Introduction
Microorganisms were traditionally detected by means of culture, microscopy and chemical reactions. Nowadays, Nucleic acid amplification by Polymerase Chain Reaction (PCR) are among the most valuable tools in biological research today and has largely replaced the traditional detection techniques. Scientists in all areas of life science – basic research, biotechnology, medicine, forensics, diagnostics, and more – use PCR techniques in a wide range of applications. For some applications, qualitative (positive or negative) nucleic acid detection is sufficient. Other applications, however, demand a quantitative (number of micro-organisms) analysis. Real-time PCR can be used to meet both demands.
The PCR Principle
Before diving into real-time PCR, it's essential to understand the basic principle of PCR.
PCR is like a molecular photocopier for DNA. It is a laboratory technique used to make millions to billions of copies of a specific segment of DNA. The process uses a few simple ingredients—DNA, primers, a special enzyme (Taq polymerase), and nucleotides—and cycles through changes in temperature to repeatedly copy the target DNA sequence. These repeated heating and cooling steps allow scientists to quickly and precisely amplify a desired piece of DNA, making it easier to study, analyze, or use in various experiments.
The following interactive guide walks you through the key steps of the PCR cycle: denaturation, annealing, and elongation.
The PCR Process
Click the "Next" and "Previous" buttons or use the arrow keys to navigate the key steps of the process.
PCR Amplification Formula
In the slides above the formula copies = 2n is shown. This applies when the PCR-mix contains one DNA template. However, we can assume that at the start of the amplification reaction, the mixture can contain more than one DNA starting template. A reaction with 40 PCR cycles can start for example with 20 viral DNA copies. The formula can then we written as:
N = N0 x 2n
N = final number of DNA copies
N0 = initial number of DNA copies (starting template)
n = number of PCR cycles
For example: N = 20 x 240
QUIZ 1
A PCR mix contains 100 Hepatitis B virus particles at the start. After 30 cycles of PCR, assuming perfect doubling at each cycle, how many total virus copies could result?
Important Note
This calculation assumes 100% PCR efficiency, meaning perfect doubling each cycle. In actual experiments, the number is lower, as factors like reagent depletion and enzyme decay reduce yield during later cycles. So, "up to" really means an estimated theoretical maximum, but practical results are always less due to real-world limitations.
If the reaction efficiency (E) is less than 100% (which is always the case in real experiments), the formula is:
N = N0 x (1+E)n
Where E = the amplification efficiency per cycle (for 100% E = 1 efficiency; for 90% efficiency, E = 0.9), usually represented as a decimal.
Using the examples in the slides, if the reaction has an efficiency of 90%, the formula would be:
N = 100 x 1.930
What is Real-time PCR?
What are the differences between conventional PCR as explained above, and real-time PCR? In conventional PCR, the amplified DNA product is detected in an end-point analysis. An example is visualizing the amplified DNA with Ethidium Bromide on an agarose gel.
In real-time PCR, the accumulation of the amplified DNA is measured as the reaction progresses, in real-time. The amplified DNA is detected by the inclusion of a fluorescent reporter molecule. The fluorescence increases by each step by the amount of amplified DNA.
The amplification process has several stages that are presented in the next block. These stages are important to remember.
Real-time PCR Amplification Curve Analysis
Click the "Next" and "Previous" buttons or use the arrow keys to learn about the different phases of real-time PCR amplification.
At each amplification cycle a 'photo' is taken of the samples. Of each sample on a plate the amount of fluorescent light is measured, emitted by fluorescent reporter molecules. Next, we will discuss two types of fluorescent reporter molecules: SYBR Green and Taqman probes.
SYBR Green
In conventional PCR where the amplified product is visualized on agarose gel, the fluorescent molecule Ethidium-Bromide was used which binds to double-stranded DNA. In modern molecular biology labs, Ethidium-Bromide is being replaced by SYBR Green. The SYBR-Green dye is considered less mutagenic and safer compared to the traditional ethidium bromide stain. Furthermore, in solutions (as in real-time PCR) the SYBR-Green dye is much more fluorescent, compared when used in agarose gel. It also doesn't inhibit the polymerase activity during the amplification process. Most real-time PCR instruments are therefore designed to detect the fluorescence wavelengths emitted by SYBR Green, and not ethidium bromide.
SYBR Green's fluorescence is quenched (diminished) when floating free in a solution, but is allowed to shine when the dye is "locked" into double-stranded DNA (dsDNA).
The figure above shows four PCR stages with the SYBR Green binding to double-stranded DNA. The dye is used to detect PCR product as it accumulates during PCR cycles. An important note is that the SYBR Green dye chemistry will detect all double-stranded DNA, including non-specific reaction products. A well-optimized reaction is therefore very important for accurate results.
Experiment
Let us do an experiment
A real-time PCR experiment is performed using SYBR Green dye. There are two samples / templates: one is from the CCHF Europe I strain, the other from the CCHF Africa II strain
Sequences of the nucleoprotein genes from the two strains are uploaded on the National Institute of Health web application: https://www.ncbi.nlm.nih.gov/tools/primer-blast/
The image displays the alignment of two CCHF virus strains, focusing on the region targeted by a PCR assay. The top section shows a graphical overview of the primer pairs mapped to the viral nucleoprotein sequence. The positions of the forward and reverse primers are marked in blue at each end of the region. (in module XX you can learn more about primer design).
The zoomed-in section gives a detailed view of the forward primer position. The DNA sequences represent the double strand sequence of the Africa strain. Red blocks/letters highlight mismatches—positions where the nucleotide bases differ between the two viral strains, making them easily identifiable against otherwise matching regions.
After PCR amplification, a melting curve analysis is conducted: the thermal cycler slowly increases the block temperature, while recording the fluorescence. As temperature rises, double-strand DNA products begin to "melt" (denature into single strands), and SYBR Green—whose fluorescence depends on binding double-stranded DNA—dissociates, causing fluorescence to decrease.
In the graph below are the melting curves of the amplified DNA from the two CCHF virus strains. With 100% helicity, all DNA is double-stranded. The percentage of double-stranded DNA drops dramatically at specific denaturing temperatures. The melting point of the African strain (blue line) is 82,2 ºC. The melting point of the European strain (purple line) is 83,4 ºC.
In the graph below are again the melting curve, but here the first derivative of fluorescence vs. temperature is displayed (often shown as −dF/dT vs. T). The derivative curve transforms the gradual decline of fluorescence into sharp, distinct peaks, with the peak position marking the melting temperature (Tm) of each strain. This makes it easy to pinpoint very subtle differences between two strains, or wild-type and mutant amplicons – even if only a degree apart.
The graphs are generated by pasting the sequences of the two strains in the online web application: https://www.dna-utah.org/umelt/quartz/dynamic.php
TaqMan Probe
Real-time PCR with TaqMan probes is an advanced technique for the detection and quantification of specific DNA sequences. TaqMan probes offer greater specificity and precision than SYBR Green assays. While SYBR Green detects any double-stranded DNA, TaqMan probes use a sequence-specific fluorescent probe to increase discrimination.
TaqMan Probe Mechanism
Click the "Next" and "Previous" buttons or use the arrow keys to understand how TaqMan probes work during PCR amplification.
Reasons why real-time PCR is superior to conventional PCR
We are now half-way of the module. You learned what the main differences are between real-time PCR and conventional PCR. Let's make a list of the advantages of real-time PCR.
Consistency
The graph below shows a real-time PCR on 96 identical samples on the same plate. The graph clearly shows variations in the plateau phase of multiple PCR reactions with the same starting DNA concentration. Note that the fluorescence values at the endpoint show a lot of variability from sample to sample. This is due to variations in the reaction kinetics. This means the conventional or end-point PCR has lots of variation, something we want to avoid in particular when we want to quantify the number of viruses in a solution. The Ct (or Cq) value remain consistent.
Sensitivity & specificity
For real-time TaqMan PCR, the optimal amplicon size is generally 50 to 150 base pairs (bp), improving the amplification efficiency. Primers are usually designed to recognize specific targets in the gene. Adding the probe binding site therefore increases specificity.
Speed and efficiency
Real-time PCR eliminates the need for post-PCR processing like gel electrophoresis, delivering results faster and with less hands-on time. It also makes it easier to automate the detection process.
Reduced contamination risk
Real-time PCR is run in closed plates. This reduces the risk of contamination from handling amplified products, which is a major issue in conventional PCR.
Broader Applications
Real-time PCR supports applications such as gene expression analysis, viral load quantification, multiple target detection and fast diagnosis of diseases – functions where conventional PCR falls short.
The next part of this module will cover some of the broader applications.
Reverse Transcriptase
PCR amplification only works on DNA. In real-time PCR (qPCR) for RNA viruses or gene expression analysis, it is essential to translate the RNA into DNA first. Reverse transcriptase is a specialized enzyme that converts RNA into complementary DNA (cDNA). The process, known as reverse transcription, allows researchers to detect and quantify RNA targets—such as viral genomes or messenger RNA—by first converting RNA to cDNA, which is then amplified and monitored in real-time PCR reactions.
One-Step vs. Two-Step Real-Time PCR
In one-step real-time PCR, both the reverse transcription (conversion of RNA to cDNA) and the PCR amplification of DNA occur in a single tube during the same reaction. This approach minimizes handling, reduces contamination risk, and is convenient for rapid diagnostics.
In two-step real-time PCR, the reverse transcription and PCR are performed in separate tubes: RNA is first converted to cDNA, which is then transferred to a new tube for amplification. This method allows greater flexibility in experiment setup, the ability to archive cDNA for future uses, and optimized conditions for each step.
Both approaches have advantages depending on the needs of the laboratory and the experiment.
One-step PCR and two-step PCR are two different approaches for performing reverse transcription polymerase chain reaction (RT-PCR), which is used to convert RNA into complementary DNA (cDNA) and then amplify specific DNA targets.
One-Step PCR
In one-step PCR, the reverse transcription (RT) and PCR amplification occur in a single reaction tube and buffer. This method combines the reverse transcriptase and DNA polymerase enzymes, allowing both processes to happen sequentially without the need to transfer the sample between steps.
Advantages
- Simplifies the workflow by reducing the number of steps and tubes
- Minimizes the risk of contamination and pipetting errors
- Suitable for high-throughput applications
- Requires less hands-on time and is faster overall
Disadvantages
- Uses gene-specific primers, limiting the analysis to specific targets
- Less flexible in optimizing conditions for RT and PCR separately
- The cDNA product cannot be stored for future use, requiring additional RNA samples for repeated assays
Two-Step PCR
In two-step PCR, the reverse transcription and PCR amplification are performed in separate reactions. First, the RNA is reverse-transcribed into cDNA in one tube. Then, an aliquot of the cDNA is transferred to a new tube for PCR amplification.
Advantages
- Allows the use of different primers for reverse transcription (e.g., oligo(dT), random hexamers, or gene-specific primers), providing more flexibility
- Enables optimization of reaction conditions for each step independently
- The cDNA can be stored and reused for multiple PCR reactions, facilitating the analysis of multiple genes from the same RNA sample
Disadvantages
- More complex workflow with additional steps and sample handling
- Increased risk of contamination and variability due to multiple pipetting steps
- Requires more hands-on time and is less suitable for high-throughput applications
Simulation Exercise: Choosing One-Step or Two-Step PCR
Scenario
You are a technician working in a diagnostic virology laboratory. You receive 10 blood samples from cattle animals with suspected Hemorrhagic Fever virus infection.
The Laboratory Requests:
Step 1:
Screen all 10 samples using the Pan-CCHF real-time PCR assay for the detection of CCHF virus RNA.
Step 2:
For any sample from a patient that tests positive, determine its virus genotype. These genotypes include:
- Genotype I: West Africa
- Genotype II: Central Africa
- Genotype III: South and West Africa
- Genotype IV: Asia I
- Genotype V: Asia II
- Genotype VI: Europe I
- Genotype VII: Europe II
Decision Point
Which reverse transcription PCR protocol should you use?
Controls
Controls are essential components of any PCR assay that help ensure the reliability and validity of results. They serve as quality assurance measures to detect potential problems in the PCR reaction and help interpret results correctly.
Types of Controls
Positive Control
Contains a known amount of target DNA/RNA that should always produce a positive result. It confirms that the PCR reaction conditions are working properly and that all reagents are functional. If the positive control fails to amplify, the entire run should be considered invalid.
Negative Control (No Template Control - NTC)
Contains all PCR reagents except the target DNA/RNA template (replaced with sterile water). It should always produce a negative result. A positive result in the negative control indicates contamination and invalidates the entire run.
Internal Positive Control (IPC)
A specific DNA or RNA sequence added to each sample that is amplified alongside the target. It monitors for PCR inhibitors in the sample that could cause false-negative results. If the IPC fails to amplify, the sample may contain inhibitors or the extraction may have failed.
Extraction Control
Used to monitor the efficiency of the nucleic acid extraction process. It can be an external control (known amount of target added before extraction) or an internal control (endogenous gene present in the sample).
Control Interpretation Guide
| Control Type | Expected Result | Interpretation if Unexpected |
|---|---|---|
| Positive Control | Positive | If negative: PCR failure, reagent problem |
| Negative Control | Negative | If positive: Contamination detected |
| Internal Positive Control | Positive | If negative: PCR inhibition, extraction failure |
| Extraction Control | Positive | If negative: Extraction efficiency problem |
CCHF-Specific Controls
For CCHF virus detection, specific controls might include:
CCHF Positive Control: Synthetic CCHF RNA or inactivated virus
Human/Animal Gene Control: β-actin or GAPDH to confirm sample quality
RT Control: For RT-PCR, confirms reverse transcriptase activity
Inhibition
Inefficient PCR amplification in real-time PCR is often caused by inhibitory substances present within the nucleic acid (NA) extract. These inhibitors can originate from the sample matrix itself, such as plant polysaccharides, proteins, or humic acids, or be introduced during nucleic acid extraction—for example, residual ethanol or guanidine. Laboratory contaminants like glove powder or ink from marker pens, as well as debris on PCR plates and residues on the detection unit, can also interfere with fluorescence detection and amplification efficiency.
Inhibitors work by affecting critical steps such as DNA denaturation or reducing the activity of the polymerase enzyme, resulting in impaired amplification. The presence of such compounds may cause an increase in cycle threshold (Ct) values or even lead to false negative results. To monitor and identify inhibition, an internal control is co-amplified in each reaction. The Ct values of these internal controls should be tightly clustered—commonly around a set value such as 27. If, for example, one internal control shifts significantly (e.g., from Ct=27 to Ct=37), this strongly suggests inhibition within that reaction. Troubleshooting strategies for PCR inhibition, as well as detailed information on the use and interpretation of internal controls, are provided in the later modules & chapters on troubleshooting and quality control.
Best Practices
Always include appropriate controls in every PCR run. Without proper controls, it's impossible to distinguish between true negative results and false negatives due to technical failures. Controls are not optional—they are fundamental to reliable PCR diagnostics.
Multiplex PCR
Multiplex PCR is a variation of PCR that amplifies multiple DNA targets simultaneously in a single reaction. This is done by using multiple sets of primers for different targets. This method is highly efficient, as it can save time and reagents by testing for several targets at once. Multiplexing is often used in pathogen detection, genetic analysis, and genotyping. With real-time PCR, different fluorescent dyes are used to label probes for each target, allowing the machine to distinguish between the different signals.
CCHFV Genetic Diversity and the Need for Multiplex PCR
CCHFV is notable for its genetic diversity, with strains grouped into six main phylogenetic genotypes based on their geographic distribution and sequence differences. These genotypes are:
- Genotype I: Found in West Africa
- Genotype II: Central Africa
- Genotype III: South and West Africa
- Genotype IV: Asia I
- Genotype V: Asia II
- Genotype VI: Europe I
- Genotype VII: Europe II
Each genotype reflects regional evolution and adaptation of the virus. The high genetic variability—up to 20-31% difference at the nucleotide level—poses a challenge for molecular detection because it can affect PCR assay performance.
Beside the detection of the CCHFv, the simultaneous detection of the internal control is also desirable. Simultaneous detection of more than one target can be achieved with 'Multiplex PCR'.
Multiplex PCR is a technique in which multiple target DNA (or RNA sequences) are amplified simultaneously in a single PCR reaction by using more than one pair of primers. This allows the detection or analysis of several genes, pathogens, or genomic regions at once, saving time and reagents and increasing the efficiency of diagnostic or research workflows. Use of reporter dyes with different wave-lengths.
Simulation Exercise: CCHFV Genotyping Workflow with Real-Time PCR
Scenario
A researcher at a diagnostic virology laboratory is using a pan-CCHFV real-time PCR assay to screen 10 animal samples for Crimean-Congo hemorrhagic fever virus (CCHFV). This assay is adapted from a protocol published in the Journal of Virological Methods for universal detection of all known CCHFV genotypes. The current approach features two main modifications: (a) the assay incorporates an internal control to monitor for extraction and amplification efficiency, and (b) it employs a two-step RT-PCR process. After nucleic acid extraction, reverse transcription is performed on each sample to generate cDNA, which is subsequently used as input for the real-time PCR. The residual cDNA is stored for follow-up testing: further multiplex real-time PCR assays or other molecular tests are conducted if required.
Step 1: Initial Pan-CCHFV Real-Time PCR Screening
- Sample Reception: The researcher receives a batch of blood or tissue samples for CCHFV testing.
- Nucleic Acid Extraction: Each sample is prepared and nucleic acids extracted.
-
PCR Reaction Setup: Each pan-CCHFV real-time PCR mix includes:
- Primers and probe targeting conserved regions of CCHFV (all genotypes)
- Internal control (e.g., EGFP RNA or unrelated sequence) to verify extraction and amplification
-
Result Interpretation: After amplification, results are interpreted:
- Negative sample: No signal for CCHFV; internal control amplified.
- Inhibited/Invalid: No CCHFV or internal control signal (possible inhibition/extraction failure; retest needed).
- Positive sample: Detectable CCHFV and internal control signals.
Multiplex PCR Results
| Sample | Pan-CCHF Result | Internal Control | Interpretation | ✓ |
|---|---|---|---|---|
| 1 | undetected | Ct 26 | ||
| 2 | Ct 27 | Ct 29 | ||
| 3 | Ct 24 | Ct 33 | ||
| 4 | undetected | Ct 38 | ||
| 5 | undetected | Ct 30 | ||
| 6 | Ct 37 | Ct 29 | ||
| 7 | undetected | Ct 36 | ||
| 8 | Ct 31 | Ct 29 | ||
| 9 | Ct 27 | Ct 33 | ||
| 10 | undetected | undetected | ||
| 11 Negative control | undetected | Ct 31 | ||
| 12 Positive control | Ct 30 | Ct 30 |
After processing clinical samples in a diagnostic virology laboratory, results are typically classified as positive, negative, inhibited, or invalid. For samples that indicate inhibition or return invalid results, further analysis is required. Detailed troubleshooting procedures for handling such samples are outlined in the course chapter on Troubleshooting. This ensures that any technical or sample-related issues are addressed.
Reporting and Advanced Investigation
Results for positive and negative samples can be reported to the requester or applicant. When necessary, positive samples may undergo further characterization. For example, determining the specific virus strain can be important during outbreaks or for epidemiological studies. To achieve this, various molecular techniques may be applied to the sample cDNA:
- SYBR Green melting curve analysis for rapid differentiation based on amplicon profiles.
- Strain-specific multiplex real-time PCR to distinguish closely related viruses in a single reaction.
- Sequencing with phylogenetic analysis to provide the highest level of detail about the viral genome, allowing robust comparison with global reference strains.
Application of Multiplex Real-time PCR in Clinical Diagnostics
Multiplex real-time PCR enables the simultaneous detection of multiple viral pathogens that can present with similar symptoms. For instance, as described by Choi & Kim (2023), assays were developed to rapidly screen for 17 viruses associated with hemorrhagic fever syndrome. Because a multiplex PCR can accurately target only a certain number of pathogens in one panel, these 17 viruses were divided into 8 panels, with design based on geographic distribution and compatibility of fluorescent detection channels.
For example, Crimean-Congo hemorrhagic fever virus (CCHFV) was grouped in the same amplification panel as Ebola virus and Marburg virus, allowing for efficient, parallel screening of high-priority pathogens.
An internal control—using synthetic plasmid DNA—was included in each reaction to validate the testing process and detect any potential PCR inhibition.
Key Learning Points
This simulation demonstrates how multiplex PCR enables simultaneous detection of multiple targets, improving diagnostic efficiency while providing specific genotype information crucial for epidemiological tracking and treatment decisions. The internal control ensures result validity, while different fluorescent reporters allow discrimination between targets in a single reaction.
Troubleshooting
Real-time PCR troubleshooting requires systematic analysis of amplification curves and understanding common issues that can affect your results. Use this interactive dashboard to identify and resolve PCR problems.
PCR Troubleshooting Dashboard
Click on any tile below to explore common PCR issues, their causes, and solutions:
🚨 Contamination
Unexpected amplification in negative controls or non-template samples
⚡ Template Degradation
Poor amplification due to degraded DNA/RNA templates
📈 High Template Concentration
Plateau phase reached early due to excessive starting material
🚫 PCR Inhibition
Reduced amplification efficiency due to inhibitors in samples
📉 Low Template Concentration
Late Ct values or no amplification due to insufficient starting material
🔗 Primer Dimer Formation
Non-specific amplification between primers creating false signals
TROUBLESHOOTING QUESTION 1
The image displays real-time PCR amplification curves for the pan-flavi-virus assay following a 10-fold dilution series of Zika virus. The red curves appear erratic during the plateau phase. What is the most likely explanation for this observation?
Select one correct answer for 100 points.
TROUBLESHOOTING QUIZ 2
A PCR master mix was dispensed into 12 wells, and each well received 5 μl of the same cDNA sample. Ideally, all wells should yield identical results. However, the last sample shows no amplification for either the target or the internal control. What could explain this outcome?
Select all that apply. Each correct answer gives 60 points.