Molecular Diagnostics Flashcards
What is molecular diagnostic testing based on?
Detection and/or amplification of nucleic acids
Includes DNA or RNA.
What are the characteristics of molecular assays?
Highly sensitive and specific, but expensive.
When is molecular testing considered?
Applied where other methods fail or have difficulty.
Give an example of a non-culturable agent.
Norovirus.
What is Nucleic Acid Extraction (NAE)?
Process to purify nucleic acids and eliminate PCR inhibitors.
What are common PCR inhibitors found in urine?
Urea.
What mechanism does polymerase chain reaction (PCR) use?
Amplification of nucleic acids.
What is the composition of a PCR reaction?
DNA template, heat-stable DNA polymerase, dNTPs, MgCl2, buffer, primers.
What is an amplicon?
Product of a PCR amplification.
What does reverse transcriptase do in RT-PCR?
Converts RNA to complementary DNA (cDNA).
What is the purpose of multiplex PCR?
Detect multiple targets in a single reaction.
How does agarose gel electrophoresis work?
Separates amplicon based on size and charge.
What is real-time PCR?
PCR with a fluorescent probe to measure amplicon production.
What does the Ct value indicate in PCR?
The cycle where fluorescence crosses the threshold.
Is real-time PCR qualitative or quantitative?
Can be both; determines presence or quantifies targets.
What are isothermal NAATs?
Nucleic acid amplification tests that operate at a single temperature.
What are some examples of isothermal NAAT technologies?
- LAMP
- NEAR
- TMA
What is a common cause of false positives in PCR?
Amplicon contamination.
What is the role of uracil DNA glycosylase (UDG) in PCR?
Degrades U-DNA to prevent contamination.
What is next-generation sequencing (NGS)?
A method to generate large amounts of sequence data quickly.
What is the main advantage of 16S rDNA PCR sequencing?
Identification of most bacteria, whether viable or not.
What is pulsed field gel electrophoresis (PFGE) used for?
Outbreak investigation of pathogens.
What is the significance of whole genome sequencing (WGS) in outbreak investigations?
Provides genetic fingerprinting for pathogen comparison.
What is the difference between DNA sequencing and PCR?
DNA sequencing uses one primer and ddNTPs, while PCR uses two primers.
What do non-synonymous mutations in genes indicate?
Reduced susceptibility or confer resistance.
What type of analysis can be performed using quantitative real-time PCR?
Monitor therapy or disease over time.
What does the term ‘amplicon’ refer to in the context of PCR?
The DNA fragment that is amplified during the PCR process.
Fill in the blank: PCR inhibitors could cause _______.
False negative results.
