Coronavirus Biology and Diagnosis Lecture PDF

Life cycle of SARS-CoV-2 Transmission, detection and diagnosis of COVID-19 by SARS-CoV-2 Outline: ❖ Overview of COVID-19 pandemics ❖ Transmission of respiratory infection ❖ Current status of COVID/respiratory infection diagnosis ❖ Concept and Evaluatio...

Life cycle of SARS-CoV-2 Transmission, detection and diagnosis of COVID-19 by SARS-CoV-2 Outline: ❖ Overview of COVID-19 pandemics ❖ Transmission of respiratory infection ❖ Current status of COVID/respiratory infection diagnosis ❖ Concept and Evaluation of testing methods ❖ Detection of de novo infection ❖ Summary A possible transmission cycle of a novel virus with pandemic potential Detection Methods for SARS-CoV-2 and other viruses  SARS-CoV-2 Infection Timeline As the virus multiplies, the concentration of viral antigens increase The immune system produces antibodies IgA, IgM, and IgG in response to infection As the immune response increases, the viral antigen concentration decreases This graph is for illustrative purposes only - each type of infection has its own specific timeline.  Common detection techniques For RNA For antigen/ proteins For antibodies Reverse transcription (RT) Antigen detection ELISA Antibody detection ELISA PCR SDS-PAGE Real-Time RT PCR Western Blotting Droplet digital PCR (ddPCR) CRISPR  Key considerations in developing a test Cost — how much does a test cost to develop and run? Development time — how long will it take to develop? Speed — Can test results be delivered at point of care (POC) or does the test require centralized analysis and sample shipment? Samples — how easy/invasive to gather samples? Instrumentation — is special equipment required? Specificity — how well does a test detect only the target of interest? Sensitivity — how well does a test detect low amounts of the target of interest? Choosing the right testing platforms: Trade-Offs Specificity vs sensitivity ◦ False positive — positive test results for someone not infected; can happen with low specificity tests ◦ False negative — negative test results for someone that is infected; can happen with low sensitivity tests Development time vs instrumentation ◦ Short development time may result in a test that requires specialized equipment Speed vs development time ◦ A quick test may require a long development time  Polymerase Chain Reaction (PCR) in diagnosis End point PCR-amplification of DNA template; analysis occurs after PCR, for example by gel electrophoresis Reverse transcription PCR (RT-PCR)-RNA is reverse transcribed into DNA which is amplified by PCR Real-time PCR-PCR amplification is monitored in real time; can be quantitative (qPCR) or combined with RT-PCR (Real-Time RT-qPCR) Droplet digital PCR (ddPCR)-PCR in which a single sample is fractionated into thousands of droplets prior to amplification, theoretically only one template per droplet; amplification either occurs or it doesn’t (1 or 0, “digital”), which enables absolute quantitation Isothermal recombinase polymerase amplification (RPA)-a modification of PCR using different enzymes that allows amplification from either RNA or DNA at a single temperature (relevant to CRISPR-based diagnostics)  Amplification of SARS-CoV-2 RNA Coronavirus contains RNA PCR-based detection relies on Reverse transcription Reverse transcription creates complementary DNA which can be amplified by PCR www.scientificanimations.com/wiki-images/, CC BY-SA 4.0 commons.wikimedia.org/w/index.php?curid=86436446  Reverse Transcription PCR (RT-PCR) Many copies of cDNA 3. PCR amplification 1. RNA isolation from sample 2. Reverse transcription cDNA Viral RNA Amplification by End-Point or Real-Time PCR Agilent Tape station END-POINT PCR REAL-TIME PCR DNA must first be separated by gel Product is measured during the PCR electrophoresis No electrophoresis required DNA staining and detection are done Can be quantitative (qPCR) when after PCR compared to a standard curve  Real-Time PCR Uses a fluorescent dye that binds to double stranded Fluorescence (correlates to #amplicons) Once reagents run out, the #amplicons stays flat DNA or a fluorescent probe that is specific to the target sequence. Exponential growth in # of amplicons (doubles with each cycle of PCR) The number of cycles required to pass the detection threshold depends the amount of starting template Amplicons are tagged with a In that way, real-time PCR can be quantitative fluorescent dye or fluorophore for detection (“qPCR”) Fluorescence detection: # of PCR cycles Requires no gel electrophoresis Higher throughput More sensitive, faster than PCR  Droplet Digital PCR (ddPCR) ddPCR: Is more sensitive that other PCR methods Fractionates a sample into 20,000 droplets Amplifies DNA target in every droplet Counting the positive droplets gives precise, absolute target quantification  ddPCR for Viral Detection Reverse-transcription ddPCR; combines reverse transcription with ddPCR -Accurate even in samples containing high background DNA or RNA or inhibitors — accuracy of ~98% -Can discriminate between nucleotide variants common to RNA viruses -Allows absolute quantification — no need for a standard curve (faster, easier, more accurate, uses fewer wells so higher throughput) RT-ddPCR useful for viral diagnostics, therapy development, and vaccine production  Designing PCR Primers T : Primer T values should be similar (+/-2°C). For 5′ nuclease qPCR assays, T values are normally m m m approximately 60−62°C. Length: Aim for 18−30 bases in length. This length typically yields a T of ~60−62°C. m GC content: Avoid runs of >4 Gs to prevent formation of G quadruplexes. GC content should range from 35−65% (ideally, ~50%). Sequence: Avoid sequences that may create secondary structures, self-dimers, and heterodimers. Use the free IDT online OligoAnalyzer 3.1 Tool to identify any such structures. Location—avoid repeated sequences: BLAST potential primer sequences and redesign them when they cross react in multiple places in the genome. Also consider primer location relative to SNPs. SNPs underlying the 3' primer end will have an impact on amplification.  qPCR Probe design T : For optimal detection of amplification, the probe T value should be 4−10°C higher than that of the m m primers. A probe with a higher T ;than the primers should be annealed to the template as amplification m begins. Length: Limit probe length to 30 bases when using dual-labeled probes designed with most common quenchers, as beyond this length quenching ability is decreased. GC content: To prevent G quadruplex formation, minimize the number of consecutive Gs. GC content should range from 30−80%. Sequence: Avoid a G base at the 5′ end as it has been observed that G bases can have a quenching effect on dyes such as FAM and other fluorescein derivatives. Location: Probes can be designed to bind to either the sense or antisense strand. Amplicon Length For typical cycling conditions, ideal amplicon size is between 70 and 200 bp. Longer amplicons can be designed, but cycling conditions should be adjusted to include longer extension times.  Pros/Cons of Real-Time RT-PCR Tests PROS CONS Detect active infections Cannot detect past infection Quick and easy (relatively) to define Best suited to centralized labs (instrumentation) targets and design primers Tests can take 4–6 hr to complete Can work from simple tissue swabs Accuracy can depend on viral load (LOD) (false (nasal or cheek) or sputum (blood not negatives) — ~80–85% required) Specificity — cross-reactivity with other viruses can High throughput be an issue Some point-of-care (POC) options POC options still require specific instrumentation and available offer lower throughput  ELISA-Based Tests ❖Virus proteins are antigens ❖The immune system produces antibodies against the viral antigens ❖ELISA uses the specificity of antibodies to detect antigens or antibodies  SARS-CoV-2 Infection Timeline As the virus multiplies, the concentration of viral antigens increase The immune system produces antibodies IgA, IgM, and IgG in response to infection As the immune response increases, the viral antigen concentration decreases This graph is for illustrative purposes only - each type of infection has its own specific timeline. Enzyme-Linked Immunosorbent Assay (ELISA) ELISA is a very common diagnostic technique A pregnancy test is based on Rapid, easy, does not require instrumentation the same principles as ELISA. Can detect active AND past infections ELISA requires specific antigens or antibodies. Lateral Flow versions (LFIA) use ELISA principles on a nitrocellulose strip. Examples include: ◦ Pregnancy, ovulation tests ◦ Rapid Strep test ◦ Flu test Detection of SARS-CoV-2 Proteins Nucleocapsid (N) Membrane (M) Envelope (E) Spike (S) The spike protein is very “immunogenic” — the human body produces antibodies against the spike protein.  Detection of SARS-CoV-2 by ELISA ANTIGEN DETECTION ANTIBODY DETECTION Screen patient for presence of the Screen patient for anti-virus antibodies. virus Sometimes called a serology test Example —Yang AS et al. (2020) used Example — Amanat et al. (2020) used monoclonal antibodies to screen recombinant S protein to screen patient patients for N-protein samples for anti-S antibodies  Antigen Detection by ELISA Antigen —the patient sample is tested for the Antigen presence of antigens from viruses, bacteria, etc. (virus) Primary antibody — binds to the antigen ○ can be produced in a lab by injecting the target antigen into an animal and then Primary harvesting the serum Antibody (rabbit anti-virus) Secondary antibody — binds to the constant region of the primary antibody ○ made by injecting the primary antibodies from one animal into a different animal Secondary Antibody ○ secondary antibodies are attached to an (goat anti- enzyme which catalyzes a color change when rabbit) substrate is added Enzyme Substrate — changes color in the presence of the enzyme, indicating a positive result Substrat e 25  Coronavirus Antibody Detection ELISA Coronavirus Antibody Detection ELISA Antigen Coronavirus Infection (lab-purified Patient Sample coronavirus S (anti-coronavirus protein) Coronavirus antibodies infects person will be present in sample if patient Secondary Antibody, Person makes was infected) goat anti-human antibodies against coronavirus Enzyme Substrate 26 EXPLORER.BIO-RAD.COM  Lateral Flow Serological Test for COVID-19 ▪Lateral flow Immunoassay (LFIA) uses the same elements as ELISA ▪Samples are added to the wells Control ▪Capillary action carries the sample through IgG regions containing the antibodies and probes in the nitrocellulose strip IgM ▪Antigen-antibody interaction changes the color S S S S Sample  Pros/Cons of ELISA Tests PROS CONS Can detect both active and past infections Longer development time — antibodies or antigens Inexpensive required for the test take time to identify and produce Does not require instrumentation; can be Can be less specific — finding antibodies that can performed at point of care (LFIA format) distinction between closely related viruses can be challenging Can work from multiple sample types, depending on the assay May not be sensitive enough during initial infection Rapid detection — within minutes Less likely to produce false negatives  Value of Detecting Previous Infections ▪ Better disease tracking ▪ More accurate mortality and hospitalization rates ▪ Can identify individuals who may be at lower risk of infection (useful for health care and other essential workers)* ▪ Can identify blood plasma donors ▪ Useful for studying and tracking vaccine efficacy * The presence of antibodies indicates possible immunity but is not proof of immunity.  CRISPR as a Diagnostic Tool Off Cas CRISPR diagnostics have two components: Protein-guide complexes — cut a target gRNA sequence, then cut other nucleic acids Target sequence indiscriminately Reporters — labeled nucleic acid molecules that produce a visible signal when cut On Reporters are only cut if the target sequence is cut first. Reporters Therefore, this system can be used to detect specific sequences. Signal  Pros/Cons of CRISPR based diagnostic tests PROS CONS ▪Highly sensitive and specific ▪Does not detect previous/past infections ▪Detects active infections ▪More computationally demanding to develop than real-time PCR ▪Rapid detection — within minutes to hours ▪Novel diagnostic test — not yet validated ▪Adaptable format — Point of Care (POC) readers or centralized instrumentation ▪Accommodates multiple sample types ▪Can be easily reconfigured for new targets  Summary of the conventional diagnostic test for microbial disease Real-Time RT-PCR ddRT-PCR ELISA CRISPR Pros Easy and fast to Absolute quantitation Specific, can detect Specific, accurate, develop previous infection rapid results More sensitive than Accuracy real-time RT-PCR Results within 20 POC possible Accuracy minutes Inexpensive Can be POC Cons Complex, requires Requires specific Results may not be Emergency instrumentation costly definitive due to the authorization to use instrumentation complexity of the Results usually in immune response hours to days Results usually in hours to days Oxford nanopore technology for the identification of Nucleic acid sequences Oxford Nanopore NGS Workflow for detection of new infections Analysis of the nucleic acid sequences_Reads Coronavirus diagnosis:PCR Vs. Next Generation Sequencing (NGS) **SARS-CoV-2 diagnosis with sequencing for accuracy at the nucleotide level Detection of sequence variation in different stains by NGS based assay Summary: COVID-19 testing methods  Overall Summary qPCR/RT-qPCR is specific and sensitive method for the diagnosis Immunoassays are useful to assess the status of infection and persistence of immunity Next Generation Sequencing (NGS) is important for detection of unknown infection and rapid changes in viral genome, leads to the development of diagnostic assays Rapidly developing single molecule NGS is important for detection of new infection Development of infrastructure is important to deal with the challenge of any future pandemics