INTRODUCTION:

Corona viruses are spherical in shape containing single, positive stranded RNA as their genetic material, with a diameter ranging from 65 to 140nm¹ and having numerous spikes made up of glycoproteins on their outer surface as shown in figure 1. Due to presence of these spikes corona virus looks like a crown, when seen under electron microscope. Corona Viruses, which are contagious to humans, were first identified in mid 1960s and classified in 7 types named 229E, NL63, OC43, HKU1, SARS-CoV, MERS-CoV, SARS-CoV 19 under four sub-groupings namely alphaCoV, beta CoV, gamma Cov, and deltaCov². Corona viruses mostly infecting humans are 229E, NL63, OC43, HKU1 types. However, sometimes, due to evolution of these viruses in animals, some new antigenic and virulent strains are developed. This theory supports the genetic similarity between SARS-CoV, MERS-CoV and HCoV-229E and bat³. All of these corona viruses are different in both antigenic determinant i.e. specificity to particular antibody and in the media requirement for their growth⁴.

This evolved SARS-CoV-2 is caused by beta-corona virus⁵ and has greater specificity for host cell than original virus so the spreading rate of this disease is 10 to 20 times higher.

Figure 1: Structure of SARS-COV-2

Mechanism of interaction of sars-cov-2 with human cells receptors:

This corona pandemic was caused by SARS-CoV-2, which enters to human cell from eyes, nose and mouth as air borne droplets^6,7. It invades the cell membrane by lock and key concept. The spike present on cell surface of SARS-CoV-2 works as a key to unlock the special protein named as angiotensin converting receptor (ACE 2) present on human cell surface^8,9. After this, SARS-CoV-2 uses another protein named trans-membrane protease serine 2 (TMPRSS2) for replication process. This protein cleaves the glycoprotein spikes of present on virus surface and activates the glycoprotein for entering in the host cell by endocytosis¹⁰. After unlocking this, SARS-CoV-2 uses release its RNA in to host cell cytoplasm for replication¹¹. SARS-CoV-2 replicates by transcription process in host cytoplasm by using host cell genetic machinery. As replication proceeds, SARS-CoV-2 kills host respiratory cells which are involved in mucus formation and protection of cells from antigens and hence weaken the immune system.

Signs and symptoms of SARS-COV-2:

COVID-19 affects different people in different ways as shown in table 1. Most infected people will develop mild to moderate illness and recover without hospitalization.

Table 1: Sign and Symptoms of SARS-COV-2

Most common symptoms

Less common symptoms

Serious symptoms

·Fever

·Dry cough

·Tiredness

· Aches and pains

· Sore throat

· Diarrhoea

· Conjunctivitis

· Headache

· Loss of taste or smell

· Rash on skin, or

· Discolouration of fingers or Toes

· Difficulty breathing or

· Shortness of breath

· Chest pain or pressure

· Loss of speech or movement

Diagnostic techniques for the detection of SARS-COV-2:

In the year 2002, many cases of severe febrile respiratory illness, also known as atypical pneumonia, appeared in Guangdong city of China spreading almost in whole Asia. This occurred due to a virus namely SARS-CoV. Later the disease was named as severe acute respiratory syndrome (SARS) by WHO^12,13. The symptoms of this disease were similar to that of other respiratory infections as the case in almost all viral diseases. To prevent and treat this pandemic, diagnosis and identification of infector are the first most important task and require a more sensitive and reliable method. In order to diagnose the disease, three methods viz. tissue culture isolation, antibody detection, and RT-PCR are used¹⁴. Among these RT-PCR is considered as the best for detection of SARS-CoV-2.

Real Time RT-PCR test method:

Real Time RT-PCR is a highly sensitive¹⁵ quantitative^{16, 17}laboratory test based on in-vitro detection of specific genetic material in disease causing microorganisms including viruses¹⁸. This method is considered as gold standard for the detection of some viruses including SARS-CoV-2 as it is highly specific and rapid¹⁹. Real time RT-PCR is based on amplification of specific DNA obtained from the variety of source such as saliva, hair, nose swab and blood²⁰. As SARS-CoV-2virus contains RNA as genetic material instead of DNA. So, scientists have to convert RNA in to DNA using reverse transcriptase enzyme and the process is known as reverse transcription. The DNA obtained from this process is known as cDNA or cloned DNA and makes cDNA libraries²¹. Later the process of amplification of DNA starts as shown in figure 2, which results in detection of particular gene. All of the above procedure is same in Real time RT-PCR and RT-PCR.

In real time RT-PCR, special DNA-binding probes or primers are used as fluorescent reporters. These probes enable the detection of DNA amplification at any time, while in case of RT-PCR this will be possible at the end of reaction. Most commonly real time RT-PCR is of two types quantitative and semi-quantitative. Both of these categories are further subdivided in non-specific dye and specific probes type. The detailed classification of real time RT-PCR is represented in Figure 3.

The dye based real time RT-PCR is also known as SYBR green based quantitative PCR, as SYBR green is most widely used DNA binding dye for real time RT-PCR. Upon binding with double-stranded DNA of the PCR products, SYBR green emits light upon excitation. When the PCR products accumulate, the fluorescence intensity increases.

The Probe based real time RT-PCR are of different categories and some examples are enlisted below²²:

1. TaqMan probes

2. Molecular beacons probes

3. Scorpion probes.

All of these probes generate a fluorescent signal for detection of PCR products. The TaqMan probes, molecular beacons and scorpions generate fluorescence based on Förster Resonance Energy Transfer (FRET) coupling of the dye molecule and a quencher moiety to the oligonucleotide substrates²³.

1. TaqMan probes:

These probes are made up of oligonucleotides that have a fluorescent probe attached to the 5' end and a quencher to the 3' end. These probes are also known as hydrolysis probes²⁴. In PCR amplification these probes get hybridize to the target sequences located in the amplicon, and as polymerase replicates the template with TaqMan bound, and cleaves the fluorescent probe due to polymerase 5'- nuclease activity²⁵. The close vicinity between the quench molecule and the fluorescent probe prevents fluorescence from being detected through FRET. The decoupling results in the increase of intensity of fluorescence, which is proportional to the number of the probe cleavage cycles.

2. Molecular beacon probes:

Molecular beacons are also based on use of FRET detection with fluorescent probes attached to the 5' end and a quencher attached to the 3' end of an oligonucleotide substrate²⁶, similar to the TaqMan probes. Molecular beacon probes remain intact and rebind to a new target during each reaction cycle, whereas the TaqMan fluorescent probes are cleaved during amplification. In solution, the close proximity of the fluorescent probe and the quencher molecule prevents fluorescence through FRET. However, when molecular beacon probes hybridize to a target, the fluorescent dye and the quencher are separated resulting in the emittance of light upon excitation²⁷. Molecular beacons are expensive to synthesize and require separate probes for each RNA target²⁸.

4. Scorpion probes:

Like molecular beacons, the scorpion probes, will not be fluorescent active in an un-hybridized state, again, due to the fluorescent probe on the 5' end being quenched by the moiety on the 3' end of an oligonucleotide. With Scorpions, however, the 3' end also contains sequence that is complementary to the extension product of the primer on the 5' end. When the Scorpion extension binds to its complement on the amplicon, the Scorpion structure opens, prevents FRET, and enables the fluorescent signal to be measured²⁹.

CONCLUSIONS:

In present scenario of COVID-19 pandemic, the detection and prevention of this disease is vital to control this emergency. In order to diagnose the disease, three methods viz. tissue culture isolation, antibody detection, and RT-PCR are used. Among these RT-PCR is considered as the best for detection of SARS-CoV-2. Real time RT-PCR is a highly sensitive quantitative laboratory test. RT-PCR has become the benchmark technology for the detection/comparison of RNA levels for several reasons:

(a) Post PCR processing is not required.

(b) A wide range (>10⁷-fold) of RNA abundance can be measured, and

(c) It provides insight into both qualitative and quantitative data. Due to its simplicity, specificity and sensitivity, RT-PCR is used in a wide range of applications and for detecting infectious agents such as the avian flu-virus and SARS-CoV-2 (2019-nCoV) (COVID-19).

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 14.07.2020 Modified on 07.08.2020

Research J. Pharm. and Tech. 2021; 14(6):3437-3440.

DOI: 10.52711/0974-360X.2021.00598