Quality Assurance & Control Flashcards

Agreement of results when an assay is performed by different operators on different days and instruments is called:

Answer: Reproducibility

Reproducibility is precision under changed conditions such as operator, day, instrument, or site.

Testing a PCR assay against closely related non-target organisms evaluates which performance characteristic?

Answer: Analytical specificity (cross-reactivity)

Analytical specificity checks that the assay does not cross-react with non-target organisms or sequences.

An extracted DNA sample has an A260/A280 ratio of 1.5. What does this suggest?

Answer: Protein or phenol contamination

Pure DNA has an A260/280 of about 1.8; lower ratios point to protein or phenol, which absorb near 280 nm.

A low A260/A230 ratio (e.g., 0.9) in a nucleic acid extract most often indicates carryover of:

Answer: Guanidine salts, carbohydrates, or phenol

Chaotropic salts such as guanidine and other organic contaminants absorb strongly at 230 nm, which lowers the ratio below the ideal ~2.0-2.2.

A patient sample shows a delayed internal control Ct (8 cycles later than expected) and no target amplification. What is the best next step?

Answer: Suspect inhibition; dilute or re-extract the sample and retest

A strongly delayed IAC signals inhibition, so the negative result is unreliable and dilution or re-extraction is needed.

In many qPCR systems, what is the purpose of the ROX passive reference dye?

Answer: To normalize non-PCR fluorescence fluctuations between wells

ROX gives a steady signal that corrects for well-to-well differences in volume, optics, or bubbles.

Where should the fluorescence threshold be set for reliable Ct determination?

Answer: Within the exponential phase, above baseline noise

Setting the threshold in the exponential phase, where amplification is most consistent, gives reproducible Ct values.