Molecular Diagnostics of COVID(Corona Virus Disease)-19.pptx

Molecular Diagnostics of COVID-19,
includes Polymerase Chain reaction, Lateral flow assays and CRISPR based SHERLOCK methods, followed by symptoms and treatment and history of Coronavirus family (CoV)
#CORONAVIRUS #MOLECULARBIOLOGY #BIOTECHNOLOGY

1. Molecular Diagnostics of COVID-19 SMSC2425101 SIES College of Arts Science & Commerce (Autonomous), SION (W), Mumbai 400 022

2. 1. INTRODUCTION COVID-19 : Corona Virus Disease-19 A respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV 2) 5' Cap : This is a modified guanine nucleotide that protects the RNA and helps in translation. S-protein : S1 domain- Receptor for binding , S2-Responsible for fusion. E-protein : Expressed in infected cells , Role in assembly and morphogenesis of virions within the cells. M-protein : Defines the shape of viral envelope N-protein : pathogenesis, replication and RNA packaging 3' UTR ( Untranslated Region) : RNA stability ACE-2 receptor : A gateway for virus entry. (Angiotensin converting enzyme) Fig.1 healthcare-in-europe.com/en/news/sars-cov-2-igg-antibody-test-receives-fda-emergency-use-authorization.html

3. 2. SYMPTOMS OF THE DISEASE Loss of taste and smell A distinguishing feature of COVID-19. This symptom is less common with other respiratory illnesses. Shortness of breath and Sore throat Lingering Symptoms Some individuals experience prolonged symptoms, even after the acute phase of the illness has passed, and can include fatigue,, joint pain, and chest discomfort Gastrointestinal Issues Nausea, Vomiting, Diarrhea.

4. 3. Outbreaks of Coronavirus family viruses around the world SARS- CoV In S.E. Asia, called Severe Acute Respiratory Syndrome Coronavirus , an unknown ( CoV ) had been identified in Bats. MERS- CoV Middle East Respiratory Syndrome, caused by a new coronavirus identified in Camels in Saudi Arabia. SARS-CoV-2 Outbreak of a new coronavirus infection (COVID-19) in Wuhan,China , indications of transmission from bats to humans. 2002/ 2003 2012 2019

5. 4. COVID-19 viral load in different specimens The samples are usually collected from upper and lower respiratory specimens such as nasal, nasopharyngeal, oropharyngeal swabs, sputum, lower respiratory tract aspirates, bronchoalveolar lavage fluid, and nasopharyngeal.  It is suggested that deep throat saliva sampling may offer early detection of SARS-CoV-2. 5. Diagnostic timing The highest pharyngeal shedding of SARS-CoV-2 is in the first few days of symptoms and peaks on day 4. Sputum viral shedding lasted till the sixth week, i.e. end of symptoms. It is possible to detect SARS-CoV-2 RNA in nasopharyngeal swab specimens one week before the onset of symptoms

6. 6. Molecular diagnostic tests 1 2 Real time (RT) – PCR Lateral Flow Assays 3 CRISPR-based Assay

7. 6.1 Real -time RT-PCR RT-PCR can amplify and detect a single copy of the specific genomic sequence and therefore, it is extremely sensitive. Real-time RT-PCR is a quantitative technique as the number of copies of RNA generated in a PCR increases exponentially and is proportional to the amount of starting material, i.e. viral load. Principle : real-time RT-PCR is used to transcribe and amplify the specific SARS-CoV-2 genomic sequence. Viral RNA is first extracted from the biological specimen collected from the nasal or nasopharyngeal swabs and is purified. Purified RNA template is converted into a cDNA (complementary DNA) by reverse transcriptase (an RNA-dependent DNA polymerase enzyme). The cDNA is subsequently amplified by the Polymerase chain reaction . 

8. Limitations : Sample Collection False Negatives Cost and Resources Advantages : High Sensitivity and Specificity Rapid Results Quantitative Fig.2 www.sciencedirect.com/science/article/pii/S2090123220301788 Probes : TaqMan (dual labeled) Fluorescent signals are detected in real time and data is plotted against Replication cycles. Cycle threshold (Ct) – No. of PCR cycles needed to report detectable fluorescence. One-step assay – Single tube & buffer for RT and PCR. ( high speed, less risk of cross contamination and human error). Two step assay – RT and PCR performed separately. ( greater sensitivity and low detection time)

9. 6.2 Lateral Flow A ssay It is a Point of care (POC) aka bed side testing. A portable tool for pathogen diagnosis. It is latest technique working on the principle of fluid flow and is employed in immune assay targeting proteins, DNA or RNA based sample. IgG and IgM Types : Antigen test : (for Active infection) Detects proteins from SARS-CoV-2 Virus. the strip has antibodies specific to SARS-CoV-2 antigen Antibody test : (for past infection) Detects antibodies produced in response to SARS-CoV-2 virus the strip contains viral antigen that captures specific antibodies if present in the sample.

10. Fig.3 www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2020.560616/full Fig.4 www.creative-diagnostics.com/Immunochromatography-guide.htm Advantages : Speed Portability Ease of use Scalibility ASSAY PROCESS : Application: The prepared sample is added to the sample pad. It then migrates through the conjugate pad, where any viral antigens or antibodies bind to the conjugated particles. Binding and Movement: The sample continues to move through the membrane strip. If viral antigens or antibodies are present, they will bind to specific capture antibodies or antigens that are fixed on the membrane, forming a visible line. Control Line: A separate control line is present on the membrane, which will also develop a visible line if the test is functioning correctly Limitations : Sensitivity Variability Proper sample collection

11. 6.3 SHERLOCK Method (Specific High-sensitivity Enzymatic Reporter unLOCKing ) This method is a molecular diagnostic technique developed for detecting specific nucleic acids, and it has been adapted to detect SARS-CoV-2, the virus responsible for COVID-19. Sample Collection and Preparation: A sample, usually a nasal or throat swab, is collected from the patient and processed to extract RNA. RNA Extraction: The RNA from the virus is extracted from the sample. CRISPR- Cas System: . In SHERLOCK, the Cas13 enzyme is guided to the target RNA by a CRISPR RNA ( crRNA ) that is complementary to a specific sequence in the SARS-CoV-2 RNA.

12. Advantages High Sensitivity and Specificity Rapid Results Multiplexing Capability Detection Reaction: When the Cas13 enzyme binds to the target RNA, it activates and cleaves nearby RNA molecules indiscriminately. This cleavage can be detected through a fluorescent signal or a colorimetric change in a test device, indicating the presence of the virus. Result Interpretation: The presence or absence of the fluorescent or colorimetric signal indicates whether SARS-CoV-2 RNA is present in the sample Limitations Complexity of Optimization Limited Availability Potential for False Negatives Fig. 5 SARS-CoV-2 pandemic: a review of molecular diagnostic tools including sample collection and commercial response with associated advantages and limitations Protospacer Adjacent Motif , which is a short, conserved DNA sequence that is crucial for the recognition and binding of the CRISPR-associated protein to its target DNA.

13. 7. TREATMENT Antiviral Medications Remdesivir ( Veklury ) It works by inhibiting viral RNA polymerase, thus interfering with viral replication. 01 03 02 04 Vaccinations COVID-19 vaccines have been shown to significantly reduce the risk of severe illness, hospitalization, and death. Boosters are also recommended to maintain immunity, especially in the context of emerging variants. Corticosteroids Dexamethasone : A corticosteroid that reduces inflammation and is used in severe cases of COVID-19 to decrease immune system overactivity and manage symptoms. Plasma therapy Plasma from recovered COVID-19 patients, which contains antibodies against the virus, has been used in some cases, though its effectiveness has been variable

14. 8. CONCLUSIONS Turnaround Time Traditional PCR tests can take several hours to days to return results, Variability in test performance and the need for standardized procedures can affect the reliability of results across different testing platforms and laboratories. Evolving Virus The emergence of new variants of SARS-CoV-2 presents challenges for detection, as some mutations can affect test accuracy. Continuous updates and validation of diagnostic tests are necessary to address these changes. Global Standardization Efforts to standardize testing protocols and ensure quality control across different regions and laboratories will help improve the reliability and comparability of results. 01 02 03

15. 9. REFERENCES Afzal , Adeel . "Molecular diagnostic technologies for COVID-19: Limitations and challenges."  Journal of advanced research  26 (2020): 149-159. Jayamohan , Harikrishnan , et al. "SARS-CoV-2 pandemic: a review of molecular diagnostic tools including sample collection and commercial response with associated advantages and limitations."  Analytical and Bioanalytical Chemistry  413 (2021): 49-71. Islam, Khursheed Ul , and Jawed Iqbal . "An update on molecular diagnostics for COVID-19."  Frontiers in cellular and infection microbiology  10 (2020): 560616 Liu, Caixia , et al. "A facile assay for rapid detection of COVID-19 antibodies."  Rsc Advances  10.47 (2020): 28041-28048.

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