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Questions and Answers
Which of the following tests is NOT commonly used for detecting RNA from SARS-CoV-2?
Which of the following tests is NOT commonly used for detecting RNA from SARS-CoV-2?
The immune system produces antibodies IgA, IgM, and IgG in response to SARS-CoV-2 infection.
The immune system produces antibodies IgA, IgM, and IgG in response to SARS-CoV-2 infection.
True
What is one key consideration when developing a test for COVID-19?
What is one key consideration when developing a test for COVID-19?
Cost, development time, speed, samples, instrumentation, specificity, or sensitivity.
During the immune response to SARS-CoV-2, as the immune response increases, the concentration of viral ______ decreases.
During the immune response to SARS-CoV-2, as the immune response increases, the concentration of viral ______ decreases.
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Match the following testing methods with their primary target:
Match the following testing methods with their primary target:
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What is a primary advantage of Real-Time PCR compared to End-Point PCR?
What is a primary advantage of Real-Time PCR compared to End-Point PCR?
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Droplet Digital PCR (ddPCR) amplifies DNA target in bulk rather than individual droplets.
Droplet Digital PCR (ddPCR) amplifies DNA target in bulk rather than individual droplets.
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What component in Real-Time PCR allows for the detection of amplicons?
What component in Real-Time PCR allows for the detection of amplicons?
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Reverse transcription is used to convert _____ into cDNA.
Reverse transcription is used to convert _____ into cDNA.
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Match each PCR method with its unique feature:
Match each PCR method with its unique feature:
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Which of the following statements is true about RT-ddPCR?
Which of the following statements is true about RT-ddPCR?
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The number of cycles in Real-Time PCR required to pass the detection threshold is independent of the starting template amount.
The number of cycles in Real-Time PCR required to pass the detection threshold is independent of the starting template amount.
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What is the primary purpose of antigen detection in ELISA tests?
What is the primary purpose of antigen detection in ELISA tests?
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ELISA tests are capable of identifying antibodies that distinguish between closely related viruses with high specificity.
ELISA tests are capable of identifying antibodies that distinguish between closely related viruses with high specificity.
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What element in the ELISA test changes color to indicate a positive result?
What element in the ELISA test changes color to indicate a positive result?
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The _______ antibody binds to the constant region of the primary antibody in an ELISA test.
The _______ antibody binds to the constant region of the primary antibody in an ELISA test.
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Match the following components of the ELISA test with their functions:
Match the following components of the ELISA test with their functions:
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What is one major advantage of Real-Time RT-PCR tests?
What is one major advantage of Real-Time RT-PCR tests?
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ELISA tests can only detect active infections.
ELISA tests can only detect active infections.
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What type of sample can Real-Time RT-PCR tests work from?
What type of sample can Real-Time RT-PCR tests work from?
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The immune system produces antibodies against viral ______.
The immune system produces antibodies against viral ______.
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Match the following types of tests with their characteristics:
Match the following types of tests with their characteristics:
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What is a significant drawback of Real-Time RT-PCR tests?
What is a significant drawback of Real-Time RT-PCR tests?
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The spike protein of SARS-CoV-2 is not considered immunogenic.
The spike protein of SARS-CoV-2 is not considered immunogenic.
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What type of antibodies does the immune system produce in response to SARS-CoV-2 infection?
What type of antibodies does the immune system produce in response to SARS-CoV-2 infection?
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ELISA tests require specific ______ or antibodies.
ELISA tests require specific ______ or antibodies.
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What time frame does Real-Time RT-PCR tests typically require for completion?
What time frame does Real-Time RT-PCR tests typically require for completion?
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Study Notes
The Life Cycle of SARS-CoV-2
- The virus is believed to be zoonotic in origin
- The virus multiplies within the host
- The body's immune system generates antibodies (IgA, IgM, and IgG)
- Viral antigen concentration levels increase then decrease as the immune system responds
- This timeline is an example only, and each infection has its own unique timeline
Transmission, Detection and Diagnosis of COVID-19 by SARS-CoV-2
- This text focuses on the diagnosis of COVID-19, the methods employed and its detection
- Includes the history of diagnostics, evaluation of different methods and considerations for their development
- The goal is to better understand and fight the pandemic
Common Detection Techniques
- Techniques use different methodologies to detect various key elements of an infection
- RNA methods include RT-PCR, real-time RT-PCR, ddPCR and CRISPR
- Antigen/protein detection is possible via techniques like ELISA, SDS-PAGE and Western Blotting
- Antibody detection is commonly performed via ELISA
Key Considerations in Developing a Test
- The development of diagnostic tests need to consider many factors to be effective
- The cost to develop and run a test is crucial
- Development time is important to quickly respond to emerging disease
- The test should be efficient and feasible for various settings - centralized labs or for point-of-care
- Sampling methods should be user-friendly and non-invasive
- The test should be sensitive, able to detect small amounts of the target, and specific, only detecting the target of interest
- PCR amplification involves isolating RNA from a sample, performing reverse transcription and generating cDNA from viral RNA
Amplification by End-Point or Real-Time PCR
- Both methods rely on PCR, but real-time PCR measures the product during PCR, while end-point PCR requires electrophoresis
- Real-time PCR can be quantitative by comparing results to a standard curve
Real-Time PCR
- Uses a fluorescent dye that binds to double-stranded DNA or a fluorescent probe that is specific to the target sequence
- The number of cycles required to pass the detection threshold depends on the starting template
- More sensitive and faster than PCR, requires no gel electrophoresis and has higher throughput
Droplet Digital PCR (ddPCR)
- A highly sensitive technique that fractionates a sample into 20,000 droplets
- DNA target is amplified in each droplet
- Counting the positive droplets yields precise, absolute target quantification
### ddPCR for Viral Detection
- Reverse-transcription ddPCR combines reverse transcription with ddPCR, allowing for accurate quantification of the target RNA
- It is accurate even with high background DNA or RNA or inhibitors and can discriminate between nucleotide variants common to RNA viruses
- ddPCR allows for absolute quantification, eliminating the need for a standard curve
- RT-ddPCR is valuable for viral diagnostics, therapy development, and vaccine production
Designing PCR Primers
- Primer T values should be similar (+/-2°C)
Pros/Cons of Real-Time RT-PCR Tests
- Pros: detects active infections, quick and easy to define targets and design primers, can work from simple tissue swabs, high throughput, some point-of-care (POC) options available
- Cons: cannot detect past infections, best suited to centralized labs, tests can take 4–6 hr to complete, accuracy can depend on viral load, specificity issues
ELISA-Based Tests
- Virus proteins are antigens. The immune system produces antibodies against viral antigens
- ELISA uses the specificity of antibodies to detect antigens or antibodies
- Can detect active AND past infections
- Lateral flow versions (LFIA) use ELISA principles on a nitrocellulose strip
### Detection of SARS-CoV-2 Proteins
- Important proteins include: Nucleocapsid (N), Membrane (M), Envelope (E), and Spike (S)
- The spike protein is highly immunogenic
### Detection of SARS-CoV-2 by ELISA
- Antigen detection screens patient samples for the presence of viral antigens
- Antibody detection screens patient samples for anti-virus antibodies
- Example: Yang AS et al.(2020) used monoclonal antibodies to screen patients for N-protein
- Example: Amanat et al.(2020) used recombinant S protein to screen patient samples for anti-S antibodies
### Antigen Detection by ELISA
- Antigen: patient sample is tested for the presence of antigens from a virus
- Primary antibody binds to the antigen
- Secondary antibody binds to the constant region of the primary antibody and is attached to an enzyme which catalyzes a color change
- Substrate changes color in the presence of the enzyme, indicating a positive result
Coronavirus Antibody Detection ELISA
- The test detects the presence of antibodies in patient samples
- Patient samples are tested against purified viral S proteins
- Antibodies bind to the S proteins if the patient was infected
- Secondary antibodies with an enzyme bind to the human antibodies
- Substrate is added to produce a coloured reaction
Lateral Flow Serological Test for COVID-19
- LFIA uses the same elements as ELISA.
- Samples added to wells are carried through the strip by capillary action.
- The strip is marked with antibodies and probes
- Antigen-antibody interaction changes the color
Pros/Cons of ELISA Tests
- Pros: can detect both active and past infections, inexpensive, does not require instrumentation, rapid detection
- Cons: longer development time, can be less specific, may not be sensitive enough
Value of Detecting Previous Infections
- Vital for disease tracking, mortality and hospitalization rates
- Can help identify individuals with lower infection risk, enabling better allocation of resources
- Identifies potential blood plasma donors and helps understand vaccine efficacy.
- The presence of antibodies does not guarantee immunity.
CRISPR as a Diagnostic Tool
- CRISPR diagnostics rely on protein-guide complexes that cut a target sequence, then cut other nucleic acids
- Reporters are labeled nucleic acid molecules that produce a visible signal only when cut by the complex
- This system can be used to detect specific sequences
Pros/Cons of CRISPR Based Diagnostic Tests
- Pros: highly sensitive and specific, detects active infections, rapid detection, adaptable format, accommodates multiple sample types, easily reconfigurable for new targets.
- Cons: does not detect previous/past infections, more computationally demanding, not fully validated
### Summary of Conventional Diagnostic Tests for Microbial Disease
- Real-time RT-PCR offers high accuracy with ease of use and development
- ddRT-PCR provides absolute quantification and more sensitivity than real-time RT-PCR
- ELISA is highly specific, can detect previous infections and is cost-effective
- CRISPR is specific, accurate, rapid, and can be used for point-of-care testing
- Each test has its own pros and cons as well as variations in cost, effectiveness, and speed of delivery.
Oxford Nanopore Technology for the Identification of Nucleic Acid Sequences
- Oxford Nanopore is a technology used for sequencing nucleic acids.
NGS Workflow for Detection of New Infections
- Next-Generation Sequencing (NGS) is a comprehensive and powerful technique for identifying and understanding new infections.
Analysis of Nucleic Acid Sequences_Reads
- Sequencing data is analyzed to identify and study the features of the detected infectious agent.
### Coronavirus Diagnosis: PCR vs.
- PCR is the gold standard for detecting the virus, but requires lab equipment and has a slower turnaround time
- Quick tests, while convenient, are less accurate and may produce false negatives
- It is critical to choose the right test depending on the need and urgency.
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Description
This quiz explores the life cycle of the SARS-CoV-2 virus, including its zoonotic origins, immune response, and unique infection timelines. It also delves into the transmission methods and various diagnostic techniques for COVID-19, covering RNA and antigen detection methods. Enhance your understanding of the pandemic and the efforts to combat it.
