Diagnosis of COVID19

Methods of Diagnosis of Corona Virus

1. D iagnostic tests for C oronavirus disease 2019 ( COVID-19) Visit for more vedio and Subscribe @ Hitesh Biochem

2. What is origin and How it spread?

3. What is COVID19? COVID-19 – Coronavirus disease-19 A respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Corona has crown-like spikes on the outer surface of the virus Coronaviruses are minute in size (65–125 nm in diameter) Single-stranded RNA as a nucleic material, size Why named Corona Virus?

4. What is structural characteristics? Spike protein: S - proteins are functionally divided into two subunits (S1 and S2). S1 domain: Responsible for receptor binding to receptor on target cell S2 domain: Responsible for fusion N protein : plays an important role in viral pathogenesis, replication, and RNA packaging CoV replication cycle Membrane (M) protein: M protein is the most abundant structural protein and defines the shape of the viral envelope. glycosylated in the Golgi apparatus Envelope (E) protein : the smallest of the major structural proteins , E is abundantly expressed inside the infected cell, Role in the assembly and morphogenesis of virions within the cell.

5. D iagnostic tests for C oronavirus disease 2019 ( COVID-19) Visit for more vedio and Subscribe @ Hitesh Biochem

6. Diagnostics procedure How paramedical staff will diagnosis of infection caused by SARS - CoV - 2? Q. What are the percussion soluble be taken for collection of sample? Sample Collection Ans. Collecting the proper respiratory tract specimen at the right time from the right anatomic site How to collect sample? Within 5 – 6 days of the onset of symptoms when high viral load s in their upper and lower respiratory tract

7. A nasopharyngeal (NP) swab or an oropharyngeal (OP) swab are often recommended OP swabs were used much more frequently in China But SARS - CoV -2 RNA was detected only in 32% of OP swabs. Nasopharyngeal (NP) is Best. The swab must be inserted deeply into the nasal cavity and should elicit “tears Where the swab to be inserted?

8. Swabs should be kept in place for 10 seconds while twirling the swab three times Personal Protective Equipment (PPE ) Swabs should be placed in viral (universal) transport medium for rapid transportation to the clinical microbiology laboratory Bronchoalveolar lavage Other Sample ???? Sputum- L ower respiratory specimens Yielded the highest SARS - CoV -2 RNA rate Virus has been detected in blood and stool,

9. Safety Measures for Specimen Processing should be done in a class II biological safety cabinet Safety cabinet should be in a negative pressure room within the laboratory

10. D iagnostic tests for C oronavirus disease 2019 ( COVID-19) Visit for more vedio and Subscribe @ Hitesh Biochem

11. Methods of Testing RT -PCR) test method: preferred testing method for the diagnosis of COVID19

12. Processing of respiratory specimen for nucleic acid extraction before real -time RT -PCR Real-time reverse-transcription polymerase chain reaction (RT-PCR) The specimen should be transferred to lysis buffer Lysis buffer = guanidinium -based inactivating agent as well as a non -denaturing detergent.

13. Real-time reverse-transcription polymerase chain reaction (RT-PCR) RNA extraction from Virus: self -enclosed systems integrating nucleic acid extraction ( GeneXpert ®) Used as a frontline test for COVID-19 as it directly tests for the presence of the virus RNA. Sensitive and reliable, capable of producing results in 3-4 hours Occurs in a closed system to minimize false -positive results

14. Real-time reverse-transcription polymerase chain reaction (RT-PCR) Positive -sense, single -stranded RNA genome that can be used for PCR assays The test kit enzymes copy the RNA to DNA, which is amplified to allow virus detection by using a PCR machine. 35 billion copies of viral DNA are made for each viral RNA strand Fluorescent markers are typically used to bind to the amplified DNA and produce light, which can be read by the machine to produce the test result

15. Real-time reverse-transcription polymerase chain reaction (RT-PCR) A cycle threshold value (Ct -value) less than 40 is defined as a positive test RT-PCR is accepted by scientists and medical staff as a robust and well documented technique.

16. Lateral Flow / Colloidal Gold Immunochromatography ‘ Antibody tests’ ----- used for pregnancy tests. Currently used to detect antibodies to disease in a patient's blood Tests can detect antibody to virus from patient blood Detect the presence of active virus by detecting virus proteins directly Require a drop of patient blood Ability to see if patients are currently infected or have recovered from COVID-19

17. Lateral Flow / Colloidal Gold Immunochromatography Label (Enzymes or nanoparticles or fluorescence dyes) coated antibody or aptamer is immobilized at conjugate pad. A primary antibody or aptamer against target analyte is immobilized over test line. A secondary antibody or probe against labeled conjugate antibody/ aptamer is immobilized at control zone.

18. Lateral Flow / Colloidal Gold Immunochromatography The antibody IgG and IgM lateral flow immunoassay tests are very simple to read: Test lines will appear if either of the antibody types are found in the sample. The appearance of lines for IgG or IgM, or both indicate a positive test

19. Enzyme-Linked Immunosorbent Assay (ELISA) An Enzyme-Linked Immunosorbent Assay (ELISA) is a common biochemical technique that can be used to detect antigens or antibodies ELISAs use enzymes linked to antibodies that can attach to the molecule that is being tested for and cause a colour change that can be measured by a specialised machine (ELISA Reader) Allowing cheap and time effective method for batch testing of large numbers of patient The most effective ELISA assays in monitoring prior infection detect antibodies against SARS-CoV-2. An ELISA detects antibodies produced in patient blood due to infection with SARS-CoV-2.

20. Enzyme-Linked Immunosorbent Assay (ELISA) A patient sample of blood or serum 2. The patient antibodies to SARS-CoV-2 stick to the SARS-CoV-2 proteins coated on the bottom 3. Antibodies with enzymes attached are added and stick to patient antibodies present in the well 4. Special colourless molecule is added to the well. Interpretation: An antibody test using ELISA would show a positive result (colour change) if the patient has antibodies to COVID-19.

21. Drug used for treatment Chloroquine approved for emergency use by US FDA ( hydroxychloroquine / Plaquenil , used to treat malaria and arthritis) Favilavir , the first approved coronavirus drug in China An anti-viral drug, as a treatment for coronavirus .

22. Vaccine under development University of Oxford’s ChAdOx1 nCoV-19 is an adenovirus vaccine vector developed by the university’s Jenner Institute  Alabama at Birmingham (UAB) to develop a single dose intranasal vaccine for COVID-19 named AdCOVID . DNA vaccine by Entos Pharmaceuticals