Quality & storage Flashcards
contamination can come from many sources
• The technologist - Including their PPE • Reagents • Work area • Instruments used • Carry over from previous reactions
• Amplification methods are able to detect even a single
copy of a target sequence!
false positives
PCR and other amplification techniques are very sensitive
• Small amounts of extracted nucleic acid or carryover amplicons from previous assays can contaminate future PCR runs
• This results in false-positive results
False positive results can also lead to reduced confidence
contamination prevention
- Laboratories that perform PCR and other amplification methods often use separate rooms for extractions, PCR reagent prep and amplification, to avoid contamination
- If this is not possible, different work-spaces within the labs and strict scheduling for testing may be implemented instead
- Sample nucleic acids and PCR amplicon should NEVER be placed in the same room or work-space as reagent preparation occurs
- Workflow should move from the cleanest room to the “dirtiest” room
- If possible, reagent set up should occur in a laminar flow hood and nucleic acid extraction should be performed in a biosafety cabinet
- Negative controls should be extensively used to ensure that the process has not been contaminated *
- Some laboratories also take routine samples from work spaces and equipment to determine contamination
When contamination occurs
• All equipment and work surfaces must be thoroughly cleaned
- Including pipettes!
• All reagents are usually replaced – including primers
• New disposable consumables must be used
pre & post amplification rooms
The ideal arrangement for a molecular diagnostic lab is to have the
pre- and post-PCR (or amplification) areas located in separate rooms
• Each room should have dedicated resources including water
source, computers, telephones, supply storage, pipettes, pipette tips, racks, etc.
- Dedicated supplies should not be moved between areas
- Lab coats and PPE should be dedicated for each area
- For highly amplified samples, disposable boot covers should be worn
extremely high performance labs or those detecting infectious agents in samples
• For extremely high-performance laboratories or those detecting
infectious agents in samples, air handlers may be required o prevent
air from circulating between rooms
- The pre-PCR lab should have a slight positive pressure
- The post-PCR lab should have slight negative pressure to prevent the escape of amplicons
decontamination if separate rooms are not available
• Reagent preparation should take place inside a laminar flow hood, equipped with UV light
- UV breaks down nucleic acids
• The walls of the hood should be wiped with fresh 10%
bleach solution before processing
- not ethanol ( wont wont remove DNA)
- Use of positive-displacement pipettes or barrier pipette tips- to prevent aresols
- Extraction should be performed in a biosafety cabinet
- Labs should consider establishing a daily schedule for performing PCR
Real - time PCR
Real-time PCR methods can help eliminate the need for two areas:
• The PCR results are generated during the reaction
• Results are available without opening post-PCR tubes
• IF NO ADDITIONAL TESTING IS REQUIRED, Post-PCR tubes can be discarded while sealed
Main tips for prevention
wear PPE
hair tied back
Pre-PCR pipettors and tips should be stored in air-tight containers to keep them free from contamination
When preparing PCR, Master Mix should be added first, followed by DNA to prevent cross-contamination
Pipette tips must be changed at each pipetting step
A used pipette tip must never be put back into a sample or reagent
Reagents should be pre-aliquoted if possible
Care should be taken when opening samples, not to contaminate the sample
• Gloves should be changed very frequently
The quality of the collection containers, storage containers, and every consumable – right down to pipette tips, must meet high standards
Other Tips to Prevent Contamination
Quality Controls
Numerous positive, negative or blank PCR samples should be incorporated into each run
• Positive controls contain the target sequence to be amplified
- Negative controls contain nucleic acid that does NOT include the target sequence( if binds; primers may not be specific enough )
- Blank PCR samples are often called “No Template Controls” ( no DNA, add water to keep volume consistent)
reagent storage
• Each kit will come with specific directions for storage of reagents
Most PCR reagents must be kept in a freezer that is between -20oC (short term) and -80oC ( for long term) • Primers • Master Mix • Controls
• PCR grade water can be kept a room temperature
- Nuclease-free should be used, especially if handing forensic samples
• “Forensic DNA grade” = free of human DNA, Dnase and Rnase
• To prevent enzymes from working before DNA templates are added:
- Once thawed, reagents should be kept on ice ( stops DNA from
breaking down)
- Master mix and primers should not be combined until ready to use
Specimen Collection and Storage
Oncology and Human Genetic specimens:
• Blood collected in EDTA tubes
• Transported at room temperature within 24 hours
• Long term storage should be 2-8 C
Tissue Samples:
• Formalin fixed and paraffin embedded tissues are acceptable
Infectious Disease specimens:
• Samples may be whole blood collected in EDTA
• Transport at room temperature
• Must be spun and separated within 6 hours
Samples may be swabs
• Transport at room temperature
• May vary based on the suspected infectious disease
• For Covid-19 testing
- Specimen should be stored in sterile medium and transported at 2-
8C
- If transport will be delayed, specimen should be frozen at -70oC and shipped on dry ice
Extracted Nucleic Acid Storage
Nucleic acids will degrade at room temperature
Nucleic acids tend to evaporate quickly if stored at 4 C
Samples should be frozen once extracted
• If a DNA sample will be use in multiple experiments, it is
recommended to make multiple aliquots and only thaw
as needed
Short term DNA storage can be in a -20oC freezer
Long term storage of DNA should be in ultra-low freezers,
typically below -80oC
