MOLBIO LAB 9

The three separate areas in a molecular biology laboratory

Reagent preparation; Sample preparation; Amplification/ Detection

Frequency of running controls in a PCR laboratory

Every new sample run

Specific sequence of nucleic acid used to ensure the reaction works as expected

Positive Template Control (PTC)

The required way PCR controls must be tested (e.g., number of times)

In duplicates

Room where a Positive Template Control should never be taken

Reagent preparation room

Alternative substance used as NTC if not provided in a kit

Nuclease-free water

Purpose of controls to evaluate if a Medical Technologist performed pipetting correctly

Procedure performance evaluation

Expected visual representation of an NTC on a graph

Never rises above the baseline

Reason why probes are not utilized in a valid NTC

No complementary DNA is present for replication

Control used to distinguish a true negative from an amplification failure

Internal Amplification Control (IAC)

The component added to the Master Mix to act as the IAC

A second set of primers unrelated to the target

Temperature for Reverse Transcription in the provided RT-qPCR protocol

50°C

Process where RNA is converted to complementary DNA

Reverse Transcription

Total number of cycles the RT-qPCR protocol repeats

40 Cycles

Stage where the action of DNA Polymerase occurs

Extension

Temperature for Initial Denaturation in the protocol

95°C

Action where primers bind to specific sequences flanking the target region

Annealing

Non-specific fluorescence in the early cycles of a reaction

Background

Phase where baseline fluorescence is determined

Baseline phase

Cycle number where sample fluorescence rises above the background

Cycle Threshold (Ct) or Crossing Point (Cp)

Phase where there is a theoretical doubling of product at every cycle

Exponential Phase

Phase where fluorescence signal no longer grows exponentially because components are limited

Linear Phase

Phase where all PCR reaction components are exhausted

Plateau Phase

The component that emits the fluorescence signal in Real-Time PCR

Reporter

The level of fluorescence based on background and peak reaction fluorescence

Threshold

Common reporter dye used for the target gene (e.g., ORF1ab)

FAM

Reporter dye often used for the Internal Amplification Control

VIC or HEX

Required shape of an amplification curve for it to be considered valid

Sigmoidal curve

Major cause of PCR contamination

Amplicons from previous testing

Equipment used to capsulize single and double strand breaks in DNA for decontamination

UV Light

Commonly used 10% chemical decontaminant in PCR labs

Bleach (Sodium Hypochlorite)

Enzymatic method used to degrade previous PCR products containing uracil

dUTP-UNG System

Specific pipette tips used to reduce nucleic acid contamination

Aerosol-resistant tips

The workflow direction required to prevent contamination

Unidirectional workflow

Action taken to prevent freeze/thaw cycles of reagents

Aliquoting

Monitoring test used to check work areas for nucleic acid interference

Wipe test

Target gene channel for SARS-CoV-2 in the example provided

FAM

Internal Amplification Control (IAC) channel

VIC/HEX

Interpretation if NTC shows no amplification in both FAM and VIC channels

Pass (Proceed to sample analysis)

Interpretation if PTC shows a sigmoidal curve with Ct < 35 (VIC) and < 37 (FAM)

Pass (Proceed to sample analysis)

Interpretation if NTC shows a sigmoidal curve in either channel

Fail (Repeat run due to contamination)

Interpretation if PTC shows no amplification or Ct above limits

Fail (Repeat run)

Interpretation for a sample: VIC Ct < 35, FAM Ct < 37

Positive for SARS-CoV-2

Interpretation for a sample: VIC Ct < 35, FAM No amplification (or Ct > 37)

Negative for SARS-CoV-2

Interpretation for a sample: VIC No amplification (or Ct > 35), FAM Ct < 37

Invalid test

Interpretation for a sample: VIC No amplification, FAM No amplification

Invalid test

Reason why a sample with FAM No amplification and VIC Sigmoidal is considered valid

The IAC (VIC) proves the reaction worked

Immediate action if a clinical sample result is Invalid

Retest by re-extracting RNA

The baseline setting for the auto threshold in the provided protocol

Cycles 3 to 15

VIC/HEX: <35

FAM: <37

Sample interpretation: positive/negative/invalid

QC run interpretation: pass/fail

1. Interpret sample run

2. What phase in PCR is affected by instrument sensitivity?

1. Negative

2. Baseline

VIC/HEX: <35

FAM: <37

Sample interpretation: positive/negative/invalid

QC run interpretation: pass/fail

1. Identify phase pointed.

2. Interpret QC run

1. Plateau phase

2. Pass

VIC/HEX: <35

FAM: <37

Sample interpretation: positive/negative/invalid

QC run interpretation: pass/fail

1. Interpret sample run

2. Interpret QC run

1. Positive

2. Pass

VIC/HEX: <35

FAM: <37

Sample interpretation: positive/negative/invalid

QC run interpretation: pass/fail

1. Interpret sample run

2. What consists of reagents for amplification, probes, primers for virus target and

internal references

1. Negative

2. Master Mix