PCR principles Flashcards

1
Q

What DNA viruses do we test for via PCR at this site?

A

CMV, EBV, HSV1 & 2, VZV (varicella-zoster), JC virus and BK virus

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2
Q

How is viral DNA extracted from pt samples for PCR ?

A

either a manual method or using MagNApure

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3
Q

What is real-time PCR? what is its other term?

A

it is quantitative, and monitors the amplification in real time

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4
Q

How is development of the target nucleic acid amplification detected?

A

using FRET, fluorescent resonance emission technology, which employs flourescently labeled probes

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5
Q

what kinds of samples can be analyzed by PCR in our lab?

A

Urine, swabs extracted into M4 media, CSF, Whole blood, tissues, respiratory and body fluids

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6
Q

Optimal transport temp and which cannot be frozen?

A

2 to 8 C (fridge); CMV

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7
Q

Each assay requires its own mastermix; these are aliquoted into microfuge tubes and stored at?

A

-20 to -30 C

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8
Q

after being thawed how long is mastermix good and what other precaution?

A

7 days; avoid light exposure to minimal

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9
Q

what 3 types of controls are run with each PCR lightcycler run?

A

a positive EXTRACTION cntrl, a negative EXTRACTION cntrl; and an AMPLIFICATION cntrl

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10
Q

What does a positive extraction cntrl and a negative axtraction cntrl ensure?

A

the positive ensures that adequate nucleic acid isolation has taken place; negative ensures no DNA contamination has occurred in either extraction or amplification phase

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11
Q

what does the amplification control ensure?

A

that the primers and probes are working as expected

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12
Q

what could the absence of a signal from the internal amplification control indicate?

A

the test could be falsely negative, there was not amplification

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13
Q

what is the Tm in PCR?

A

it is the melting temp of the PCR product, defined as the temp at which 50% of the product helix is dissociated

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