39 - Diagnostic Microbiology (Part 3)

“End Point” Polymerase Chain Reaction (PCR)

Exponentially amplifies segments of DNA with very little starting material needed (enables detection of pathogens/antibiotic resistance genes/virulence factors): relies on thermal cycling

Who invented “End Point” PCR?

Kary Mullis (1983) and later received a nobel prize

Why do they call it “End Point” PCR?

The test result happens AFTER the PCR test is conducted 

DNA Template / DNA / Template

Sample being tested

DNA Polymerase / Taq Polymerase

Enzyme that polymerizes new DNA strands

Forward and Reverse Primers

Specific to the area you want to amplify

dNTPs (Deoxynucleoside Triphosphates)

Building Blocks of DNA (you need materials to make the area being amplified)

Bivalent Cations (typically Mg)

Activates the polymerase = required cofactor

What can “End Point” PCR be used for?

Tissue typing (important for transplants), identifying the specific subtype of a cancer, viral identification, or slow growing bacteria

Advantages and Disadvantages of “End Point” PCR

Advantages → Cheap, results tend to be easy to interpret, well-established method
Disadvantages → Need to have a specific amplicon in mind for primer design, susceptible to contamination

“Real Time” PCR (RT-PCR)

Used to determine viral load as well as percent of a pathogen, can view amplification in real time and quantify DNA with proper controls (uses a thermal cycler attached to a computer)

Why is it called “Real Time” PCR?

You can see the test result in real-time

Advantages and Disadvantages to RT-PCR

Advantages → Faster, no need to pour/run gel, differentiate chemistries available for the primers
Disadvantages → More controls needed, more precise pipetting needed, more expensive supplies/equipment

Multiplex PCR

Multiple regions can be amplified in a single reaction, can be end-point or RT-qPCR based, saves time if multiple tests are needed

Sequencing 

Determining nucleotide order in a DNA sample

Sanger Sequencing Requirements

DNA template, DNA primer, DNA polymerase, dNTPs, modified dNTPs with radioactive or fluorescent reporters (ddNTPs)

Sanger Sequencing

4 reactions set up where only 1 of the 4 types of ddNTPs are added and effectively PCR is performed. Products are then denatured and separated via gel electrophoresis

WGS Sequencing

Allows whole genomes to be sequenced rapidly (not needed or ideal in a clinical setting), becoming widely available and cost is decreasing (but still time-consuming and more expensive) 

Antibodies

Formed during infections to combat future infections (they can also help us in the clinical lab)

How are antibodies helpful in the clinical lab?

Extreme specificities for their antigens → this concept can be used in designing clinical testing

Serology

Testing of serum to gain insights into the immunologic status of patient

Primary Basis of Most Immune Testing

Formation of Antigen/Antibody Complexes

What are Antibody/Antigen reactions often used to determine?

Titer (Quantity of antibodies in the serum)

Agglutination Tests

Antibodies crosslink the antigens to form visible clumps (used for blood typing)

Precipitation Tests

Soluble antigen is precipitated by an antibody (used for VDRL test)

Agglutination and Precipitation Reactions

Both produce “chunks” because both form complexes to form an insoluble 3D aggregate large enough to come out of solution

Difference Between Agglutination and Precipitation

Agglutination → Antigens = whole cells with determinants on their surface
Precipitation → Antigen = soluble molecule

Lateral Flow Test / Assay (LFT or LFA)

Commonly used for flu, COVID, and pregnancy which is a kit-based method so it does not require specialized training and contains an internal control as a rule

Western Blotting / Immunoblotting

Semi-quantitative technique used to probe for the presence of specific proteins in a sample (sample is run on a gel and incubated with antibodies before undergoing washes and imaging)

What does Western Blotting / Immunoblotting test for?

Lyme Disease and HIV

Immunoassays

Extremely sensitive methods that permit rapid/accurate measurement of trace amounts of antibody/antigens

Enzyme-Linked Immunosorbent Assay (Elisa)

Contains an enzyme-antibody complex that can be used as a colorimetric indicator for antigen-antibody reactions

HRP (Horse-Radish Peroxidase) and Alkaline Phosphatase Release

Dye when exposed to their substrate 

Indirect ELISAs are able to

Capture antibodies in the serum samples (common screening method for HIV/Hep A and C, Helicobacter, Lyme Disease)

Capture ELISAs

Have antibodies in the walls and can detect unknown antigens (common screening method for hantavirus, rubella, taxoplasma)

What indicates antibody is present in an ELISA sample?

Color Development