2 - Nucleic Acid Testing Flashcards
Define “Nucleic Acid Testing”
It is an amplification technology which detects the viral genome/nucleic acid sequence of pathogens such as viruses or bacteria. It s highly sensitive as it multiplies the copies of the viral RNA/DNA targets prior to detection.
Define “window period”
It is the the time between being infected and detectection of the infection with laboratory tests
Define yield donors
“Yield” donor tests negative by serology but positive by NAT (infectious in the window period)
Define elite controllers
Patients and donors who are HIV negative by NAT but found to be anti-HIV positive with healthy CD4 and CD8 lymphocyte counts.
This means they have resolved the infection (do not have any actively replicating genome) and made antibodies to the virus
What is the principle of NAT?
- Increases test sensitivity by amplification - multiplying copies of the RNA/DNA viral targets of the pathogen prior to the detection phase.
- Testing is a multiplex assay. HIV 1 and 2, HBV and HCV are tested for in the same tube (multiplex assay). If positive, tests are repeated using specific primers for each of the viruses - HIV, HBV and HCV primers used separately.
- Testing is done in a fully automated analyser therefore no need for physical pre and post PCR rooms
Describe the multiplex Nucleic Acid Test (NAT) used by New Zealand Blood Service to screen blood donors for viruses.
You may use diagrams in your answer [14 marks]
1. Extraction and Capture
Extraction of viral target sequences takes place using heat at 64*C and TER (Tareget Enhancement Reagent) detergent treatment which break open the viral particle to expose the core particle containing DNA/RNA nucleic acid material. Exposure of the nucleic acids allows for effective attachment of probes.
Magnetic microparticles have attached probes which identify and capture the specific target (viral nucleic acid) sequences - the RNA of HIV and HCV, and the DNA of HBV. Capture allows for effective washing of other material to be removed, leaving only the intended target genetic material.
2. Amplification
what are the 3 NAT stages?
- Extraction and capture
- Amplification
- Detection
Heat is used during extraction (NAT) at what temperature?
64*C
What does TER stand for
Target Enhancement Reagent
What does the detergent (NAT) do?
The detergent is a TER that breaks open the viral particles to expose the core particle and outer coat protein, releasing DNA and opening nucleic acid strands more effectively, allowing the probes to find the exposed genetic material
How does the magnetic microparticle probes work?
Magnetic microparticles have attached probes which capture nucleic acid sequences specific to RNA of HCV and HIV, and DNA of HBV allowing for subsequent effective washing of test solution which gets rid of all but the viral target sequences.
Explain what an internal control is for during the amplification step of NAT?
An internal control is used in the amplification of viral genomes of the NAT technique and it is a piece of non-viral DNA added to the sample solution to see if the amplification process is working. The DNA is usually a sequence that all humans possess such as the human growth hormone gene. Internal control primers will specifically bind to the complementary DNA sequence of the internal control.
What are some differences in Transcription Mediated Amplification of RNA versus DNA?
TMA of DNA uses a displacer primer in addition to a T7 primer.
DNA target is denatured to a single strand before TMA takes place.
Two reverse transcriptases are utilised to extend the displacer primer sequence and the T7 primer sequence.
TMA of RNA: after the RT makes a complementary DNA strand on the original target RNA, RNAse H activity of RT degrades the original strand, whereas in the TMA of DNA, displacer primer elongation displaces the T7 DNA primer product, releasing a single-stranded DNA product with T7 primer attached (T7 DNA product).
Why is a single stranded DNA product made using displacer primer instead of using heat to make the DNA single stranded?
Becuase the temperature cannot be changed - the whole process takes place at one temperature (41.5*C)
