Real-Time Polymerase Chain Reaction: Current Techniques, Applications, and Role in COVID-19 Diagnosis

Abstract

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 currently one of the most powerful molecular approaches and is widely used in biological sciences and medicine because it is quantitative, accurate, sensitive, and rapid. Current 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. This review aims to provide an overview of the current applications of the real-time PCR technique, including its role in detecting emerging viruses such as SARS-CoV-2.

Keywords: COVID-19; SARS-CoV-2; molecular diagnosis; polymerase chain reaction; quantitative PCR; real-time PCR.

Figure 1

Representation of a single amplification plot of real-time quantitative PCR. ∆Rn = fluorescence emission of the amplicon at each time point minus fluorescence emission of the baseline. Ct = threshold cycle. Baseline refers to the PCR cycles in which the fluorescent signal of a reporter accumulates. However, it is below the limits of detection of the instrument (adapted from Arya et al. [5]).

Figure 2

Mechanism of action of SYBR Green 1 dye. SYBR Green 1 probe is a double-stranded DNA-intercalating agent which exhibits very little fluorescence whilst free in solution. In the time of primer elongation and polymerization, SYBR Green 1 molecules become inserted into the double-stranded amplicons, causing an increase in detectable fluorescence [5].

Figure 3

Mode of action of TaqMan probe. The TaqMan probe is a hydrolysis probe with a fluorescent reporter dye bound to its 5′ end and a quencher dye at its 3′ terminus. Whilst the probe is intact, fluorescence resonance energy transfer (FRET) occurs, and the fluorescence emission of the reporter dye is absorbed by the quenching dye. In the presence of the target sequence, the fluorogenic probe anneals downstream from one of the primer sites. It is cleaved by the 5′ nuclease activity of the Taq polymerase enzyme during the elongation step of the real-time PCR. Cleavage of the probe by Taq polymerase during PCR segregates the reporter and quencher dyes, thereby producing a fluorescence signal (Adapted from Arya et al. [5]).

Figure 4

Action mode of dual hybridization probe. The dual hybridization probe consists of two hybridization probes, one brings a donor fluorophore at its 3′ end, and the other is labeled with an acceptor fluorophore at its 5′ terminus. After the denaturation phase, both probes attach to their target sequence in a head-to-tail arrangement during the annealing step. This causes the two dyes in close proximity to facilitate fluorescence resonance energy transfer (FRET). The donor dye in one of the probes transmits energy, facilitating the other one to dissipate fluorescence at a distinct wavelength (Adapted from Arya et al. [5]).

Figure 5

Mechanism of action of the molecular beacon. Molecular beacons contain covalently linked fluorescent and quenching dyes at either end of a single-stranded DNA molecule. Whilst free in solution, the probe is maintained in a hairpin conformation by complementary stem sequences at both ends of the probe, which brings the fluorescent dye and the quencher in close proximity. This causes fluorescence resonance energy transfer (FRET) to occur, which suppresses reporter fluorescence. The loop part of the molecule is complementary to the target nucleic acid molecule. In the presence of a target sequence, the loop hybridizes to the complementary target sequence throughout the annealing step, resulting in a conformational alteration that forces the reporter and quencher dyes to separate, and fluorescence is emitted (Adapted from Arya et al. [5]).

Figure 6

Mechanism of action of Scorpion probe. The scorpion probe adopts a stem-and-loop conformation held by complementary stem sequences on the 5′ and 3′ sides of the probe. A fluorophore is attached to the 5′ end, and a quencher is linked to the 3′ end of the probe. A specific probe sequence is held within the hairpin loop, which is linked to the 5′ terminus of a PCR primer sequence by a PCR stopper. This chemical variation hinders PCR from amplifying the stem-loop sequence of the scorpion primer. In the course of PCR, scorpion primer is elongated to generate an amplicon. In the annealing phase, the specific probe sequence in the scorpion tail curls back to hybridize with the complementary target sequence in the amplicon, hence opening up the hairpin loop. This prevents the fluorescence from being quenched, and a signal is detected (Adapted from Arya et al. [5]).

Figure 7

Schematic diagram of molecular structure, genome organization, and relative positions of amplicon targets on the SARS-CoV-2 genome. On the SARS-CoV-2 virion, together with membrane protein and envelope protein, the spike protein glycoprotein projects from a lipid bilayer, giving the virion a distinctive appearance. SARS-CoV-2 virion (top): M: membrane protein; E: envelope protein; S: spike protein; N: nucleocapsid protein. The SARS-CoV-2 genomic RNA is associated with the nucleocapsid protein forming the ribonucleoprotein with a helical structure. The SARS-CoV-2 genome encodes structural (S, M, E, N) and nonstructural proteins. The relative positions of amplicon targets (RdRp, E, N) are shown. SARS-CoV-2 genome (bottom): Orf1a: open reading frame 1a; Orf1ab: open reading frame 1ab; S: spike protein gene; M: membrane protein gene; RdRp: RNA-dependent RNA polymerase gene; E: envelope protein gene; N: nucleocapsid protein gene (adapted from Corman et al. [3]; Artika et al. [55]).