**1. Introduction**

Coronavirus outbreak first case found in Wuhan, China in December 2019 [1]. Further, it became a pandemic, affecting the whole world. On February 2020, World Health Organisation (WHO) announced an official name for Coronavirus spread disease as COVID-19 [2]. It primarily targets a respiratory system in humans, as the appearance of symptoms depends on the incubation period, which further relies on the patient's age and the immune system [3].

SARS-CoV-2 as a public health emergency was declared by WHO and thereafter it became essential to find diagnostic tests for early detection and early treatment [4]. Normally the best way to detect any virus from the sample is its isolation and further confirmation by various molecular techniques. But Center for Diseases Control and Prevention (CDC) recommended not to isolate the SARS-CoV-2 as it is a new virus and this practices could be a risky approach and suggested to use patients sample directly for diagnosis [5]. Most of the diagnostic methods are molecular-based hence for diagnosis virus genome study become necessary. On January 2020 the ssRNA - 29870 bp whole sequence of SARS-CoV-2 was reported with GenBank accession number MN908947 [6, 7]. Thus, the basic information

of genome sequence helpful for molecular based detection method development specially Polymerase chain reaction (PCR) based methods.

Main focused was on PCR as it is a rapid detection method. Among that Reverse Transcription Polymerase Chain Reaction (RT-PCR) is the most commonly used and still be using the method as it is exactly showing the positive results with great -sensitivity. RT-PCR was said to be a gold-standard for testing coronavirus [8]. RT-PCR can detect the virus from throat, nasopharyngeal swab as well as from stool sample [9]. Early detection using PCR prove to be most sensitive way as negative sample cannot be ruled out. One cohort study found that patients even if not having any symptoms still may be the carrier for viruses thus RT-PCR can detect those at genetic level. Thus, it helps in prevention of nosocomial infection and further spread unknowingly [10].

Sample collection methods also affect the diagnosis results. Thus, WHO and CDC recommended the use of nylon, rayon or any synthetic fibre swabs for sample collection. But shortage of such swabs hammered the testing numbers. Hence, some researchers decided to study the effect of cotton tipped plastic swabs on PCR results and it found that no inhibition effect was seen on PCR [11]. Another method related to heating of sample known as Loop-mediated Isothermal Amplification (LAMP) this works by skipping the RNA extraction process. RNA RT-LAMP sensitivity found to be 97%, while specificity was 99% [12]. Study tried the three viral transmission medium heating treatments named as directly without additives, in a formamide-EDTA buffer and in a RNAsnap™ buffer. Basically, this method skips the RNA extraction process of RT-qPCR by replacing it with heating treatments thus reduces the time for the test and also gives the same result as normal RT-PCR protocol [13]. Similarly, in one study RNA extraction process was skipped and on that place RT-qPCR master mixes were used. Now this helps under such situation where reagent shortage occurs in hospitals. It also expanded the testing capacities [14]. Similar way direct RT-qPCR was used in one of the study found to be an alternative for classical method without RNA extraction [15]. But there are certain limitations in remote areas and portable diagnosis will be preferable using mini PCR based diagnosis kits. One study combined the mini PCR and multi-well plate reader for convenient and portable diagnosis under pandemic situation [16].

As per the available RNA sequences and need of diagnosis method for COVID-19, several RNA extraction kits are available in the market. But due to pandemic situation and rising number of COVID patients, it was under shortage. Thus, magnetic beads base RNA extraction methods were used by various researchers. Silica beads were found to yields RNA and comparable with commercially available QIAcube viral RNA extraction kit which were determined by RT-qPCR and RT-LAMP [17]. Another study due to complexity of protocol was done, in which rather than using transport medium and RNA extraction it was decided to study the use of direct elution of swab and performing RT-qPCR. Elution of dry swabs were done directly in simple TE buffer and tested. Thus, this process simplifies the pre RT-qPCR preparation [18].

Commercial RT-PCR kits are already available in the market and few of them are manufactured by Altona Diagnostics (Hamburg, Germany), BGI (Shenzhen, China), CerTest Biotec (Zaragoza, Spain), KH Medical (Gyeonggi-do, Republic of Korea), Primer Design (Chandler's Ford, UK), R-Biopharm AG (Darmstadt, Germany), and Seegene (Seoul, South Korea). All these kits are the best for detection without any cross-reactivity with another virus and hence can be used for regular diagnosis of SARS-CoV-2 [19]. But the limit of detection (LoD) of kits matters the quality. Earlier China National Medical Products Administration (NMPA) approved 6 PCR kits, but due to time shortage optimisation was not done so LoD might get affected. Thus

examination of LoD of this 6 PCR kits with real RNA of the virus was carried. All kits showed different LoD and the poorest LoDs has high chances of giving false negative results. The lab should confirm the performance of the kits and the utilise [20]. Some of the primers and probes for CoV detection has been shown in **Table 1**.

Currently, the RT-PCR test has a 95% specificity and 70% of the sensitivity rate [28, 29]. Self-collected saliva specimens was tested with 6 different molecular diagnostic tests like RTqPCR LDT, SARS-CoV-2 RAT, 3 direct RT-qPCR kits, and RT-LAMP and all tests showed a excellent results. Thus, based on these results, molecular diagnostic method has a great scope ahead [30]. Dr. Aneesh Mehta said that saliva test for coronavirus by PCR is the new type of diagnosis, as it is not invasive method but just spit is required as a sample [31]. This test is said to be a 'SalivaDirect' test developed by a scientist of the Yale School of Public Health, authorised by Food and Drug Administration (FDA) [32].

Most countries have been used Indian Council of Medical Research (ICMR, India) suggested Rapid Antigen Test (RAT) which was available as a kit and direct antigen identification from nasal swab [33]. However several countries found that the negative findings by RAT were shown to be positive by RT-PCR. Thus, according to Dr. Balram Bhargava, Director of ICMR, the RAT was found to offer false-negative outcomes. One of the reports says that around 11% of people found negative by RAT in Delhi, after testing with RT-PCR found to be positive [34]. The Indian Health Ministry and ICMR provided a guideline to re-test the RAT tested negative patients and those who develop symptoms after a few days of the test by RT-PCR [35]. Until now, RT-PCR is the recommended test for all organisations and has been followed. Still, many experts are trying to carry out a number of inventions and studies to make it simpler, more practical and more cost-effective for the whole planet. This book chapter summarised the upto date basics and applied study


#### **Table 1.**

*Primers and probes recorded for SARS-CoV-2 real-time reverse transcription PCR assays.*

of PCR based diagnosis of SARs CoV-2 including current challenges of diagnosis, protocols and future prospects.

## **2. Challenges for SARs CoV-2 diagnosis**

For treatment of SARs CoV-2, no vaccines is available but it was only the option left out to healthcare sectors that to prevent transmission as soon as possible [26]. So most the countries invested in isolation and detection [26]. The diagnosis of COVID-19 was difficult as it shows symptoms similar to those of flu viruses, thus it was crucial to find the diagnosis as early as possible for management purpose [36]. One of the most important problems in analytical chemistry was highlighted by the COVID-19 pandemic outbreak: the discrepancies between the testing technique (TT) and the testing method (TM) are a common confusion in the clinical field. In addition to the research procedure, TT consists of many steps, such as the collection of specimens, their preservation, storage, transport, labelling and distribution. Pre-test planning procedures for patients are also part of the process. Previously, these procedures, also known as preanalytical variables, have been identified as the key causes of laboratory testing errors. The most acceptable TT for the TM must be validated during the production of the TM for the identification of the target analyte; otherwise the analysis is performed within a wide range of analytical errors [37]. Successful detection of the virus also depends on time of testing, early or late detection, viral load, sample collection etc. [38].

Further challenge was during the false negative results. It is easy to understand and interpret a perfect test for a disease; the test would only be positive if the disease was present, and it would only be negative if it were absent. However, since all studies have false positives and false negative results, diagnostic tests are not flawless. Test results do not definitively state whether or not there is a disease (or virus). This does not mean that the test is not beneficial; it merely implies that the test results must be probabilistically tested on the basis of test output characteristics, patient data, and disease prevalence [39]. To interpret the results of incomplete tests, two main metrics that characterise the test are needed: diagnostic sensitivity and diagnosis specificity [40]. At present, for commonly used SARS-CoV-2 samples, there is minimal information on these values. To accurately interpret an incomplete test, the approximate probability that the person being evaluated has the disorder must also be considered [39].

The diagnosis was completely relied on two ways Molecular and serological testing as shown in **Figure 1**. Different test among these two categories was tried during a pandemic situation [41].

Currently, RT-PCR is commonly used method by many laboratories due to specificity and fast detection [40]. Some test commonly use for CoV detection has been shown in **Table 2.**

**Figure 1.** *Two ways of COVID-19 diagnosis.*


**Table 2.**

*Diagnostic testing for SARS-CoV-2.*

#### **3. Real-time RT-PCR basics**

RT-PCR stands for real-time reverse transcription-polymerase chain reaction (RT-PCR). It is a technique use to determine the nucleic acid (DNA/RNA) from sample specifically from a virus or bacteria. Currently used for COVID-19 testing. It combines the reverse transcription of RNA into DNA It uses radioactive isotope marker or fluorescent dyes for detection of the targeted gene. It can detect genetic material from nose, throat, stool and sputum sample [45].

#### **4. Real-time RT-PCR protocol**

SARS-CoV-2 testing solely depends on patients' health and concern, if the patient is getting some symptoms like cough, fever, headache or related, then the patient can personally take a test by visiting COVID center. If patient is already under medical treatment related to some other health issues, then doctor can recommend a COVID test to that patient for an early treatment. After that the most important part of RT-PCR covid testing is a collection of sample. It is most

important as if the sample is not collected properly, it can affect the result as seen in many research [46–48]. CDC has provided the protocol for sample collection and all labs are following this same. Along with that, instruction regarding virus isolation has been given, it is not suppose to be done by an unless it is performed in the BSL-3 laboratory. Sample recommended for collection are nasopharyngeal (NP) and oropharyngeal (OP) swab for RT-PCR [49]. An expert technician can collect the sample from the nose and throat of patient separately by using swab which is individually wrapped. Swab has to be made up of synthetic fiber like plastic or wire shafts. Technician has to follow certain rules while collecting sample like 6 feet of separation, personal protective equipment (PPE) kit, proper gloves and faced covered lid. The sample is to be collected deeply. Once collected it has to be transferred to viral transport medium (VTM). VTM is made up of 2% FBS, 100 μg/mL Gentamicin and 0.5 μg/mL Amphotericin B. Among this 3 ml of media is transferred to sterile screwed capped bottles in which swab is put and stored before further testing [50]. Sample can be stored at 2–8 °C for 72 hours only. If the sample has to be transported for a long distance, then it should be carried in icepack [51].

Once the sample has been collected, then next important and time consuming process is RNA extraction means purification of RNA samples. Most of RNA extraction kits are available in the market shown in **Table 3**, which can be used directly contain lysis buffer and other chemicals which will lyse the virus and RNA will get into solution. First, in order to release the genetic material, the patient's sample is


#### **Table 3.**

*Virus extraction and detection kits.*

#### *Development of RT-PCR Based Diagnosis of SARS-CoV-2 DOI: http://dx.doi.org/10.5772/intechopen.96823*

mixed with a solution which lyses the cells. In order to purify RNA, inactivation of RNA activity, denaturation of nucleoprotein complexes and elimination of contaminating DNA and proteins must take place by (phenol which will attract the other protein and break it down, guanidine isothyocynate is also a protein denaturant, RNase inhibitors to inactivate the ribonuclease enzyme) [52]. The resulting cellular debris will then proceed to the RT-PCR stage with the extracted RNA. Further RNA purification is carried by using kit or by centrifugation and solid phase extraction by using column Centrifuge and spin column, which is to be placed in a clean collection tube to collect the supernatant and filtrate is discarded. Again, it has to be washed and centrifuge. Further by using elution buffer RNA is purified.

After purification of viral RNA, the next step is the preparation of the reaction mixture for PCR amplification. In this step master mix has to be used which is premixed concentrated solution that consists of buffer, Reverse transcriptase enzyme nucleotide, forward primer, reverse primer, TaqMan probe, DNA polymerase. Finally, the RNA template to be added and mixed by pulse vortexing. Then load the reaction mixture into a PCR plate which generally contain 96 wells, allowing analysis of several samples at a time. Then place this plate in PCR machine (thermal cycler). Real time RT-PCR is used for detection of new Coronavirus 2019 by amplification of target sequences in Rdrp genes, E gene and N gene. The choice of the target depends on primers and the probe sequences. The first step in RT-PCR is reverse transcription. The first strand complementary DNA synthesis is primed with the PCR reverse primer which hybridizes the complementary part of the virus RNA genome. Reverse transcriptase then add DNA nucleotides onto the 3-prime end of the primer synthesizing DNA complementary of the viral RNA. Then denaturation takes place. Thus PCR consists of a series of cycles consisting of Denaturation, annealing and elongation. In cycle 1, DNA denaturation at 95 °C occurs. The next step at 58 °C allows the annealing of forward primer to complementary part of DNA. In elongation step, DNA polymerase synthesize a new strand complementary to the DNA template by adding nucleotides from the reaction mixture. In 2nd cycle, DNA denaturation form ssDNA, then annealing of primers


**Figure 2.**

*RT-PCR methodological performance flowchart.*

and Taq-man probe to its complementary part of the target DNA. Taq-man probe consist of a fluorophore covalentaly attached to the 5′ end of oligonucleotide probe, the fluorescence is emitted by the fluorophore when is excited by the cyclers light source. Also, this probe consists of quencher at 3′ end. The close proximity of the reporter to the quencher prevents detection of its fluorescence. When polymerase reaches the Taqman probe its endogenous 5′ nuclease activity cleaves the probe, separating the dye from the quencher, with each cycle of PCR more dyes are released, resulting in an increase in fluorescence intensity proportionately to the amount of amplicon synthesized. For the fluorescence signal a tungsten-halogen lamp, an excitation filter, lens, emission filter and charged coupled device (CCD) camera are use which converts light into digital data. A flowchart of the protocol has shown in **Figure 2** [24].

### **5. Recent development of RT-PCR for SARS-CoV-2 diagnosis**

In addition to prevention methods (e.g., hygiene, social distance, isolation of infected individuals, and travel restriction), rigorous community infection testing is essential to track the transmission of the disease as well as educating public policies [53]. Nations that have implemented large research strategies at an early stage like South Korea, Vietnam, and New Zealand have been better able to restrict the spread of the disease. Tests should ideally be simple to sample and evaluate, precise, reliable, scalable and inexpensive. Often, point-of-care tests (POCT) based on antibodies match this definition. However, rapidly emerging epidemics due to novel viruses do not allow antibody-based tests to evolve in a timely manner [54]. Because of the simple adaptability to the nucleotide sequence of the target, viral load tests based on real-time, quantitative RT-PCR (referred to as RTqPCR) are thus an ideal test [55]. This RT-qPCR is currently a reliable test commonly used for the diagnosis of SARS-CoV-2 infected symptomatic and asymptomatic patients [56]. Several scientific and clinical institutions around the world have produced molecular assays to diagnose SARS-CoV-2 and have made RT-qPCR primers and sample sets available to the public [57].

As the genetic code of coronaviruses consists of RNA, its purification of the test samples is a crucial step in RT-qPCR protocols. Officially, institutions in some countries have suggested unique RNA isolation kits for SARSCoV-2 detection [57]. Various virus extraction and detections kits are available in market as shown in **Table 3**. The qRT-PCR has the benefits of high sensitivity, high precision, and a wide variety of sample types that can be use but there are however several factors that influence qRT-PCR outcomes, such as repeated washing, purification and separation of viral nucleic acids, which cause a substantial loss of nucleic acid and increase the risk of fragmentation and hydrolysis of nucleic acid; the number of virus replications below the qRT-PCR detection threshold would also cause false negative effects on the early stage of SARS-CoV-2 infection. So this problem was faced by many laboratories [58]. Some started focusing on RT-LAMP—reverse transcriptase loop-mediated isothermal amplification [59]. Other PCR, which was found to be more potent to detect low viral load was digital droplet PCR (ddPCR) as compared to qRT-PCR but due to limited availability ddPCR instrumentation and lab expertise mostly qRT-PCR is being used [60]. At this point, superior resources are concentrated in all parts of the country to reinforce scientific research. It is assumed that our ability to diagnose and treat patients with new coronavirus pneumonia will develop further with the enhancement and advancement of detection technology.
