Real-Time Polymerase Chain Reaction

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1		I Made Artika	--	3587	2022-12-23 17:47:08	\|
2	format change	Peter Tang	-1 word(s)	3586	2022-12-26 02:22:46	\|

This entry is adapted from the peer-reviewed paper 10.3390/genes13122387

Successful detection of the first SARS-CoV-2 cases using the real-time polymerase chain reaction (real-time PCR) method reflects the power and usefulness of this technique. 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 synthesize copies in multiple amounts of a selected DNA region for various purposes. Real-time PCR is one of the most powerful molecular approaches and is widely used in biological sciences and medicine because it is quantitative, accurate, sensitive, and rapid. Applications of real-time PCR include gene expression analysis, mutation detection, detection and quantification of pathogens, detection of genetically modified organisms, detection of allergens, monitoring of microbial degradation, species identification, and determination of parasite fitness. The technique has been used as a gold standard for COVID-19 diagnosis. Modifications of the standard real-time PCR methods have also been developed for particular applications.

polymerase chain reaction real-time PCR quantitative PCR molecular diagnosis COVID-19 SARS-CoV-2

1. Introduction

The polymerase chain reaction (PCR) was first used to amplify particular DNA sequences and has since been extended into one of the most robust research tools in biological sciences and medicine. Its extension to RNA studies was based on using a reverse transcriptase enzyme to first make complementary DNA (cDNA) and then employing this in the process of PCR amplification, a method termed reverse transcription PCR (RT-PCR) ^[1]. However, as the standard PCR cannot be reliably used for accurate quantification, the technique was refined, giving the powerful analytical tool we now call real-time polymerase chain reaction (real-time PCR) ^[2].

At the end of 2019, the COVID-19 pandemic, due to the novel SARS-CoV-2, hit the globe and gave rise to a great challenge to public health laboratories. The gold standard diagnosis for SARS-CoV-2 infection is a nucleic acid amplification test (NAAT), and real-time PCR assay is the major platform that was applied ^[3]. COVID-19 also forced Indonesia to increase the number of laboratories with the capacity for COVID-19 detection. In the beginning, the government assigned only one lab. However, due to the increasing number of COVID-19 cases, by 29 April 2020, as many as 89 laboratories were officially appointed ^[4]. The fact that the real-time PCR platform is a multipurpose platform and can be applied in various fields of application is worthy of exploration. The technique can be used for basic molecular research right through to an approved molecular diagnostic assay. The exploration of the current wide range of applications of the real-time PCR method is critical, including its feasibility in low-middle income countries.

2. Basic Principles

Real-time polymerase chain reaction (real-time PCR), also known as quantitative PCR, is a modification of the PCR strategy which allows monitoring of the PCR progress in real-time PCR itself is an enzymatic process used in vitro for the amplification of a selected DNA region through several orders of magnitude, generating thousands to millions of copies of a specific DNA segment. Ingredients needed include template DNA, primers, nucleotides (dNTPs), and thermostable DNA polymerase ^[5]^[6]. In addition to improved accuracy, sensitivity, and rapidity, one of the principal advantages of the real-time PCR over basic PCR is that this technique provides a reliable quantification relationship between the number of starting target sequences (before the amplification by PCR) and the amount of amplicon accumulated in a particular PCR cycle ^[5]. This is of paramount importance for the precise quantification of the target nucleic acids, which is critical for mRNA quantification in gene expression analysis ^[7] and the determination of the viral load of a clinical specimen ^[8]. Moreover, there is no need for post-PCR processes, thus minimizing the chance of cross-contamination due to previous amplicons ^[5]. This real-time PCR technique, therefore, has revolutionized the detection and quantification of target nucleic acids and gained a wide range of applications ^[9].

2.1. Quantification

The number of DNA molecules available in the starting mixture determines the quantity of amplicon generated following a set number of PCR cycles. If only a few DNA molecules are present at the start of the PCR process, relatively little amplicon will be synthesized. On the contrary, if there are large amounts of starting molecules, then the amount of product will be higher. This relationship permits the use of PCR to calculate the number of DNA molecules present in samples by measuring the amount of product that is generated. However, using conventional PCR, in which the amplicons are measured after finalizing the PCR process (end-point detection), the quantitative correlation between the starting DNA molecules and the PCR product becomes imprecise as large differences in the number of starting DNA cause relatively small differences in the resulting PCR products. This is due to factors such as the presence of inhibitors of the polymerase reaction, reagent limitation, and the accumulation of pyrophosphate molecules. The ability to monitor the PCR product in real-time, especially during the exponential phase, makes real-time PCR a reliable quantitative method because, during this phase of the PCR reaction, a precise quantitative relationship between the amount of starting DNA and the quantity of PCR product can be established. By detecting the amount of amplicon during the exponential phase, it is possible to extrapolate back to the quantity of the starting DNA in the mixture, hence, the concentration of the nucleic acids in the original sample ^[2]^[5].

Plotting the amount of PCR product (amplicon) versus the number of reaction cycles produces a representative real-time PCR amplification curve. Major phases of the amplification curve include linear (at the start), exponential (logarithmic-linear), and plateau phases. Throughout the initial cycles of the PCR process, the values of the fluorescence emission of the product represent the linear ground phase and do not exceed the baseline. During the exponential phase, PCR gains its optimum amplification period, doubling the product after each cycle. The ideal reaction conditions are achieved during this phase, with none of the reaction components being limiting. Fluorescence intensity in the exponential phase is used for data calculation. Although theoretically, PCR itself is an exponential process, and the number of DNA molecules should double after each cycle because reaction components eventually become limiting, so the rate of target amplification decreases, and the PCR reaction reaches a plateau. The fluorescence intensity at the plateau phase is, therefore, not useful for data calculation ^[5]^[10].

There are several terms used related to the amplification curve of real-time PCR. The baseline is defined as the number of PCR cycles in which a fluorescent reporter signal accumulates but is below the limits of detection. Threshold refers to an arbitrary value selected based on the variability of the baseline to reflect a statistically significant increase of signal over the baseline, hence distinguishing a relevant amplification signal from the background. It is generally set at 10× the standard deviation for the average signal of the baseline fluorescence. A fluorescent signal detectable above the threshold is assumed to be a real signal used to define the threshold cycle (Ct) for a sample. Ct refers to the fractional PCR cycle number in which the reporter fluorescence level is higher than the minimum detection level, the threshold. The availability of more nucleic acid templates at the beginning of the reaction results in fewer cycles required to reach the position at which the fluorescent signal is substantially higher than the background. Nucleic acid quantification can then be performed by comparing the Ct values of the samples at a particular fluorescence value with similar data obtained from a series of standards by constructing a standard curve ^[5]^[11]^[12]. A standard curve can be generated based on a serial dilution of a starting amount of known nucleic acids, such as a plasmid for the gene of interest or a chemically synthesized single-stranded sense oligonucleotide for the whole amplicon. Alternatively, a standard curve can also be generated based on a cell line with a known copy number or expression level of the gene of interest. In the absence of standard curves, relative quantification can be carried out by comparing the Ct values of the samples with that of a reference control ^[5].

Theoretically, real-time PCR can only be applied to the amplification of templates in the form of DNA molecules. How, then, to detect and quantify an RNA sample? For these purposes, the RNA molecule is first reverse-transcribed into a complementary DNA (cDNA) using reverse transcriptase, followed by conversion of the generated single-stranded cDNA to double-stranded DNA. This double-stranded DNA is then amplified using standard PCR. This procedure is known as real-time reverse transcription polymerase chain reaction (real-time RT-PCR) ^[6]. The real-time RT-PCR can be carried out using either a one-step or a two-step method. In one-step real-time RT-PCR, the RT step is coupled with PCR. In this process, RNA is reverse transcribed to cDNA and then amplified in one reaction. The main advantages of this method are rapidity of set-up, cheapness, and involving less handling of samples to reduce pipetting errors and contamination. However, as this method employs gene-specific primers for both the RT and PCR occurring in one reaction tube, other genes of interest cannot be amplified for later analysis ^[13]. In two-step real-time RT-PCR, the process consists of two separate steps. The initial step is an RT reaction to construct cDNA. The second step is the cDNA amplification using traditional real-time PCR. The main advantage to two-step RT-PCR is that the cDNA is typically generated using random hexamer- or oligo-dT primers, which allow complete conversion of the messages in the RNA sample into cDNA, hence, permitting future analysis of other genes ^[13].

2.2. Probes

Real-time PCR systems employ a fluorescent reporter of the probe for detection and quantification. In general, they are classified into two main groups depending on the fluorescent agent used and the specificity of the PCR detection. The first class is based on double-stranded DNA intercalating molecules such as SYBR Green I and EvaGreen, allowing the detection of both specific and non-specific amplicons. For the second group, fluorophores are linked to oligonucleotides. Thus, they only detect specific amplicons ^[14]. This group includes hydrolysis probes (such as the TaqMan probe), dual hybridization probes, molecular beacons, and scorpion probes ^[5]. Other types of probes, such as those which belong to analogs of nucleic acids, have also been described ^[14]. A fluorophore is a fluorescent molecule that absorbs light energy at a particular wavelength and then re-emits light at a longer wavelength. There are two kinds of fluorophores: donor or reporter and acceptor or quencher. If a donor fluorophore absorbs light energy, it raises its energy level to that of an excited state. The process of a return to the ground state is accompanied by the emission of energy as fluorescence. This emitted light energy can be transmitted to an adjacent acceptor fluorophore when the two fluorophores are present in proximity. This transfer of excited-state energy from a fluorescence-reporter to a quencher is termed “fluorescence-resonance-energy transfer” (FRET) ^[14]. It should be noted that there are two distinct FRET mechanisms depending on how the energy is passed on to the acceptor fluorophore and dissipated, called FRET-quenching and FRET. The phenomenon of FRET quenching occurs when the energy of the quencher (a non-fluorescent molecule) is released as heat rather than emitted as light. FRET happens when the transferred energy is emitted as fluorescent light due to the acceptor molecule being a fluorocompound ^[14].

SYBR Green 1 is the most commonly used double-stranded DNA intercalating agent. It is a dye that attaches to the minor groove of double-stranded DNA, regardless of its sequence. It only fluoresces when inserted into double-stranded DNA. The strength of the fluorescence signal is therefore dictated by the quantity of double-stranded DNA existing in the reaction. The superiorities of SYBR Green 1 are low cost, convenience, and sensitivity. The major drawbacks of this probe are that they are not specific because the probe interacts with all double-stranded DNAs synthesized in the course of the PCR process, including the nonspecific amplicons and primer-dimers ^[5]^[14]. Considering that nonspecific products, including primer-dimers, are able to be generated during the PCR process, it is highly recommended to perform a melting curve analysis to determine the specificity of the amplified DNA sequences ^[14]. Notably, by optimizing the SYBR Green technique, its performance and quality can be as good as the specific TaqMan assay ^[15]. Other DNA-binding dyes available commercially include ethidium bromide, YO-PRO-1, SYBR® Gold, SYTO, BEBO, BOXTO, and EvaGreen ^[14]. The SYBR Green has recently been employed as a probe in a quantitative PCR platform to detect SARS-CoV-2 ^[16].

The TaqMan Probe is a very popular hydrolysis probe, which is designed to attach to a specific sequence of the target DNA. The mechanism of its action depends on the 5′–3′ exonuclease activity of Taq polymerase, which hydrolyzes the attached probe throughout PCR amplification. The TaqMan probe has a fluorescent reporter dye linked to its 5′ end and a quencher dye at its 3′ terminus. While the probe is intact, the reporter and quencher stay in close proximity, and excitation energy are quenched, prohibiting the emission of any fluorescence. In the presence of the target sequence, the TaqMan probe binds downstream from one of the primer sites. During PCR, when the polymerase replicates a DNA sequence on which a TaqMan probe is bound, the 5′ exonuclease activity of the polymerase cuts the probe. This sets apart the fluorescent and quenching dyes, and excitation energy is released as fluorescent light. Fluorescence intensity increases in each cycle in proportion to the rate of cleavage of the probe ^[5]^[14]. The TaqMan probe has been used to develop a multiplex real-time PCR method for the concurrent detection of novel swine coronaviruses to improve animal and public health ^[17].

The dual hybridization probe system consists of two hybridization probes. One carries a donor fluorophore at its 3′ terminus, and the other harbors an acceptor fluorophore at its 5′ end. Following the denaturation step, both probes hybridize to their target sequence in a head-to-tail formation during the annealing step. This makes the two dyes in close proximity mediating the energy transfer process (FRET). The donor dye in one of the probes absorbs light. It transmits energy, permitting the other one to dissipate that energy as fluorescence at a higher wavelength. As the fluorescence from the acceptor probe only happens if both the donor probe and the acceptor probe anneal to the PCR product, the detected fluorescence is directly proportional to the amount of DNA formed during the PCR process. The specificity of this reaction is therefore increased because a fluorescent signal is only happened upon two independent probes hybridizing to their specific target sequence ^[5]^[18]. The dual hybridization probe has been applied in a real-time PCR technique for rapid identification of Bacillus anthracis in environmental swabs based on the amplification of a special chromosomal marker, the E4 sequence. The method may contribute to strengthening the biodefense system ^[19].

The molecular beacon is another hybridization-based probe suitable for real-time PCR. This probe also contains attached fluorescent and quenching dyes at either end of a single-stranded DNA molecule. However, it is intended to form a stem-and-loop structure when free in solution so as to bring the fluorescent dye and the quencher in close proximity, and, as a result, resonance energy is quenched. The loop segment of the molecule is complementary to the target nucleic acid molecule, and the stem is formed by the annealing of complementary arm sequences on the termini of the probe sequence. When the probe sequence in the loop attaches to a complementary nucleic acid target sequence during the annealing step, a conformational change takes place that forces the stem apart. This leads to the separation of the fluorophore from the quencher dye.

The scorpion probe is another fluorescence-based method developed for the specific detection of PCR products. Similar to molecular beacons, the scorpion probe adopts a stem-and-loop configuration due to the presence of complementary stem sequences on the 5′ and the 3′ sides of the probe. A fluorophore reporter molecule is attached to the 5′ end and a quencher molecule is joined to the 3′ end of the probe. The specific probe sequence is kept within the hairpin loop, linked to the 5′ terminus of a PCR primer sequence by a non-amplifiable monomer called a PCR stopper. The function of the PCR stopper is to prevent PCR from amplifying the stem-loop sequence of the scorpion primer. During PCR, scorpion primers are extended to generate amplicons. During the annealing phase, the specific probe sequence in the scorpion tail curls back to hybridize with the complementary target sequence in the amplicon. This hybridization event opens up the hairpin loop and prevents the reporter molecule’s fluorescence from being quenched, and therefore a light signal is emitted. As the tail of the scorpion and the amplicon become part of the same strand of DNA, the interaction is intramolecular. This is beneficial as it leads to an effective instantaneous reaction giving a much stronger signal compared with the bimolecular interaction used in TaqMan or molecular beacon techniques ^[5]. The scorpion probe has been employed in a real-time PCR method to detect Escherichia coli in dairy products for food safety monitoring ^[20].

3. Applications

Apart from offering great sensitivity and specificity, real-time PCR can be applied for both qualitative and quantitative analysis. Therefore, it has become the method of choice for the rapid and sensitive detection and quantification of nucleic acid in biological samples for many diverse applications such as gene expression analysis, detection of mutation, determination of cancer status, microRNA analysis, detection of genetically modified organisms, bacterial detection, bacterial quantification, viral detection, and viral load measurement. Due to its versatility and usefulness, the real-time PCR technique has been employed in many research areas, including biomedicine, microbiology, veterinary science, agriculture, pharmacology, biotechnology, and toxicology ^[14]. Selected examples of the application of real-time PCR are presented in Table 1.

Table 1. Examples of application of real-time polymerase chain reaction.

Field	Application	References
Gene expression analysis	Analysis of wireless fidelity radiofrequency radiation on the expression of E. coli genes that potentially alter its pathogenic traits	^[21]
	Examination of plant gene expression impacting lignin synthesis for plant cell wall structure	^[22]
	Analysis of gene expression as a potential biomarker for early-stage diagnosis in colorectal tumor and cancer patients	^[23]
	Examination of microRNA expression profile in response to viral infection	^[24]
Detection of mutation	Detection of mutation patterns in human cancer cells	^[25]^[26]
Detection of mutation	Detection and quantitative analysis of mutation for monitoring drug resistance	^[27]^[28]
Food Analysis	Detection of genetically modified organisms (GMO)	^[29]^[30]^[31]
	Detection of allergens in food	^[32]
	Detection of pork in food products	^[33]
Bioremediation monitoring	Monitoring microbial degradation	^[34]^[35]^[36]^[37]
Detection and quantification of pathogens	Detection of pathogenic bacteria	^[38]
	Identification of microbial species as etiology of a disease	^[39]
	Molecular bacterial load assay (i.e., Mycobacterium tuberculosis)	^[40]
	Determination of growth fitness of plasmodium parasites	^[41]
	Detection of pathogenic RNA viruses	^[3]^[42]^[43]
	Diagnosis of pathogenic DNA viruses	^[44]
	Analysis of viral load associated with clinical features of the disease	^[8]^[45]^[46]

4. Detection and Quantification of SARS-CoV-2

Together with the DNA sequencing method, the real-time RT-PCR technique was successfully applied to detect and identify for the first time the newly emerged 2019 novel coronavirus (2019-nCoV) in Wuhan, China in December 2019 by employing primers that targeted a consensus RNA-dependent RNA polymerase (RdRp) region of pan β-CoV ^[47]. The Coronaviridae Study Group of the International Committee on Taxonomy of Viruses (ICTV), renamed “severe acute respiratory syndrome coronavirus 2” (SARS-CoV-2) ^[48]. As the etiologic agent of the coronavirus disease 2019 (COVID-19) pandemic, SARS-CoV-2 has caused over 618.5 million human cases with more than 6.5 million deaths globally ^[49]. SARS-CoV-2 is an enveloped virus with a positive-sense single-stranded RNA genome that encodes structural, nonstructural, and accessory proteins ^[50]. All along the COVID-19 pandemic, the real-time RT-PCR procedure has been adopted by the WHO as a standard method for confirmation of acute SARS-CoV-2 infections due to its sensitivity and specificity ^[51]. Primers and probes for real-time RT-PCR detection of SARS-CoV-2 were designed to target and detect the genes encoding RdRp, envelope (E), and nucleocapsid (N) proteins ^[3].

A cycle threshold value <40 is interpreted as a positive detection of SARS-CoV-2 RNA. Nasopharyngeal/oropharyngeal swabs have typically been used to confirm the clinical diagnosis. Notably, SARS-CoV-2 could also be detected in specimens from other sites such as bronchoalveolar lavage fluid, sputum, fiber bronchoscope brush biopsy, feces, and blood ^[52]^[53]. The real-time PCR method was applied to detect SARS-CoV-2 in more than 64,000 specimens collected from COVID-19 suspects or individuals in contact tracing programs in Jakarta and neighboring areas, Indonesia, within the first year of the COVID-19 pandemic ^[52]. In order to assess environmental contamination with SARS-CoV-2 in a hospital setting, swab samples were collected from hospital surfaces such as intensive care unit (ICU) floors, medical floors, heating, ventilation, and air conditioning (HVAC) and then used for diagnosis of SARS-CoV-2 using RT-qPCR ^[54]. Similarly, environmental samples from surfaces of university classrooms, libraries, computer rooms, gymnasiums, and common areas have also been employed for the detection of SARS-CoV-2 RNA to evaluate the potency of SARS-CoV-2 transmission through indirect contact mediated by SARS-CoV-2 contaminated objects and surfaces ^[55]. To monitor the occurrence of SARS-CoV-2 RNA in wastewater as an indication that the community members shedding SARS-CoV-2 RNA in their stool, influent, secondary, and tertiary treated effluent water samples were collected and used for SARS-CoV-2 RNA detection using RT-qPCR ^[56].

In relation to COVID-19, the real-time RT-PCR assay has been applied to analyze the viral load dynamics in sputum and nasopharyngeal swabs of patients. The nucleotide sequences targeted for amplification were the SARS-CoV-2 open reading frame 1ab (ORF1ab) and N protein gene fragments. The viral load in the sputum was found to be higher than that in the nasopharyngeal swab at the time of disease presentation ^[46]. In addition, the viral load in the sputum samples decreased more slowly than in the nasopharyngeal swab samples as the disease progressed, primarily in patients with another underlying disease, such as hypertension or diabetes. These data suggested the value of using sputum specimens for SARS-CoV-2 detection to reduce the spread of COVID-19 within the community ^[46].

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