PCR principles Flashcards
What DNA viruses do we test for via PCR at this site?
CMV, EBV, HSV1 & 2, VZV (varicella-zoster), JC virus and BK virus
How is viral DNA extracted from pt samples for PCR ?
either a manual method or using MagNApure
What is real-time PCR? what is its other term?
it is quantitative, and monitors the amplification in real time
How is development of the target nucleic acid amplification detected?
using FRET, fluorescent resonance emission technology, which employs flourescently labeled probes
what kinds of samples can be analyzed by PCR in our lab?
Urine, swabs extracted into M4 media, CSF, Whole blood, tissues, respiratory and body fluids
Optimal transport temp and which cannot be frozen?
2 to 8 C (fridge); CMV
Each assay requires its own mastermix; these are aliquoted into microfuge tubes and stored at?
-20 to -30 C
after being thawed how long is mastermix good and what other precaution?
7 days; avoid light exposure to minimal
what 3 types of controls are run with each PCR lightcycler run?
a positive EXTRACTION cntrl, a negative EXTRACTION cntrl; and an AMPLIFICATION cntrl
What does a positive extraction cntrl and a negative axtraction cntrl ensure?
the positive ensures that adequate nucleic acid isolation has taken place; negative ensures no DNA contamination has occurred in either extraction or amplification phase
what does the amplification control ensure?
that the primers and probes are working as expected
what could the absence of a signal from the internal amplification control indicate?
the test could be falsely negative, there was not amplification
what is the Tm in PCR?
it is the melting temp of the PCR product, defined as the temp at which 50% of the product helix is dissociated
