper respiratory specimens, enabling detection limits as low as 3.9 copies per reaction for the E gene.^[71] These assays, including WHO-recommended protocols and emergency use authorized kits like the CDC 2019-nCoV panel approved in early 2020, quantify viral loads from 10^3 to 10^8 copies per milliliter, supporting both diagnosis and variant tracking.^[72]^[73] The diagnostic workflow typically begins with sample collection, such as nasopharyngeal swabs for respiratory pathogens or blood for systemic infections like HIV, followed by nucleic acid extraction and amplification in a thermocycler.^[74] Limits of detection (LOD) for most assays range from 10 to 100 copies per reaction, depending on the target and extraction efficiency, allowing reliable identification even in low-viral-load scenarios.^[75] Post-amplification, fluorescence data is analyzed to confirm positivity via cycle threshold values. Beyond individual diagnostics, real-time PCR has expanded into epidemiological surveillance, particularly through wastewater monitoring for early outbreak detection since 2020.^[76] This approach concentrates viral RNA from sewage samples and applies RT-PCR to track community-level prevalence of pathogens like SARS-CoV-2, providing unbiased insights into transmission trends weeks before clinical cases surge.^[77] Such sentinel systems have been implemented globally, correlating wastewater signals with hospitalization rates and informing public health responses.^[78]

Genotyping and GMO Identification

Real-time polymerase chain reaction (qPCR) is widely employed for single nucleotide polymorphism (SNP) genotyping, enabling the discrimination of alleles through allele-specific probes such as TaqMan assays, which utilize fluorogenic probes that hybridize to target sequences and are cleaved during PCR amplification to produce distinct fluorescent signals for each allele.^[79] These assays achieve high accuracy in single-base discrimination, with validation studies demonstrating 100% concordance in genotyping challenging loci like CYP2D6 variants.^[79] For simpler variants, high-resolution melting (HRM) analysis following qPCR amplification allows genotyping by detecting differences in melting temperatures of DNA duplexes, offering a cost-effective alternative that requires only unlabeled primers and a DNA intercalating dye.^[80] In genetically modified organism (GMO) identification, qPCR targets common transgenic elements such as the Cauliflower mosaic virus (CaMV) 35S promoter or the nopaline synthase (NOS) terminator to detect and quantify engineered sequences in food and feed samples.^[81] These methods meet regulatory requirements, including the European Union's 0.9% threshold for labeling authorized GMOs per ingredient, with sensitivities often reaching 0.1% or lower for unauthorized events to ensure compliance and safety monitoring.^[81]^[82] qPCR also supports forensic and environmental applications, such as species identification through DNA barcoding of mitochondrial genes like cytochrome c oxidase subunit I (COI), where real-time detection confirms the presence of target species in trace samples from wildlife trade or biodiversity surveys.^[83] Copy number variation (CNV) assays using qPCR, often with TaqMan probes, quantify genomic duplications or deletions relative to a reference gene, providing insights into structural variants associated with traits in agriculture or disease susceptibility.^[84] Multiplexing in qPCR genotyping accommodates up to four targets simultaneously for haplotype analysis, as demonstrated in assays for pharmacogenetic loci like CYP2C9, enhancing throughput without compromising specificity.^[85] Integration with digital PCR variants offers absolute quantification of genotypes, particularly useful for low-abundance variants in heterogeneous samples.^[86]

Advantages and Limitations

Benefits Compared to End-Point PCR

Real-time PCR provides superior quantitative precision compared to end-point PCR by monitoring amplification during the exponential phase of the reaction, allowing accurate measurement of the initial template concentration through fluorescence signals proportional to product accumulation, whereas end-point PCR relies on semi-quantitative analysis via gel electrophoresis after the reaction reaches a plateau where differences in starting material are obscured.^[87]^[8] This enables real-time PCR to achieve precise quantification using cycle threshold (Ct) values that correlate linearly with log input amounts, offering a dynamic range often spanning five to seven orders of magnitude.^[88] In terms of speed and throughput, real-time PCR typically completes a run in 1-2 hours, including automated detection, eliminating the need for post-amplification steps like gel preparation and electrophoresis that extend end-point PCR workflows to 3-4 hours or more, while automation further reduces hands-on time from hours to minutes per sample.^[89]^[8] This efficiency supports high-throughput processing of hundreds of samples in a single run on modern instruments, enhancing laboratory productivity for routine diagnostics and research.^[90] Real-time PCR demonstrates enhanced sensitivity and specificity, capable of detecting fewer than 10 target copies per reaction due to its ability to signal amplification early in the process, surpassing the detection limits of end-point methods that often require visible bands on gels representing at least 10^9 molecules.^[91] The closed-tube format of real-time PCR minimizes carryover contamination by preventing aerosolization of amplicons during handling, as amplification and detection occur without opening reaction vessels, thereby improving specificity and reducing false positives compared to open-tube end-point protocols.^[88]^[8] The versatility of real-time PCR extends to both absolute and relative quantification methods, where absolute quantification uses standard curves for direct copy number determination and relative quantification compares targets to reference genes for normalized expression analysis, capabilities not feasible with end-point PCR's qualitative outputs.^[87] It also supports multiplexing for simultaneous detection of multiple targets using spectrally distinct probes, increasing informational yield per reaction without compromising accuracy.^[92] Although initial instrument costs are higher, real-time PCR offers long-term savings through reduced reagent use, automation, and elimination of labor-intensive post-PCR analyses.^[8]

Common Challenges and Error Sources

Real-time PCR assays are susceptible to inhibition by contaminants commonly present in biological samples, such as heme from blood and humic acids from soil or environmental extracts, which can bind to DNA polymerase or interfere with amplification, resulting in increased cycle threshold (Ct) values by 2-5 cycles and reduced sensitivity.^[57]^[93] These inhibitors often co-purify with nucleic acids during extraction, leading to incomplete amplification or false negatives; for instance, heme at concentrations as low as 1% v/v blood can significantly impair Taq polymerase activity.^[94] Mitigation strategies include using specialized extraction kits with silica-based or magnetic bead purification to remove inhibitors, which can recover up to 90% of target DNA while minimizing loss, or adding bovine serum albumin (BSA) at 0.2-1 mg/mL to the reaction mix, as BSA binds inhibitors and enhances polymerase tolerance without altering specificity.^[57]^[95] Suboptimal primer design remains a primary cause of amplification inefficiency in real-time PCR, where issues like secondary structures, primer-dimers, or non-specific binding can reduce efficiency below 80%, leading to inaccurate quantification and skewed standard curves with slopes deviating from the ideal -3.1 to -3.6.^[96]^[97] For example, primers forming stable hairpins or dimers compete with target annealing, particularly at lower temperatures, resulting in delayed Ct values and variable melt curves. Validation through serial dilution standard curves is essential to assess efficiency, where plotting Ct against log(input) reveals deviations; efficiencies under 90% often necessitate redesign using tools that predict secondary structures and optimize melting temperatures (Tm) around 60°C.^[61]^[98] Inter-run variability in real-time PCR, often exceeding 5% coefficient of variation (CV) for Ct values, arises from pipetting inconsistencies, thermal gradients across the reaction plate, or fluctuations in reagent quality, compromising reproducibility across experiments.^[99]^[100] Pipetting errors, such as air bubbles or uneven volumes, can introduce up to 10% variation in low-concentration samples, while edge effects from thermal unevenness in cyclers amplify this in multi-well formats. Implementing robotic liquid handling systems reduces manual pipetting variability to below 2% CV, and including no-template controls (NTC) alongside positive standards on each plate helps normalize for run-specific drifts, as recommended in established guidelines for qPCR reporting.^[101]^[50] A key limitation of real-time PCR is its inability to differentiate between nucleic acids from live and dead cells, as it amplifies DNA or RNA regardless of viability, potentially overestimating pathogen loads in diagnostics without additional steps like viability dyes (e.g., propidium monoazide or ethidium monoazide), which penetrate compromised membranes to block amplification from dead cells.^[24] Probe-based detection methods, while offering higher specificity than intercalating dyes, incur higher costs due to the need for fluorescently labeled oligonucleotides (up to 10-fold more expensive per reaction) and require precise optimization to avoid multiplexing interference. In reverse transcription qPCR (RT-qPCR), RNA degradation from RNases or improper storage further exacerbates errors, reducing template integrity and efficiency by up to 50% if samples are not handled with stabilizers like RNasin.^[102]^[103]

References

[1]
Polymerase Chain Reaction (PCR) - StatPearls - NCBI Bookshelf - NIH
Jul 7, 2025 · Real-time PCR is a widely used method for analyzing small DNA segments, offering advantages such as elimination of post-PCR processing, use of ...Introduction · Testing Procedures · Results, Reporting, and... · Clinical Significance
[2]
What is Real-Time PCR (qPCR)? - Bio-Rad
In real-time PCR, the accumulation of amplification product is measured as the reaction progresses, in real time, with product quantification after each cycle.What Is Real-Time PCR? · How Does Real-Time PCR... · RNA Isolation
[3]
The real-time polymerase chain reaction - PubMed
This new technique is a refinement of the original Polymerase Chain Reaction (PCR) developed by Kary Mullis and coworkers in the mid 80:ies.
[4]
PCR past, present and future - PMC - PubMed Central
Aug 20, 2020 · Here we review the history of PCR development and the technologies that have evolved from the original PCR method.
[5]
Simultaneous Amplification and Detection of Specific DNA Sequences
Apr 1, 1992 · 1992. Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications. Submitted. Higuchi, R. 1989.
[6]
Kinetic PCR Analysis: Real-time Monitoring of DNA Amplification ...
Sep 1, 1993 · Higuchi, R., Dollinger, G., Walsh, P.S. and Griffith, R. 1992. Simultaneous amplification and detection of specific DNA sequences. Bio/ ...Missing: paper | Show results with:paper
[7]
Real-time polymerase chain reaction - QIAGEN
Real-time PCR, or qPCR, is a molecular biology technique for quantifying nucleic acids by monitoring amplification through fluorescence.
[8]
Real-Time Polymerase Chain Reaction: Current Techniques ...
Real-time PCR is a variation of the PCR assay to allow monitoring of the PCR progress in actual time. PCR itself is a molecular process used to enzymatically ...
[9]
Simultaneous amplification and detection of specific DNA sequences
We have enhanced the polymerase chain reaction (PCR) such that specific DNA sequences can be detected without opening the reaction tube.Missing: seminal paper
[10]
A Basic Guide to Real Time PCR in Microbial Diagnostics - NIH
Feb 2, 2017 · Real time PCR (quantitative PCR, qPCR) is now a well-established method for the detection, quantification, and typing of different microbial agents.Introduction · Qpcr Parameters In Microbial... · Precision Of Qpcr
[11]
The MIQE guidelines: minimum information for publication ... - PubMed
MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial ...
[12]
New COVID-19 Rapid-test Technology Performs PCR Faster than ...
Jul 25, 2022 · Researchers at Columbia Engineering and Rover Diagnostics team up to develop a low-cost, portable platform that gives RT-PCR results in 23 minutes that match ...<|control11|><|separator|>
[13]
Real-time PCR Instruments and Reagents 2025-2033 Trends
Jun 19, 2025 · Miniaturization and portability: Development of smaller, more portable instruments ... COVID-19 diagnostic tests led to significant market growth.
[14]
What is Real-Time PCR (qPCR)? - Bio-Rad
The measured fluorescence is proportional to the total amount of amplicon; the change in fluorescence over time is used to calculate the amount of amplicon ...What Is Real-Time PCR? · How Does Real-Time PCR... · RNA Isolation
[15]
Essentials of Real-Time PCR | Thermo Fisher Scientific - ES
Real-time polymerase chain reaction (qPCR) is the ability to monitor the progress of the PCR as it occurs in real time. Data is therefore collected ...
[16]
Kinetic PCR analysis: real-time monitoring of DNA amplification ...
We describe a simple, quantitative assay for any amplifiable DNA sequence that uses a video camera to monitor multiple polymerase chain reactions (PCRs) ...Missing: paper | Show results with:paper
[17]
Polymerase chain reaction (PCR) - Hamamatsu Photonics
Our photodetectors and imaging systems are used in PCR machines and equipment that require precise detection and analysis of fluorescence signals.
[18]
Understanding Fluorescent Quantitative PCR (qPCR) Curves
Baseline. The baseline refers to the region in the amplification curve where the fluorescence signal remains relatively stable due to the background ...
[19]
How to Interpret qPCR Amplification Curves - Patsnap Synapse
May 9, 2025 · The curve typically consists of three phases: the baseline phase, the exponential phase, and the plateau phase. Each phase offers specific ...
[20]
What is Real-Time PCR (qPCR)? - Bio-Rad
The amplification plot shows two phases, an exponential phase followed by a non-exponential plateau phase. During the exponential phase, the amount of PCR ...
[21]
The Purpose of ROX Reference Dye in Real-Time PCR (qPCR)
Jun 3, 2021 · ROX dye is a low-effort tool that provides a way to diagnose problems with a qPCR run and normalize data to make it more precise.
[22]
Normalization of Real-Time PCR Fluorescence Data with ROX ...
ROX passive reference dye is an inert additive that provides a constant fluorescent signal for sample normalization throughout the real-time qPCR assay.
[23]
[PDF] Using Passive Reference Dyes for Normalization and ... - Illumina
Adding ROX to the qPCR Master Mix is the most popular incarnation of passive reference normalization; it is so common, in fact, that passive reference ...
[24]
A Basic Guide to Real Time PCR in Microbial Diagnostics - Frontiers
Feb 1, 2017 · These two approaches were developed in parallel (Holland et al., 1991; Higuchi et al., 1992) and are used in pathogen detection; however, probe- ...
[25]
https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/qpcr/quantitative-pcr
[26]
Methods to determine limit of detection and limit of quantification in ...
Apr 29, 2017 · Here we present methods to determine the limit of detection (LoD) and the limit of quantification (LoQ) as applicable to qPCR.
[27]
Quantitative PCR (qPCR) Data Analysis | Thermo Fisher Scientific - US
Real-Time PCR instruments can support a large dynamic range. For example, a 20 µL PCR volume should provide a dynamic range potential of approximately 9 logs.
[28]
https://doi.org/10.3390/genes13122387
[29]
https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/qpcr/sybr-green-qpcr
[30]
SYBR Green I - an overview | ScienceDirect Topics
The fluorescence of the SYBR® Green I dye molecules increases up to 1000-fold after binding to double-stranded DNA (Morrison et al., 1998; Wittwer et al., 1997 ...
[31]
Molecular Beacons: Probes that Fluoresce upon Hybridization - Nature
Mar 1, 1996 · We have developed novel nucleic acid probes that recognize and report the presence of specific nucleic acids in homogeneous solutions.
[32]
qPCR Protocol for SNP Genotyping - Thermo Fisher Scientific
SNP genotyping using fluorescent dual-labeled probe technology such as TaqMan ® probes requires two PCR primers as well as two allele-specific probes that ...Introduction · Materials · General Protocol · Support Protocol 4 - Real Time...
[33]
Real-time PCR thermal cyclers - Inagea
Peltier system to control temperature, precision and uniformity of ±0.1 °C; Gradient range: 1–40 ºC; Software functions: qualitative analysis, absolute and ...
[34]
Introduction to qPCR System | Bio-Rad
Six filtered LEDs and six filtered photodiodes are used for excitation and detection. Light is filtered in a narrow bandwidth, ensuring that only data from the ...
[35]
[PDF] Real-time PCR handbook - Gene-Quantification
In real-time PCR, the amount of DNA is measured after each cycle via fluorescent dyes that yield increasing fluorescent signal in direct proportion to the ...<|control11|><|separator|>
[36]
MicroAmp™ Optical 96-Well Reaction Plate 10 Plates | Buy Online
In stock $86 deliveryContains ten 96-well reaction plates. Will require either caps or full plate cover to seal (not included). May be stored at room temperature.
[37]
Eppendorf twin.tec® <i>real-time</i> PCR Plates - qPCR Plates
Films and foils provide your PCR plate with a tight seal. Optically clear films are the ideal sealing material of choice for qPCR and other optical assays.
[38]
[PDF] StepOne and StepOnePlus Real-Time PCR Systems - Fisher Scientific
A complete line of reagents, including TaqMan master mixes,. SYBR Green master mixes, and consumables, including. 96-well plates, is available for use with the ...
[39]
Development of a portable multi-step microfluidic device for point-of ...
Jan 22, 2025 · This study aims to develop a cost-effective, portable, and practical microfluidic nucleic acid PoCT device for rapid virus diagnosis.
[40]
A Finger-Actuated Microfluidic System for Point-Of-Care Detection of ...
Aug 18, 2025 · In this study, we developed a finger-actuated visual microfluidic chip with a colorimetric detection system based on reverse transcriptase loop- ...
[41]
A Compact Real-Time PCR System for Point-of-Care Detection ...
We present a compact and cost-effective real-time PCR system designed for point-of-care testing (POCT), utilizing a PCB-based disposable chip and an open- ...
[42]
Real-Time PCR (qPCR) Basics - Thermo Fisher Scientific
Real-time PCR combines PCR amplification and detection into a single step. This eliminates the need to detect products using gel electrophoresis.Efficiency of Real-Time PCR · Benefits of FAST Real-Time PCR · US
[43]
PCR Cycling Parameters—Six Key Considerations for Success
May 16, 2016 · If the DNA input is fewer than 10 copies, up to 40 cycles may be required to produce a sufficient yield. More than 45 cycles is not recommended ...
[44]
[PDF] The Ultimate qPCR Assay Design Guide - Bio-Rad
8 TIPS FOR DESIGNING PRIMERS. 1. Design amplicons that are 70–150 bp long. 2. Design primers that have a GC content of 40–60%. 3. Avoid sequences with long (4 ...
[45]
https://www.promega.com/-/media/files/resources/protocols/product-information-sheets/g/pcr-master-mix-protocol.pdf
[46]
[PDF] PCR Master Mix (2X), #K0171 - Thermo Fisher Scientific
PCR Master Mix is a 2X concentrated solution of Taq. DNA polymerase, dNTPs and all other components required for PCR, except DNA template and primers. This pre- ...
[47]
[PDF] pcr-master-mix-protocol.pdf - Promega Corporation
PCR Master Mix is a premixed, ready-to-use solution containing Taq DNA polymerase, dNTPs, MgCl2 and reaction buffers at optimal concentrations for efficient ...
[48]
https://pmc.ncbi.nlm.nih.gov/articles/PMC7786041/
[49]
MIQE Guidelines | Thermo Fisher Scientific - US
What are MIQE guidelines? MIQE guidelines are a set of standards aimed at improving the quality and transparency of quantitative real-time PCR experiments.<|separator|>
[50]
qPCR and qRT-PCR analysis: Regulatory points to consider ... - NIH
Reactions for qPCR are loaded into a standard 96-well plate, sealed with optical caps or film, and centrifuged to remove any droplets from the side or top of ...
[51]
Insights into qPCR: Protocol, Detection Methods, and Analysis
Nov 8, 2023 · Key qPCR cycle conditions ; Denaturation: DNA strands separate at a high temperature (94-98°C). ; Annealing: Primers bind to the specific target ...
[52]
Minimum Information for Publication of Quantitative Real-Time PCR ...
MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial ...Abstract · Conceptual Considerations · Research vs Diagnostic... · qPCR
[53]
MIQE and RDML Guidelines - Bio-Rad
In this section we discuss MIQE guidelines that define the minimum information that needs to be provided when publishing qPCR experiments.Minimum Information for... · Practical MIQE Tools for...
[54]
A new method for robust quantitative and qualitative analysis of real ...
The real-time PCR curve is a fluorescence response with a roughly sigmoidal shape that correlates to the growth of amplified product during the PCR ...
[55]
qPCR Analysis | Bio-Rad
Baseline occurs from cycles 0 through 10, where the initial concentration of template is low. At this level, the fluorescence intensity is too low to be ...
[56]
Accurate quantification of DNA using on-site PCR (osPCR) by ... - NIH
May 17, 2023 · The ct values are then calculated using a threshold of 10 times standard deviation of baseline cycles. For all other samples, we use cycles ...
[57]
Use and Misuse of Cq in qPCR Data Analysis and Reporting - NIH
In the analysis of quantitative PCR (qPCR) data, the quantification cycle (Cq) indicates the position of the amplification curve with respect to the cycle axis.
[58]
Analysis of relative gene expression data using real-time ... - PubMed
The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments.
[59]
PCR inhibition in qPCR, dPCR and MPS—mechanisms and solutions
Feb 12, 2020 · In qPCR, the slopes of the amplification curves were not affected by IgG, but delayed amplification (higher Cq values) was observed. This ...Missing: artifacts | Show results with:artifacts
[60]
Product differentiation by analysis of DNA melting curves during the ...
A microvolume fluorometer integrated with a thermal cycler was used to acquire DNA melting curves during polymerase chain reaction by fluorescence monitoring.
[61]
Trust your SYBR Green qPCR Data - Thermo Fisher Scientific
Nov 3, 2022 · The nonspecific nature of SYBR Green as a fluorophore makes melt-curve analysis one of the very few ways to confirm that target-specific ...
[62]
High-resolution genotyping by amplicon melting analysis ... - PubMed
High-resolution melting analysis of PCR products amplified in the presence of LCGreen can identify both heterozygous and homozygous sequence variants.Missing: HRM seminal paper
[63]
[PDF] Real-Time PCR Applications Guide - Bio-Rad
Determination of the absolute copy number of β-actin in unknown samples A and B using the absolute quantification method. The equation of the linear ...
[64]
Twenty-Five Years of Quantitative PCR for Gene Expression Analysis
May 16, 2018 · This review examines the current state of qPCR for gene expression analysis now that the method has reached a mature stage of development and implementation.
[65]
https://anatomypubs.onlinelibrary.wiley.com/doi/10.1002/dvdy.23972
[66]
Quantitative real-time PCR: a powerful ally in cancer research
Quantitative real-time PCR (qrt-PCR) technology has recently reached a level of sensitivity, accuracy and practical ease that supports its use as a routine ...Missing: seminal paper
[67]
Validation of novel reference genes for RT‐qPCR studies of gene ...
Apr 5, 2013 · In our study, our goal was to identify reference genes for gene expression analysis during Xenopus tropicalis embryonic and post-embryonic ...Results · Rna Extraction And Cdna... · Rt-Qpcr Data Analysis
[68]
Shedding light: The importance of reverse transcription efficiency ...
Feb 12, 2019 · Here we explore the problem of reverse transcription efficiency using digital PCR and the RT method's impact on subsequent data analysis.
[69]
New insight in molecular detection of Mycobacterium tuberculosis
Jun 21, 2024 · In this review, we endeavor to provide an overview of molecular assays for the diagnosis of tuberculosis with their properties (utilization, challenges, and ...
[70]
RealTime HIV-1 Viral Load Assay - Abbott Molecular
The Abbott RealTime HIV-1 assay is an in vitro reverse transcription-polymerase chain reaction (RT-PCR) assay for the quantitation of Human Immunodeficiency ...
[71]
Evaluation of a real-time PCR assay performance to detect ...
May 9, 2024 · RT-PCR itself has been accepted as a robust method for detecting Mycobacterium tuberculosis (MTB), making its implementation highly recommended ...Introduction · Methods · Results · Discussion
[72]
Development and validation of multiplex real-time PCR for ...
Feb 7, 2024 · Our multiplex quantitative RT-PCR assays are fast and simple, but sensitive and specific in detecting six bacterial pathogens of LRTIs and their antimicrobial ...
[73]
Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT ...
... detection end point determined in preliminary dilution experiments. The resulting LOD from replicate tests was 3.9 copies per reaction for the E gene assay ...
[74]
[PDF] Protocol: Real-time RT-PCR assays for the detection of SARS-CoV-2
This protocol uses real-time RT-PCR for SARS-CoV-2 detection targeting two RdRp targets (IP2 and IP4) and an E gene assay, with RNA extracted using NucleoSpin ...
[75]
[PDF] CDC 2019 nCoV Real-Time RT-PCR Diagnostic Panel - FDA
Mar 7, 2023 · A preliminary LoD for each assay was determined testing triplicate samples of RNA purified using each extraction method. The approximate LoD ...
[76]
Multiplex Real-Time Reverse Transcription PCR for Influenza ... - CDC
May 26, 2021 · Multiplex detection of RNA from influenza A virus, influenza B virus, and SARS-CoV-2 can increase testing capacity and reduce use of reagents.<|separator|>
[77]
Direct Comparison of SARS-CoV-2 Analytical Limits of Detection ...
We determined analytical limits of detection for seven SARS-CoV-2 assays using serial dilutions of pooled patient material quantified with droplet digital PCR.
[78]
Wastewater Surveillance for COVID-19 - NCBI
Wastewater surveillance provides a method to passively assess trends in COVID-19 burden in a community unbiased by the availability of testing or test-seeking ...
[79]
Wastewater surveillance of pathogens can inform public health ...
Sep 8, 2022 · Wastewater monitoring has been used to identify SARS-CoV-2 outbreaks and track new variants. This sentinel system should be expanded to monitor other pathogens.Missing: post- | Show results with:post-
[80]
Wastewater-based surveillance as a tool for public health action
Dec 14, 2023 · Wastewater-based surveillance (WBS) has undergone dramatic advancement in the context of the coronavirus disease 2019 (COVID-19) pandemic.
[81]
SNP genotyping using TaqMan® technology: the CYP2D6*17 assay ...
Mar 19, 2015 · The CYP2D6 gene is challenging to genotype due to the highly complex nature of its gene locus. TaqMan® technology is widely used in the clinical and research ...
[82]
Genotyping of single-nucleotide polymorphisms by high-resolution ...
SNP genotyping by high-resolution melting analysis is simple, rapid, and inexpensive, requiring only PCR, a DNA dye, and melting instrumentation.
[83]
A highly sensitive and specific method for the screening detection of ...
Aug 4, 2015 · Different countries have established different thresholds, such as 0.9% in the European Union (EU) and 5% in Japan, whereas the USA has ...
[84]
[PDF] Real-time PCR-based Screening and Quantification of Genetically ...
The threshold for labeling food and feed with authorized GMO content in EU countries was set to 0.9% in April 2004. With regards to the US market, the FDA ...
[85]
Three-gene PCR and high-resolution melting analysis for ... - Nature
Mar 16, 2020 · However, forensic identification of vertebrate tissue relies on sequencing of the mitochondrial cytochrome oxidase I (COI) 'barcode' gene, which ...
[86]
Quantitative Analysis of Copy Number Variants Based on Real-Time ...
The assay is performed on a qPCR instrument and data are collected in real time. The following is a protocol for the assay. Materials. Samples of interest ...
[87]
(PDF) Improved real-time PCR assay for homogeneous multiplex ...
Improved real-time PCR assay for homogeneous multiplex genotyping of four CYP2C9 alleles with hybridization probes ; ing from coding single-nucleotide.
[88]
Digital PCR for Genotype Quantification: A Case Study in a Pasta ...
May 9, 2021 · Digital polymerase chain reaction (dPCR) is a breakthrough technology able to provide an absolute quantification of the target sequence ...Missing: integration counts
[89]
Application of Reverse Transcription-PCR and Real-Time PCR in ...
Real-time PCR allows for the detection of PCR amplification in the exponential growth phase of the reaction and is much more quantitative than traditional RT- ...
[90]
Real-Time PCR in Clinical Microbiology: Applications for Routine ...
The closed system for amplification and detection used with real-time PCR virtually eliminates the amplicon contamination caused by the opening and closing of ...
[91]
Benefits of FAST Real-Time PCR | Thermo Fisher Scientific - US
Under current standard cycling conditions, a typical run takes approximately 2 hours, limiting the number of samples that can be processed to only four runs per ...want to process more samples... · just want the best-performing...
[92]
Clinical Applications of Quantitative Real-Time PCR in Virology - PMC
Real-time PCR provides a highly valuable tool for screening, diagnosing, or monitoring diseases, as well as evaluating medical and therapeutic decision points.
[93]
Detection and monitoring of virus infections by real-time PCR - PMC
Due to the exponential amplification of the target sequence, it has exquisite sensitivity. Less than 10 copies of any transcript can be readily detected, even ...
[94]
Determination of Advantages and Limitations of qPCR Duplexing in ...
Multiplexing of any PCR expands its detection capability as more than one target gene is simultaneously amplified and detected.
[95]
[PDF] Analysis of the Effect of a Variety of PCR Inhibitors on the ...
This study will examine the effects of a variety of PCR inhibitors using the Plexor® HY qPCR assay while monitoring amplification efficiency and slope, CT, and ...
[96]
Pitfalls of Quantitative Real-Time Reverse-Transcription Polymerase ...
There are numerous components within blood and tissue that can inhibit RT-PCR assays. Mammalian blood, especially the heme compound, is well known for ...
[97]
https://pmc.ncbi.nlm.nih.gov/articles/PMC6647679/
[98]
Poor PCR Efficiency | Thermo Fisher Scientific - US
The efficiency of any PCR can be evaluated by performing a dilution series experiment using the target assay. We recommend a 10-fold dilution series.
[99]
Troubleshooting fine‐tuning procedures for qPCR system design - NIH
Fine-tuning involves identifying optimal primer concentrations, avoiding primer hairpins and dimmers, and avoiding binding sites in stable secondary structures.Missing: issues | Show results with:issues
[100]
qPCR primer design revisited - ScienceDirect.com
We provide a concise overview of the main primer-related issues that confront anyone wanting to design a qPCR assay, consider the main criteria that have an ...
[101]
Review The Ultimate qPCR Experiment: Producing Publication ...
This review article describes and illustrates the critical pitfalls and sources of error in qPCR experiments, along with a rigorous, stepwise process.
[102]
Simulation of between Repeat Variability in Real Time PCR Reactions
Besides influences of more obvious factors such as camera noise and pipetting variation, changing efficiencies within and between reactions affect PCR results ...Missing: inter- gradients
[103]
Robotic automation performs a nested RT-PCR analysis for HCV ...
Aug 6, 2025 · This study demonstrated a user-programmed robotic pipetting system could successfully automate a complex PCR assay without contamination. Our ...
[104]
Top Ten Most Common Real-Time qRT-PCR Pitfalls
Degraded or impure RNA can limit the efficiency of the RT reaction and reduce yield. RNA should either be prepared from fresh tissue, or from tissue treated ...
[105]
Recent Methods for the Viability Assessment of Bacterial Pathogens
Sep 16, 2022 · For viability assessments, PI is usually coupled with dyes that can penetrate and stain nucleic acids of live and dead bacteria, thereby ...