Molecular methods validation and Laboratory design

Question 1

Explain why molecular diagnostic laboratories require special design and workflow.

Answer 1

Due to Amplicon contamination. Amplicon contaminate the air, gloves, skin, clothing, environment, new DNA solution,. And can lead to false positive by one contaminating amplicon

Question 2

Methods for controlling contamination in engineering controls.

Answer 2

• UV lights- UV irradiates samples and surfaces: this crosslinks TT & TC
• Pipette use- decontaminate once a week with 2N HCL, use disposable pipette barrel, use filtered/barrier tips to trap aerated DNA
• Physical separation of PCR steps

Question 3

List physical separation of PCR steps in engineering controls.

Answer 3

-isolate DNA (sample prep)
-Set up PCR reaction and reagent prep
-analyze PCR product (high copy number)
Uni-directional work flow

Question 4

Methods for controlling contamination in procedural and chemical controls within the assay.

Answer 4

• open only one tube at a time
• use low level positive controls
• use UNG

Question 5

Define UNG and mechanism

Answer 5

• UNG (n-uracil glycosylase–marketed as AmpErase (cary over prevention kit)
• UNG destroys uracil
• When UNG is used, the master mix is differnt from typical master mix (dTTP—-dUTP)

Question 6

Methods for controlling contamination in surveillance

Answer 6

-Wipe testing: reinforces need to disinfect the environment daily and can improve contamination control in the lab
-monitoring negative controls
monitoring prevalence rates

Question 7

List three challenges in validating an LDT

Answer 7

-no packages insert to guide you
-no controls provided
-need to find out whether you must pay for use of patented materials
development of procedural streps by trial and error.

Question 8

The range of values found in individuals who do not have the disease or condition that is being assayed by the test. To establish this, test specimens from healthy donors to show that they produce a negative results.

Answer 8

reference range

Question 9

The range of values that will generate a positive result from the specimens assayed by the test. It depends on whether the test is qualitative or quantitative.

Answer 9

Reportable range

Question 10

The ability of an assay to produce the same result for a given sample when repeatedly tested over time.

Answer 10

Precision

Question 11

Three levels of precision.

Answer 11

Within run, between run, and between day

Question 12

The measurement of reproducibility when multiple aliquots of a sample are analyzed in parallel during one assay run

Answer 12

within run

Question 13

reproducibility when multiple aliquots of a sample are analyzed in separate assay runs on the same day

Answer 13

between-run

Question 14

reproducibility when multiple aliquots of a sample are analyzed over a period of more than one day.

Answer 14

between day

Question 15

The ability of the test to produce a correct results compared with an external standard

Answer 15

accuracy

Question 16

This refers to the lowest measurable amount of target that can be detected. For molecular tests this may be measured as copies/mL. and sometimes called detection limit.

Answer 16

Analytical sensitivity

Question 17

The degree by which the assay detects the specific target yet does not produce a positive result in the presence of nonspecific targets.

Answer 17

Analytical specificity.

Question 18

What works for eliminating contaminating DNA amplicons?

Answer 18

10% bleach (freshly made), nucleolytic agents, some acids (HCl)