Real-Time PCR

What is the Real-Time PCR technique?

The Real-Time PCR technique is a widely utilized molecular method in various fields such as basic science, biotechnology, medicine, forensic medicine, and infectious disease diagnosis. Unlike conventional PCR, which necessitates post-reaction analyses like agarose gel electrophoresis to detect amplified sequences, Real-Time PCR enables detection of amplified fragments during the reaction. With the advent of Real-Time PCR, including its various forms such as Real-Time Reverse Transcription PCR (Real-Time RT-PCR), has facilitated the examination of gene expression. This method allows for simultaneous reproduction of fragments and detection in a single step, t which is due to the use of fluorescent compounds in the reaction. These fluorescent molecules indicate an increase in the quantity of replicated DNA by emitting a greater fluorescence signal. The device can measure and record this signal, which can then be visualized as graphs.

There exist two distinct approaches for employing fluorescent molecules in the context of Real-Time PCR reaction. These approaches encompass the utilization of fluorescent molecules that bind to DNA, such as Cybergreen, as well as the utilization of sequences that are labeled with fluorescent molecules, such as primers and probes. Both of these strategies were developed in parallel and are implemented in the identification of pathogens. However, due to the fact that labeled primers and probes specifically recognize the target sequence, their application is preferred.

The fluorescence signal that is detected within the device is directly proportional to the quantity of amplified product during each cycle of the reaction. The most significant advantage of Real-Time PCR in comparison to conventional PCR is its ability to determine the number of primary copies of the template sequence. Consequently, the quantity of sample required to carry out the reaction can be extremely small. Generally, Real-Time PCR has the capability to be qualitative, indicating the presence or absence of the desired sequence, as well as quantitative, indicating the number of DNA copies. The quantitative Real-Time PCR is commonly referred to as qPCR.

With the accumulation of information regarding the genome sequencing of living organisms, it is now possible to design a Real-Time PCR reaction in order to detect any microorganism. The utilization of this methodology engenders precise and expeditious detection and quantification of the target sequence in various matrices. The concomitant nature of amplification and result observation reduces the reaction time, and furthermore, owing to the absence of post-amplification measures, the likelihood of cross-contamination is exceedingly low in this method.

As all stages of the Real-Time PCR reaction are conducted within a sealed tube, the risk of contamination is significantly diminished when compared to conventional PCR. Consequently, the occurrence of false positive results is averted. The qPCR technique is also employed in the identification of pathogens, gene expression studies through examination of RNA with the RT-qPCR methodology, allelic differentiation examination, strain typing determination, genetically modified organism examination, antibiotic resistance determination, toxin production assessment, and more.

Different phases in the Real-Time PCR reaction

Different phases in the Real-Time PCR reaction can be categorized into four distinct phases. These phases encompass various aspects of the reaction process.

Linear ground phase

At the beginning of the PCR reaction, the amount of product is very low and consequently exhibits low fluorescence. At this stage, the replication of components commences, resulting in stabilization of the response. During this period, the curve may exhibit minor peaks referred to as “noise” or background signal. This phase typically occurs between cycles 1 and 15.

early exponential phase

In this phase, the reproduction is done exponentially and the device receives the signals.

log-linear or exponential phase

In this phase, the speed of the Real-Time PCR reaction gradually decreased and the reaction materials and their efficacy are in a reduced state.

plateau phase

In the fourth phase, the device no longer detects any change in the level of fluorescent light, which indicating the loss of compounds in the reaction.

Upon completion of the PCR product amplification, the fluorescent signals accumulated during the reaction are measured using a cycle threshold (Ct) to determine the outcomes. The Ct value signifies the cycle number at which the fluorescent amount of a PCR product surpasses the background signal. This value is directly proportional to the amount of PCR product present in the reaction. Consequently, a lower Ct value indicates a higher quantity of PCR product. This correlation arises from the fact that a higher initial sample necessitates a lower number of PCR cycles to detect the product through the background signal. For instance, a reaction Ct value below 29 suggests a substantial amount of the sample. Ct values ranging from 30 to 37 correspond to average sample quantities, while Ct values between 38 and 40 represent minimal amounts of the target nucleic acid.

Various types of fluorescent dyes in Real-Time PCR

In the Real-Time PCR technique, two types of fluorescent dyes are commonly used. The first group consists of fluorescent dyes, such as SYBR Green and EvaGreen, which bind to double-stranded DNA. These dyes non-specifically bind to both strands of the DNA in the reaction medium. The second group comprises fluorescent dyes that are attached to oligonucleotide sequences, known as probes. These dyes can only attach to specific sequences, thus providing specificity. Examples of such hydrolytic probes include TaqMan probes and beacons.

Cybergreen and EvaGreen

Fluorescent dyes known as Cybergreen and EvaGreen are widely utilized in various tests. SYBR® Green, exhibits minimal fluorescence when in a free solution. However, when it binds to double-stranded DNA (dsDNA), its fluorescent signal experiences a significant increase, up to 1000 times. Consequently, the overall fluorescence signal in a reaction is proportionate to the amount of dsDNA present. As the target sequence is amplified and more PCR products accumulate, the intensity of the signal received by the device amplifies.

DNA-binding dyes can be categorized into two groups: non saturating and saturating.

Saturating dyes, like SYBR® Green, hinder PCR reactions when used above a specific concentration. Conversely, non-saturating dyes, such as EvaGreen, do not inhibit the PCR reaction and can be employed in a sufficient concentration to cover all the vacant binding sites on dsDNA. However, saturated dyes are preferable for high-resolution melt assays due to the more distinct signal change observed after DNA denaturation.

The advantages of utilizing dsDNA-binding dyes include:

simple primer design: Only two specific DNA primers are needed for sequencing, so probe design is not necessary.
the ability to test multiple genes quickly without requiring multiple probes.
lower cost
the capability to perform melt curve analysis to assess the reproduction process of fragments.

TaqMan probes and beacons

This group of fluorescent dyes are used in conjunction with the probe. The TaqMan hydrolysis assay comprises a sequence-specific fluorescently labeled oligonucleotide probe and a sequence-specific PCR primer. Heat-resistant polymerases, like Taq polymerase, are utilized in the hydrolysis assay due to their 5’→3′ exonuclease activity. TaqMan probes are labeled with a fluorescent reporter at the 5′ end and a quencher at the 3′ end. In an intact and healthy probe, the reporter fluorescent signal is suppressed due to its proximity to the quencher.

However, when the probe connects to the target sequence and the amplification process commences, the reporter becomes separated from the quencher by the 5’→3′ exonuclease activity of the Taq pol enzyme. Consequently, the signals corresponding to the amplification rate of the PCR product are recorded by the device.

One if the most advantages of using TaqMan probes include high specificity, a high signal-to-noise ratio, and the ability to conduct multiplex reactions. Nevertheless, one drawback of this method is the initial cost of the probe, which may be substantial.

In contrast, the beacon probe is a sequence of nucleotides, typically ranging from 25 to 40, that adopts a hairpin structure. The 5′ and 3′ ends of this probe possess complementary sequences of 5-6 nucleotides, forming the stem structure. The loop section of these probes is designed to be complementary to a sequence of 15 to 30 nucleotides derived from the target sequence. In the design of these probes, a fluorescent molecule that acts as a reporter is strategically placed at the 5′ end, while a quencher is placed at the 3′ end. Consequently, through the formation of a series pin structure, the Reporter and Quencher molecules are close together, resulting in the absence of any fluorescent signal being emitted.

Real-Time PCR applications in infection diagnosis

It is not feasible to utilize classical diagnostic techniques such as cultures and immunoassays to identify numerous infectious diseases. nowadays, the Real-Time PCR technique has supplanted these methods. This technique enables prompt and highly accurate identification of infectious diseases through the direct detection of pathogen’s DNA or RNA. Consequently, it has been widely employed in laboratories as a means to diagnose various infectious diseases in recent times.

When diagnosing and quantifying pathogens such as viruses, bacteria, and parasitic agents, various factors should be considered, including the purpose of DNA or RNA detection, the viability of culturing the microorganism, the clinical relevance, and the interpretation of reaction results. Given the time-consuming or unfeasible nature of cultivating clinical pathogenic viruses and the relatively low sensitivity and specificity of the ELISA test, qPCR techniques play a crucial role in the diagnosis, quantification and typing of these pathogens.

Viral disease diagnosis

Viruses are infectious agents that have the ability to replicate exclusively within living cells and can induce infection in all living organisms, encompassing plants, animals, and microorganisms. Viruses are categorized based on the nature of their DNA or RNA genome, whether it is single-stranded or double-stranded and the manner in which it undergoes replication. As previously mentioned, viruses can solely propagate and replicate within living cells. Consequently, the infectivity of viruses occurs subsequent to their entry into the body and subsequent penetration into its cellular structures. Certain viruses elicit cytopathic effects and, in some cases, cellular death.

In contrast, certain viruses remain dormant and inactive within the host cell. In some viruses such as the papilloma virus and Epstein-Barr virus have the potential to induce carcinogenesis in the host cell. The genomic composition of numerous pathogenic viruses, including HIV and COVID-19, consists of RNA. To facilitate their detection, the RNA isolated from the virus is initially transcribed into complementary DNA (cDNA) via Reverse Transcriptase enzyme, followed by continuation of the qPCR reaction.

This technique is referred to as RT-qPCR and is employed not only in the detection of RNA viruses but also in gene expression studies. The conventional microscopic method employed in the detection of single-celled parasites is also a laborious and time-intensive approach that necessitates skilled personnel. Therefore, Real-Time PCR represents a powerful tool in the detection, quantification and identification of single-celled parasites.

In general, the diagnosis of pathogenic viruses in humans can be achieved through several methods, including serology, virus-antigen detection, virus culture and viral genome replication. In recent years, the Real-Time PCR method has gained widespread usage in the diagnosis of viral infections due to its high precision in DNA/RNA detection and quantification. Although viral culture is considered the gold standard for diagnosing culturable viruses, such as adenoviruses, cytomegalovirus, herpes and influenza, the confirmation of results requires more than 5 to 9 days. Additionally, a viral genotyping determination is not feasible using this method.

Bacteria detection

Unlike viruses and parasites, numerous bacteria have the capacity to be cultivated. Consequently, the process of cultivation is regarded as the principal means of detecting these pathogens. However, in critical cases the conventional approach proves to be a laborious and multi-step method that fails to yield timely outcomes. In such instances, Real-Time PCR can deliver accurate results within a shorter timeframe. Nonetheless, it is imperative to take into account the phenotypic and biochemical attributes of the bacteria.

Cancer diagnosis

Advancements in the realm of biological science and technology have unveiled molecular targets for cancer diagnosis and treatment. Today for cancer diagnosis, a plethora of molecular markers are currently employed for accurate diagnosis, precise prognostication and resistance analysis to various treatment modalities. The utilization of the Real-Time PCR technique is one of the pragmatic approaches in the realm of identifying deletions or duplications, exploring markers associated with cancer and investigating alterations in gene expression within tumor cells. To elucidate, breast cancer stands as the most prevalent form of cancer among women globally and represents the primary cause of mortality.

The findings of a research study suggest that the analysis of HER-2 and topoisomerase IIα (topo IIα) genes is of great importance in the prognosis and treatment of breast cancer. Approximately 20% of individuals diagnosed with breast cancer exhibit amplification of the HER-2 gene at the DNA level, resulting in an excessive expression of its corresponding protein. In contrast, the topo IIα gene is situated in close proximity to HER-2 at the chromosomal position 17q12-q21, a region frequently mutated in breast tumors. This investigation utilized real-time PCR technique and labeled probes to assess the variants of HER-2 and topo IIα genes in comparison to the albumin gene (serving as a control) within DNA samples derived from both healthy and tumor tissues. As anticipated, the tumor tissues exhibited a conspicuous overexpression of HER-2 and topo IIα genes when compared to their normal counterparts.