Covid-19 Test Detection by Real Time RT-PCR

 

Saxena Pranjal¹*, Goswami Raksha¹*, Pandey Haymanshu², Kumawat Deepak², Chandy Steffy Mary²

¹Department of Pharmacology, Faculty of Pharmacy, Department of Pharmaceutical Chemistry,

Faculty of Pharmacy, Oriental University, Indore (M.P.) – India.

²Acropolis Institute of Pharmaceutical Education & Research, Indore (M.P.) – India.

 

 

ABSTRACT:

Corona Virus is a disease caused by Novel coronavirus is initially called 2019-nCoV. On 11th February 2020 WHO declared an officially name called COVID-19. Corona Virus is a large family of virus there are two more virus which can also declared as PANDEMIC.  Namely SARS and MERC.SARS in 2002 to 2003 and MERC in 2012. These diseases which infects Human and Bats. WHO declares COVID-19 is a global pandemic? COVID-19 affect more than 30 lakhs people over the world. The main symptoms are is fever, cough and shortness of breath. This review shows that the vaccine and dectetion of COVID-19 by RT-PCR. RT-PCR is a technique widely used in biotechnology in this technique we make copies of billions of DNA and RNA. Only DNA is Synthesis in PCR. But Reverse Transcription Polymerase Chain Reaction is slightly different than PCR. In this we have synthesis DNA and RNA both. This RTPCR technique is used in the determination of COVID-19 in human being. This technique widely used in Biotechnology.

 

 

 

INTRODUCTION:

COVID-19 is a disease caused by Novel coronavirus termed as SARS-CoV-2 by International committee of Taxonomy of Virus(ICTV).

 

WHO Declared COVID-19 as global pandemic on 11th February 2020. It effect more than 200 countries across the World. First identified in Wuhan, city in Hubei, China. First reported by WHO on December 31,2019. COVID-19 affect more than 50 lakh peoples all over the World till now and many death reported till now [1].

 

In India, it affect more than 1 lakh peoples across the country[2]. From 2002 to 2003, the severe acute respiratory syndrome coronavirus (SARS-CoV) rise an epidemic started in China later spread to more than 24 countries across the world. Approximately 8000 cases and 800 deaths were reported. In 2009, Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 was first detected in Saudi Arabia and caused about 2,500 cases and 800 deaths. This is also a family member of Novel corona virus. The mortality rate of severe acute respiratory syndrome coronavirus (SARS-CoV) was reported up to 10%.The potential for this SARS-CoV-2 virus to grow to become a pandemic worldwide presents a serious public health hazard. With reference to COVID-19, the WHO raised the threat to the CoV epidemic to the "very high" level, on February 28, 2020. At the same time, scientists of all over the world works  hardly to gathering information and scientific evidence about the etiology, pathophysiology, epidemiology, clinical manifestations and diagnosis, therapeutic strategies, and management of this novelcoronavirus disease. Progressive reduction of cases was observed in China implementing determined isolation measures in the last few days [4].

 

Etiology and Pathophysiology:

Corona-viruses are positive single-stranded RNA viruses. When observed under an electron microscope, coronaviruses have a crown-like appearance owing to the presence of spike glycoproteins on their envelope that infects humans along with a wide range of animals. In 1965, two scientists Tyrrell and Bynoe, isolated a virus from the nasal washings of a male child. The child had typical symptoms and signs of a common cold and the washing was found to be able to induce common colds in presented a detail explanation of coronaviruses after cultivating viruses from patients suffering from common colds [5]. On the basis of their morphological observations as spherical virions with a core-shell and projections on the surface of enveloping that look like a solar corona, these viruses were called as coronavirus [6] (Latin: corona = crown). The Coronaviridae family (order Nidovirales) diverges further into subfamilies of which subfamily Orthocoronavirinae further classified into four genera of CoVs:  Alphacorona-virus (alphaCoV), Betacoronavirus (betaCoV), Deltacoronavirus (deltaCoV) and Gammacorona-virus (gammaCoV). Members of this huge family of viruses are found responsible for causing respiratory, enteric, hepatic, and neurological ailments in various animal species and humans[7]. There are seven human CoVs (HCoVs) having the capability of infecting humans has been recognized. Broadly, the estimate suggests that healthy carriers of a CoV constitute 2% of the total population[8]. Among these seven types of human coronaviruses that can communicate a disease to humans, the beta-coronaviruses are known to cause severe illness and epidemics[9].

 

Detection of COVID-19 by RTPCR:-

COVID-19 test is detection by RTPCR for a qualitative detection of nucleic acid form SARS nCoV-2 in lower and upper respiratory tract of human respiratory system. This test is most used for the Home collection kit to self collect Nasal swab specimens at home by any person which is determined by Healthcare professionals The SARS-CoV-2 RNA is generally detectable in respiratory specimens during the acute phase of infection. Positive results are indicative of the presence of SARS-CoV-2 RNA; clinical correlation with patient history and other diagnostic information is necessary to determine patient is suffering from infection. Positive results do not rule out bacterial infection or co-infection with other viruses. The agent detected may not be the definite cause of diseases. Testing of COVID-19 by RT-PCR test is only used by trained healthcare professionals, laboratory personnels specifically instruction and training in the techniques of real-time polymerase chain reaction. In response to this need, three types of diagnostic tests for SARS-nCoV-2 were quickly developed: Tissue culture isolation, Antibody detection, and reverse transcription-polymerase chain reaction (RT-PCR) assays. In this pandemic situation various types of kits are manufactured including a commercial ready-to-use RT-PCR kit. Early RT-PCR assays based on conventional design which was time consuming. Real-time RT-PCR assays based on detecting and quantitating by fluorescent signal generated during amplification. This RTPCR is based on real-time format, based on hydrolysis technology (Applied Biosystems), has been shown to provide sensitive, accurate, and quantitative results in viral diagnostic assays. It has been used to successfully detection of virus infections. In this pandemic situation of COVID-19 public health emergency, we used real-time RT-PCR technique based on three main targets in the detection of SARS-nCoV-2.

 

MATERIALS AND METHODS:

Clinical Specimens

A total number of clinical specimens collected in which some persons with confirmed or suspected SARS-nCoV-2 infection were used in this study[20]. Samples of suspects included oro and nasopharyngeal swabs in upper and lower tract of respiratory system (dry and in viral transport media), sputum, nasal congestions, and lung tissue specimens are collected. Specimen processing was performed in a classII biological safety cabinet using BSL3 (biosafety level three) work practices. Three 100-µL aliquots of each sample were distributed to vials each containing 900 µL of NucliSenslysis buffer and stored at –70°C until testing.

 

Virus Culture:-

Virus cells were inoculated with the help of clinical samples and observed for cytopathic effect, consisting of cell rounding with a refractive appearance followed by detachment from the flask surface[12]. Plaque titrations were conducted by standard methods[21].

 

Nucleic Acid Extraction:-

After successful culture of virus Nucleic acids were recovered from clinical samples using the automated NucliSens extraction system. Specimens received in NucliSenslysis buffer were incubated at 37°C for 30 min with intermittent mixing, and 50 µLof silica suspension, provided in the extraction kit, was added and mixed. Then the material of the tube were then transferred to a nucleic acid extraction cartridge and processed on an extractor workstation. Approximately 40-45 µL of total nucleic acid was recovered into nuclease-free vials and tested immediately or stored at –70°C.

Primers and Probes:-

After cultivation of virus and Nucleic acids extraction Multiple primer and probe sets were added from the strain of SARS-nCoV-2 polymerase and nucleo capsid gene sequences[22] by using Primer Express software (Applied Biosystems) with the following default settings: primer melting temperature set at 60°C; probe melting temperature set at approximately 70°C; and no guanidine residues permitted at the 5′ probe termini. All primers and probes were synthesized by various techniques at the Biotechnology Core Facility at the Centers for Disease Control and Prevention (CDC). Probes were labelled at the 5′-end with the reporter molecule 6-carboxy-fluorescein and at the 3′-end with the quencher Blackhole Quencher. Optimal primer and probe concentrations were determined by cross-titration of serial dilutions of each primer against a constant amount of purified SARS-nCoV-2 RNA. Primer and probe concentrations that gave the highest amplification were selected for further study[Table 1].

 

Assay of Real-Time RT-PCR

The real-time RT-PCR assay was performed by using the Real-Time One-Step RT-PCR Applied Biosystems. Each 25-µL reaction mixture contained 12.5 µL of 2X Master Mix, 0.625 µL of the 40X MultiScribe and RNase Inhibitor mix, 0.25 µL of 10 µM probe, 0.25 µL each of 50 µM forward and reverse primers, 6.125 µL of nuclease-free water, and 5 µL of nucleic acid extract. Amplification was carried out in 96-well plates on an Real-Time Detection System. Conditions for thermocyclic consisted of 30 min at 48°C for reverse transcription, 10 min at 95°C for activation of the DNA polymerase, and 45 cycles of 15 s at 95°C and 1 min at 60°C. Each run included one SARS-nCoV-2 genomic template control and at least two template to check for contamination during sample processing and one template control for the PCR-amplification step. For controlling PCR inhibitors, and to monitor nucleic acid extraction efficiency, each sample was tested by real-time RT-PCR for the presence of the human ribonuclease (RNase). Use of the following primers and probe: forward primer 5′-AGATTTGGACCTGCGAGCG-3; reverse primer 5′-GAGCGGCTGTCTCCACAAGT3′; probe 5′TTCTGACCTGAAGGCTCTGCGCG-3′. The assay was performed identically to described above except that primer concentrations used were 30 µM each. Fluorescence measurements were taken and threshold cycle value for each sample was calculated by determining the point at which fluorescence exceeded a threshold limit set at the mean plus 10 standard deviations above the baseline. A result of  test was considered positive if two or more of the SARS genomic targets showed positive results (Threshold cycle <45 cycles) and all positive and negative control reactions gave expected values. Clinical samples submitted to  Centre for Disease Control(CDC) for SARS-nCoV-2 testing that gave positive results were confirmed with real-time RT-PCR assay based on three different primer and probe sets. As shown in [Table 1]. This assay was performed independently in a separate laboratory using newly extracted nucleic acid. The confirmatory assay used the SuperScript One-Step RT-PCR and the Mx4000 Multiplex Quantitative PCR system.

 

 

Synthesis of RNA Transcription:-

Template for the nucleocapsid gene RNA was plasmid DNA containing a full-length copy of the open reading frame for the SARS-nCov-2 nucleocapsid gene oriented behind a T7 promoter. The template for the polymerase RNA was a RT-PCR product generated by using the following primers:Cor-p-F2-T7, 5′GTAATACGACTCACTATAGGGCTAACATGCTTAGGATAATGG-3′ and Cor-p-R2, 5′-CCTATTTCTATAGAGACACTC-3′. Approximately 1 µg of RNA from cells that infected with SARS-nCoV-2 was used in RT-PCR reactions performed by using the SuperScript RT-PCR kit according to the kit manufacturer’s instructions; both templates were purified by phenol chloroform extraction and ethanol precipitation before being used for in vitro transcription. RNA was synthesized in vitro by using the MegaScript kit according to the standard measures. Synthetic RNA was treated with RNase-free DNase before being purified by phenol-chloroform extraction and ethanol precipitation. The concentration of RNA was determined by use of UV spectroscopy. Synthetic RNA was positive sense and 1,369 nt in length for N and 325 nt in length for polymerase.

 

RESULTS:

Real-time RT-PCR Sensitivity and Reproducibility

Polymerase and nucleocapsid RNA transcripts were tested to assess the copy detection limits and dynamic range of our optimized real-time RT-PCR assays. Limit of detection was approximately 2 transcript copies per reaction for COVID-19. The confirmatory assays, which employe three different primer and probe sets (N3, 3′NTR, and M),showed potential limits of detection similar to the assays. Linear correlations were obtained between CT values and transcript quantity over at least a 6-log range from approximately 10² to 10⁵[11].

 

Prevention Strategies:

Since, it is spreading and there is no cure treatments available for this COVID-19 disease, prevention is essential. The infection even before the onset of symptoms in the incubation period, transmission from people to people, non-specific features of the disease, long-lasting period of the disease and transmission even after clinical improvement and sensitivity of transmission through mucosal surfaces such as the nose are few characteristics of novel coronavirus 2019 that prevention are difficult. Therefore, Isolation of confirmed or suspected cases with mild illness at home or at the hospital is the most prevention strategy. The house should be properly ventilated with sunlight access to allow for the destruction of the virus. Appropriate measures should be adopted throughout the diagnosis and during the clinical care of an infected patient. Droplet contact and airborne safety measures must be taken up during samples collection, and sputum induction should be avoided. Healthcare workers attending for infected persons should take care of contact and airborne infection by utilizing Personal protective equipment, "PPE" such as N95 masks, eye protection, gowns, and gloves to minimize the exposure of contamination and transmission of the pathogen form suspected or infected person[3].

 

CONCLUSION:

This novel coronavirus, which originated from a viral family Coronaviridae has a public health emergency, according to the World Health Organization. The COVID-19 outbreak, which has now declared as „Pandemic‟ by World Health Organization, has confronted the economic, medical, and public health infrastructure of many countries, including China, the United States of America, Italy, France,and all over the world. In India,COVID-19 newer cases are being detected daily. India is continuing to face this situation in a well-planned approach so far. The Indian government quarantined India from the rest of the world, but the detection of COVID-19 by RT-PCR techniques is very easily in this Pandemic situation Real Time RT-PCR is very useful.

 

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Received on 01.02.2021          Modified on 03.02.2021

Accepted on 07.02.2021     ©AandV Publications All right reserved