**4. Molecular testing**

These diagnostic assays detect the virus through the presence of their genetic material, which is amplified to produce a detectable signal. To fully comprehend the mechanism of these assays, it is essential to first understand the central dogma (**Figure 3**). The common genetic material is DNA (deoxyribonucleic acid), a comparatively more stable nucleic acid than RNA. Generally, DNA is a double-stranded (ds) molecule composed of plus (+) and minus (−) strands [18]. It is made of deoxyribose sugar molecules as backbone and are attached with bases or nucleotides A (adenine), T (thymine), G (guanine) or C (cytosine) [18]. The complementary nature of the bases, i.e. their ability to pair specifically, provides the ds structure. The same feature allows faithful replication of the DNA and syntheses of RNA, thus enabling truthful relaying of the message. The base pairings are A-T and C-G [18]. The deoxyribose (and ribose) sugars provide a directionality to the nuclei acids

#### **Figure 3.**

*Central dogma. The message in the double stranded DNA is converted to single stranded RNA by transcription, through complementary base pairing. The code in RNA is converted to proteins by a process called translation (three bases constitute a codon, which represents one amino acid). Amino acids are the building blocks of proteins. RNA can be converted back to double stranded DNA (cDNA) through reverse transcription.*

with their chemical groups, 5′ end and 3′ end [19]. All DNA strands are synthesized by DNA polymerases in 5′ - > 3′ direction [19]. The two strands, however, run in opposite directions i.e. the 5′ end of plus strand is closest to the 3′ end of minus strand, while the 3′ end of the plus strand is opposite to the 5′ end of the minus end [19]. Based on the ATGC code (sequence) carried by the plus strand, the minus complementary strand is built [19]. For example, 5′-AGGCTC-3′ sequence on the plus strand will be paired with 5′-GAGCCT-3′.

#### **4.1 DNA to RNA**

The information in the genetic material needs to be converted to proteins, which act at the functional level. In this process, a key intermediate is the RNA. The code in the DNA is first converted to RNA through transcription by RNA polymerases [20]. The complementarity of the bases is used to transfer the information faithfully into RNA. Usually, RNA is a ss molecule, with the same complementary base pairings as the DNA. In RNA, T is replaced by U (uracil) which also pairs with A (A-U) [19]. In some viruses, ds RNA serves as the genetic material. However, the rules of complementation and the ss RNA intermediate for protein synthesis remain the same.

#### **4.2 RNA to protein**

The code carried by RNA is used for the synthesis of proteins, through translation [20]. Proteins are composed of amino acids building blocks. A codon in the RNA, which is composed of three bases in a specific order, codes for a particular amino acid [20]. So, the sequence of the amino acids in the protein are built according to the sequence of the codons in the respective RNA. Proteins are the macromolecules which acts as support structures, catalyze reactions, relay signaling information, and many other functions.

The dependency of protein synthesis on RNA has been ingeniously employed in the current Pfizer and Moderna vaccines [21]. These vaccines carry an mRNA (messenger RNA) which carries the code for an antigenic fragment of the CoV-2 S-protein [21]. The host cells produce the S-protein fragments which elicit an appropriate immune response. This leads to the production of antibodies that can identify the CoV-2 S-protein upon an actual infection.

#### **5. Polymerase chain reaction (PCR)**

PCR is the process of photocopying a specific region (target region) of the DNA, achieved through base complementation. This amplification process is used to produce a detectable signal, which can be correlated to the presence and amount of target DNA present in the reaction (**Figure 4**). In PCR, a specific target region in the DNA sample is demarcated through primers, which are short stretches of DNA ranging from 8–20 nucleotide bases. Short DNA strands are termed as oligonucleotides, where primers are a sub-group which are used in PCR reactions. Primers are complementary to the boundaries of the target region in the DNA sample. In PCR, two primers are required to bind at the 5′ end boundaries, one each for the plus and minus strands. The primers provide a pre-requisite platform for the DNA polymerase to bind and extend the new complementary strands [22], one for each of the two original template strands (**Figure 4B**).

When the starting material for PCR is RNA, as in the case of CoV-2, the RNA template is first reverse transcribed to complementary DNA (cDNA) (**Figure 4A**).

#### **Figure 4.**

*RT-PCR. (A) Viral ss RNA is converted to ds DNA by the reverse transcriptase enzyme. (B) In PCR, the cDNA acts the template (1). The two strands of the cDNA are separated (denatured) through high heat (2). This allows the annealing of the primers (forward and reverse) and the probe with their complementary regions on the template DNA strands (3). The DNA polymerase extends the primers to synthesize new DNA strands. In this process, the polymerase displaces and separates the fluorescent reporter (F) and the quencher (4). (C) Explanation of the read-out signal generation. At the ground state, the quencher is in close proximity to the reporter, and thus suppresses its emission. Upon cleavage by the polymerase (during extension step), the reporter is released. This relieves its inhibition thereby producing a fluorescent signal. Thus, with an increase in amplification of the template DNA, there is a corresponding increase in the detectable fluorescent signal.*

The cDNA then serves as a template for PCR amplification using targeted primers. The conversion of RNA to DNA is achieved through naturally occurring RNAdependent DNA polymerases, aptly called reverse transcriptase (RT). PCR reactions which depend on the RT enzyme are generally categorized as RT-PCR.

The amplified DNA region was traditionally detected using color or fluorescent agents which bind to ds DNA products [23]. Thus, with an increase in amplification there is an increase in the color/fluorescent signal. PCR read-outs are of two types: quantitative and qualitative. The former, quantitative, yields the amounts of template DNA, either absolute or relative values. On the other hand, qualitative PCR provides information on whether the template DNA is present in a sample. For CoV-2 RNA detection, a simple answer on its presence or absence is essential.

The general types of PCR techniques employed or developed for COVID-19 diagnosis are discussed below.

#### **5.1 Reverse transcriptase-polymerase chain reaction (RT-PCR)**

The first diagnostic kit developed to detect the CoV-2 infection was based on RT-PCR. As described above, the viral genomic RNA in the specimen is reverse transcribed to cDNA, followed by PCR amplification of a target gene using primers (**Figure 4**). There are two variants of this reaction: 1-step and 2-steps [3]. In 1-step, reverse transcription and PCR are conducted in the same tube, in tandem. This minimizes the chances of contamination by reducing handling. The 2-steps variation separates the RT reaction which provides cDNA in a separate tube for retention. This is useful since it is easier to store and handle DNA than RNA, and the cDNA can be used for further testing of other genes, if needed.

Choosing the target gene and region within that gene (usually PCR is directed at amplifying only a small region within a gene) is important, as it needs to be specific to the virus, excluding any overlap with the host genome or any other parasite/ virus. For CoV-2 PCR, regions within the N, E, RdRP, S and ORF1ab genes have been successfully used as targets for RT-PCR [3]. It was recommended to use PCRs directed at amplifying at least two target regions for higher specificity. In addition to the viral genes, the human RNase P gene which is present ubiquitously in all cells is also amplified separately [3]. Detection of RNase P ensures that the PCR reaction did receive the specimen. This is important especially in determining negative results for viral gene targets.

During the PCR, the two strands of DNA template (plus and minus strands) are separated using high temperature to break the complementary pairs (reversible). This step is termed as "denaturation" in PCR, which is required to expose the bases for the primers to bind. This is followed by an "annealing" step which is ambient for primer binding to the complementary regions. Entailing this step is "extension" of the primers by DNA polymerase to synthesize complementary product strands. These three key steps are repeated multiple times in the same order to amplify the signal (referred to as PCR cycles). The newly synthesized DNA fragments can then themselves act as templates in the following PCR cycles, thereby giving an exponential amplification pattern. Thus, even a small amount of starting DNA is sufficient to generate a positive signal. Although, the annealing and extension temperatures can be synchronized through appropriate designing of the primers, denaturation requires a higher temperature. This demands the use of thermocyclers for RT-PCR, which can change temperatures of the reaction cyclically. Further, RT-PCR read-out is generally a fluorescent signal which also requires a specific detection instrument. These limit the use of this assay at point-of-care (POC), i.e. use by medical practitioners for instant results to make informed and immediate decisions.

A variation of this conventional assay, TaqMan PCR, was employed as a primary technique for CoV-2 diagnostics [24]. It involves the addition of another oligonucleotide called the probe. This probe, which is complementary to the plus strand is usually positioned towards the center of the target region i.e. between the two opposing primers. Probe is flanked by a fluorescent reporter molecule at its 5′ end and a quencher molecule at the 3′ end (as explained earlier, all oligonucleotides have a direction imparted by the backbone sugar molecules). The fluorescent reporter signal is suppressed by the quencher due to their close proximity. When the probe binds to the plus strand of the template (after denaturation), the DNA polymerase starts synthesizing the new strand from the 3'end of the forward primer (the quencher molecule in the probe 3′ end will not allow the polymerase to start at the probe). In this process, the polymerase cleaves the probe and releases the fluorescent and quencher molecules separately. Due to this irreversible separation, the signal from the fluorescent reporter is uninhibited and detectable. Thus, the level of signal from the reaction is proportional to the amount of new DNA products. TaqMan PCR retains the need for a thermocycler and a fluorescence reader, but provides more specificity than the traditional technique.

During the early stages of the COVID-19 pandemic, samples from multiple individuals were pooled together to reduce the testing times [25]. Upon detecting a positive result, the samples from that pool were individually tested to identify the infected individual/s.

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#### **5.2 Recombinase polymerase amplification (RPA)**

This assay works on the same principle as the RT-PCR but bypasses the need for temperature variations for DNA amplification. It eliminates the denaturation high temperature step, and then combines the annealing and extension steps to a single temperature [28]. The assays which use a single temperature to complete all the reactions are termed as 'isothermal', thus eliminating the need for a thermocycler.

RPA achieves isothermal amplification through the inclusion of a few key components in the reaction mixture (**Figure 5**). The first is the recombinase enzyme, which is incubated with the primers to form a complex, in the presence of a crowding agent (increases viscosity of the solution). The recombinase-primers complex is then added to the reaction with the cDNA derived from viral RNA. Thus, this assay also depends on RNA isolation and RT reaction. The recombinase allows the invasion of the ds cDNA by the primers to bind to their complementary regions. The ss DNA regions (or loops) that are created due to this invasion are stabilized by the binding of ss DNA-binding proteins (SSBPs). This prevents the re-binding of the original template strands (plus and minus). The recombinase enzyme is then displaced from the DNA by a strand-displacing DNA polymerase. This polymerase opens the template DNA structure as it synthesizes new DNA strands emanating from the primer (i.e. strand-displacing). All these components of RPA aim towards the elimination of the denaturation step in the PCR cycle, thus making it isothermal. This assay holds the capacity to be carried out in solid-phase, i.e. on a dry surface with immobilized components [28]. Although the load-of-detection (LOD) and time are compromised in solid-phase RPA [28], this feature can enable the designing of lyophilized kits with minimal storage and transport requirements.

End-point detection of RPA has been vastly calibrated to fit the lateral flow assays (LFA) [28]. This assay yields rapid results in a visual read-out format (**Figure 6**). To adapt RPA to LFA, three different oligonucleotides (2 primers and 1 probe) and a *nfo* nuclease are required. Similar to the TaqMan assay described above, the probe is flanked with a 5′ end antigenic label (usually

#### *Molecular Biology of PCR Testing for COVID-19 Diagnostics DOI: http://dx.doi.org/10.5772/intechopen.96199*

#### **Figure 5.**

*Recombinase polymerase amplification (RPA). The primers and the recombinase enzyme are allowed to form a complex (1). The recombinase opens up the cDNA template, while the primers bind to their complementary regions (2). The ss DNA regions generated due to the opening of the template structure are stabilized through the binding of the single-strand binding proteins (SSBPs) (3). The DNA polymerase binds to the primers attached to the template DNA, detaching the recombinase (4). The polymerase extends the primers, while opening the ds template on its way. It also moves the SSBPs while synthesizing the new strands (5). New strands are synthesized by the polymerase (6). New ds DNA products which can act as templates for the following amplification cycles (7).*

6-Carboxyfluorescein i.e. FAM) and a 3′ end blocking group [28]. The 3′ end group inhibits the DNA polymerase from extending the probe (remember that the polymerase can only add nucleotides at the 3′ end). In addition to these end groups, the probe is also equipped with an abasic nucleotide (tetrahydrofuran) which does not pair with any of the standard bases (A, T, G or C) [28]. This abasic nucleotide creates a fold in the probe that is bound to the complementary template DNA. The *nfo* nuclease recognizes this fold and nicks the probe at this position [28]. The abasic nucleotide is strategically positioned in the probe, such that the nick by the nuclease releases the 3′ end blocking group from the probe/template DNA complex [28]. This opens up the 3′ end for the polymerase to extend the new DNA strand from

#### **Figure 6.**

*Lateral flow assay. (A) The strip consists of the regions depicted in the figure. The sample is loaded onto the sample pad. The sample then moves upwards, towards the absorption pad due to capillary action. Sample comprises of the amplified ds DNA products with tags FAM and biotin at two separate ends. From the sample pad, the sample first moves to the conjugation pad which is pre-loaded with Ab1 (antibodies against FAM). The Ab1 antibodies bind to the FAM-DNA-biotin products. These complexes move upward and are captured at the test line by the immobilized biotin-binding proteins. This produces a visible test line. The unbound free Ab1 antibodies (excess) from the conjugation pad also move upwards. These antibodies, however, move past the test line as they do not possess any biotin for interaction at this line. Upon reaching the control line, these antibodies are captured by Ab2 (antibodies that can bind Ab1). This interaction produces a visible control line. (B) The appearance of the positive, negative and invalid results. Positive result should show two lines, since it ensures the working of all components of the LFA strip. A conclusive negative result will only produce a control line which is generated by the interaction of immobilized Ab2 with free unbound Ab1. All other results are considered invalid.*

the probe. The reverse primer (the primer that will bind to the opposite strand) is tagged with a 5'end ligand (usually biotin) [28]. The main feature of these two 5′ end tags (FAM and biotin) is the availability of strong binding proteins or antibodies against them. The binding proteins or antibodies are immobilized on LFA strips (dipsticks) at two separate lines, test and control. The test band is coated with biotin-binding proteins (which will capture the 5'end tag of the reverse primer), while the control band is stacked with Ab2 antibodies (which capture to unbound Ab1 antibodies, see below). Once the stick is exposed to the sample (either through immersion of the sample pad or loading of the sample) on the sample pad, the sample moves across the conjugation pad which has lyophilized antibodies against FAM (Ab1). Here the RPA ds DNA products which carry both the tags will be bound by Ab1. From the conjugation pad, the complex containing Ab1-RPA products will move further up the strip (due to capillary action) towards the test band. At this

#### *Molecular Biology of PCR Testing for COVID-19 Diagnostics DOI: http://dx.doi.org/10.5772/intechopen.96199*

juncture, only DNA products which have the biotin tag will be captured by the biotin-binding proteins, showing a positive result. Further movement of these complexes is restricted as the binding proteins are immobilized onto the test line. Along with the complexes, the unbound free Ab1 antibodies also move up from the conjugation pad. These free antibodies move further up from the test line as they do not carry any biotin. They are captured by the Ab2 antibodies in the control line. Hence, a positive result should yield two distinct lines in the strip. In case of a negative sample, there is no fruitful conjugation of Ab1 antibodies on the conjugation pad. However, due to capillary action of the sample, the Ab1 move up the strip. Although these antibodies will not be captured at the test line, they will be immobilized by Ab2 on the control line. Thus, a reliable negative result should show one control line on the strip. All other combinations would indicate inconclusive results. Advantages:


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## **5.3 LAMP (loop-mediated isothermal amplification)**

LAMP is another isothermal amplification technique which produces long, selfcomplementary looping DNA strands to generate a detectable signal. This technique employs an engineered DNA polymerase *Bst* 2.0 with strand-displacing feature [29]. This enzyme can separate the two template DNA strands (plus and minus) as it builds the new strand, thus removing the denaturation step from PCR. LAMP is conducted at a single temperature (60–65 °C) [29], conducive to both the annealing and extension steps. Recent modifications include addition of the engineered RT enzyme along with the *Bst* 2.0 polymerase, thus making it a 1-step protocol [29]. Again, this assay still requires RNA isolation from the sample.

Generally, a set of 4 (or 6) specific primers are used in LAMP assay. These primers cover at least six distinct regions, flanking the entirety of the target region. A glimpse of the assay steps is described in **Figure 7**. The self-complementary regions of the primers promote the formation of looped DNA products. This allows LAMP to yield concatemers of various lengths, which are long DNA strands with multiple copies of the target region aligned back to each other [29]. Concatemers multiply the read-out signal at a much faster rate than RT-PCR. The mechanism is explained

**Figure 7.**

*LAMP. Six specific regions (A, B, C, D, E, F) flanking the target DNA are chosen to design the primers (1). The forwards inner primer (FIP) invades the target DNA. This primer encompasses a self-complementary region (C) on its 5′ end (2). The new strand synthesized with FIP is displaced by the forward primer (3). The DNA products synthesized by the forward primer is similar to the original cDNA template (4). Due to the self-complementarity region on the FIP, it folds on itself forming a loop. This strand is invaded by the backward inner primer (BIP) and backward primer (5). The product formed by BIP on the FIP strand leads to two loops at the ends, forming a dumbbell shaped structure (6). These strands can be extended to form concatemeric DNA products (7). Concatemeric products of variable lengths (8).*

in the **Figure 7**. A review article by Thompson and Lei [29] is recommended for further reading on this assay. Here, I would like to highlight the modifications made to RT-LAMP to progress COVID-19 diagnostics.

This detection assay was developed to provide a visible color read-out. A colorimetric detection was incorporated using a simple pH-sensitive dye such as phenol red [29]. The color of this dye is closer to red when the pH is neutral (pH 7.0), and changes towards the yellow spectrum with acidic pH (pH < 7.0). The byproducts of DNA synthesis are acidic, which reduce the pH of the solution. This color change can be easily noted by eye, without any instrument. However, this detection method is limited by the baseline differences in the pH of collected specimens. To circumvent this, fluorescent dyes such as GeneFinder have been employed [29]. This dye produces green color under blue light illuminator to report positive results. Another

variation of this assay excluded RNA isolation step to find comparable amplification of the N-protein gene under laboratory conditions [29]. This modification still needs more testing and evaluation with patient samples, prior to field use. After calibration, this assay could significantly reduce the TAT.

Moreover, individual samples can be tagged with specific barcodes in LAMP assay which allow tracing in a pooled sample [29]. A common method of barcoding is the transposase Tn5-adapter system [30]. The original article on this barcoding method is recommended to readers for understanding its mechanism and potential in diagnostics [30]. The barcodes, however, have to be read through NGS (nextgeneration sequencing), which requires specific lab equipment. This adds time for obtaining the results. It is possible that the bargain between time saved by pooling samples and the time added by NGS could be a deciding factor for the field-use application of this assay.

An important feature of LAMP is its amenability to be paired with other PCR techniques to combine their advantages. So far, the efficacy of LAMP has been tested after merging with RPA, using lab samples [29]. The combined assay is found to have increased sensitivity. RT-LAMP when integrated with CRISPR-Cas12 assay (described below) was shown to reduce detection time considerably [3].

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#### **5.4 CRISPR-based assays**

CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated nuclease) technology has been recently tested for its potency in the field of diagnostics. This technique, which was given recognition through the Nobel Prize in Chemistry 2020, is a modified biological process of the bacterial (prokaryotic) adaptive immune system [1]. Here a ss guide RNA (sgRNA or crRNA) leads/guides the CRISPR-Cas complex to the target nucleic acid region [1], owing to its complementarity to this region (**Figure 8**). Upon binding, Cas nuclease cleaves the template nucleic acid [1], along with non-specific cleavage of nearby ss DNA/ RNA. This feature is called "collateral cleavage" activity [1], and has been used in

#### **Figure 8.**

*CRISPR-Cas cleavage system. Viral RNA is reverse transcribed to ds cDNA, which can be further transcribed to amplified RNA through an in vitro system. The cDNA can be processed either through Cas9 or Cas12a, while the ss RNA can be cleaved through Cas13a. The single-guide RNA (sgRNA) or crRNA is complementary to the region of interest, and thus guides the Cas nuclease to the site. After target cleavage, collateral cleavage activity cleave and releases the reporter from quencher inhibition. This produces a detectable fluorescent signal, which is proportional to the amount of target DNA in the reaction.*

designing read-out methods for diagnostic tests. This assay is equipped with ss DNA or RNA probes with a fluorescent or traceable reporter molecule, which produces a detectable signal only upon cleavage through collateral activity.

The target for CRISPR-Cas cleavage complex can be modified by changing the crRNA strand sequence, similar to the primers/probes in RT-PCR. Based on the Cas nuclease paired with the crRNA in the assay, the template can vary. For example, Cas13 targets ss RNA, while Cas9 or Cas12 target ds DNA [1]. Thus, in case of CoV-2, the virus ss RNA will need to be reverse transcribed to cDNA which will be either amplified as DNA products or through *in vitro* transcription as RNA products. CRISPR-Cas complexes cannot amplify nucleic acids, and hence rely on other amplification techniques for this (such as previously described methods).

A recently invented SHERLOCK (Specific high-sensitivity enzymatic reporter unlocking) technique includes the crRNA-Cas13a complex to target RNA molecules [1]. This technique uses RPA amplification assay and a non-targeting RNA strand tagged with a fluorescent dye [31]. Patchsung *et al.* [32] used SHERLOCK for CoV-2 diagnostics, targeting the S and ORF1ab genes. This assay has been further modified to suit LFA detection methods, i.e. using paper strips. Another variation of CRISPR-Cas technique is DETECTR, where it is combined with RT-LAMP amplification. This has been tested for CoV-2 E and N genes [33].

Ding *et al.* [34] developed an All-In-One Dual CRISPR-Cas12a (AIOD-CRISPR) assay where all the reactions components are incubated at 37 °C together. This simplifies the diagnostic assay protocol. The AIOD-CRISPR was then modified for a visual color detection in LED blue light illuminator.

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