COVID-19 PCR Laboratory Setup: Introduction, Design, Test Requirements, Laboratory Practices, Decontamination Approaches, Validation/Verification Study, Quality Control Plan, quality Indicator, Proficiency Testing, and Keynotes

Introduction of COVID-19 PCR Laboratory

COVID-19 PCR Laboratory is the most common laboratory which was used in the COVID-19 pandemic for the diagnosis of COVID-19 (detecting genes of SARS-CoV-2) as well as prognosis COVID patients. To my knowledge, there is no country where there is no any COVID-19 PCR Laboratory. To run PCR, there should be at least four separate rooms for RNA extraction, Template addition, master mix, and PCR analysis respectively. Tests for COVID-19 are rapid antibody test, antigen test, loop-mediated isothermal amplification (LAMP), nucleic acid amplification test (NAAT), and reverse transcription-polymerase chain reaction (RT-PCR). Among them, RT-PCR is the gold standard assay.

COVID-19 PCR Laboratory Setup Pictures

Sample Aliquoting and RNA Extraction Room

Template Addition Room

Master Mix Room

PCR Analysis Room

Biomedical Waste Management Room

Design for COVID-19 PCR Laboratory Setup

To design COVID-19 PCR Laboratory is really a hard task due to the following point of view-

• The laboratory should be compatible with mechanical barriers to prevent contamination which is the most common issue in the molecular laboratory.
• The spatial separation of pre-and post-amplification work areas
• Each area should be fascinated with adequate requirements.
• Unidirectional Flow
• Maintenance of air pressure
• Temperature and humidity requirements
• Exhaust ventilation
• Water quality
• Electric outlet
• Back-up power system, etc.
• Note: The above design is modified on the following guidelines-

1. CLSI MM19-A Establishing Molecular Testing in Clinical Laboratory Environments -Mitchell P. S. et al. Nucleic Acid Amplification Methods: Laboratory Design and Operations, 2004, In “Molecular Microbiology: Diagnostic Principles and Practice, edited by D. H. Persing et al” 99. 85-93.
2. http://www.roche-applied science.com/ campaigns/ DeveloperTips/ pcr/Physical-separation.html
3. http://fx.damasgate.com/the-pcr-laboratory/

Requirements of COVID-19 PCR Laboratory Setup

Requirements of the molecular laboratory are as follows-

• Mechanical barriers to prevent contamination
• The spatial separation of pre-and post-amplification work areas-Area 1: Sample Aliquoting and RNA Extraction Room, Area 2: Master Mix Room, Area 3: Template Addition Room and Area 4 – Amplification Room/ PCR Analysis
• Physically separated and, preferably, at a substantial distance from each other
• Unidirectional Flow

1. Both personnel, including cleaning personnel, and specimens
2. Amplification product-free to product-rich
3. Remove PPE before leaving one area
4. Avoid or limit the reverse direction
5. Reusable supplies in the reverse direction need to be bleached.

• Features of the 4 Areas

1. Each area has separate sets of equipment and supplies
2. Refrigerator/freezer (manual defrost)
3. Pipettes, filtered tips, tubes, and racks
4. Centrifuge, timers, vort
5. Lab coat (color-coded), disposable gloves, safety glasses, and other PPE
6. Cleaning supplies
7. Office supplies
8. Ventilation system

• Dead airbox with UV light – serves as a clean bench area
• Air pressure

1. Reagent Preparation – Positive
2. Sample Preparation – Negative
3. Post Amplification – Negative

• Reagent Prep – Single entrance, reagents used for amplification should not be exposed to other areas
• Specimen Prep – Specimens should not be exposed to post-amplification work areas
• The size of each area should consider space for equipment and bench space needed for preparation

• Alternative to Spatial Separation

1. Class II biological safety cabinet
2. Dedicated areas for each work phase
3. Unidirectional
4. Automated specimen processing station/closed tube amplification and detection system

• Other Laboratory Design Considerations

1. Temperature and humidity requirements
2. Exhaust ventilation
3. Water quality
4. Electric outlet
5. Back-up power system
6. Eyewash
7. Ergonomic assessment

Laboratory Practices for COVID-19 PCR Assay

Proper design and adequate requirements of the laboratory are not only sufficient without good laboratory practices and thus following points are useful for laboratory practices-

• Use of positive displacement pipettes and disposable filtered pipette tips
• Avoid production of aerosols when pipetting
• Use of sterilized single-use plasticware
• Use of cleanroom sticky floor mats
• Minimizes the risk of amplicon carry-over on clothing, hair, and skin

1. Hairnet
2. Dedicated safety glasses
3. Disposable lab coat/gown, color-coded preferred
4. Gloves, need to change periodically
5. Shoe covers

• Clean punches between samples
• Use of nuclease-free or autoclaved water
• Aliquot oligonucleotides – multiple freeze thaws will cause degradation
• Always include a blank (no template) control to check for contamination
• Use of electronic data system (flow of paper)
• Wipe test (swab test)-

1. Monthly
2. Detect, localize, and remove contamination
3. Identify the source of the contamination

Decontamination Approaches for COVID-19 PCR Laboratory

• Clean the work area & equipment routinely

1. Clean the PCR workstation at the start and end of each workday/run (UV light, 70% ethanol, fresh 10% sodium hypochlorite, DNA Away)
2. Clean the exterior and interior parts of the pipette
3. Clean the equipment
4. Clean the doorknobs, handle of freezers

Chemical and Enzymatic Controls: Work stations should all be cleaned with 10% sodium hypochlorite solution (bleach), followed by removal of the bleach with ethanol and water.
Ultra-violet light irradiation: UV light induces thymidine dimers and other modifications that render nucleic acid inactive as a template for amplification

Enzymatic inactivation with uracil-N-glycosylase: Substitution of uracil (dUTP) for thymine (dTTP) during PCR amplification. New PCR sample reactions pre-treated with Uracil-N-glycosylase (UNG) – contaminating PCR amplicons are degraded leaving only genomic DNA available for PCR.

When is a Validation/Verification Study Required?

• Introduce a new testing system

1. New analyte
2. Analyte previously measured/detected on an alternate system

• An analyte added to a test system
• A modification to a test system
• Applies to

1. Unmodified, FDA-cleared, or approved method
2. Modified, FDA-cleared, or approved method
3. In-house method
4. Standardize methods such as textbook procedure

• Determine the analytic performance of an assay

Quality Control Plan

Monitor all steps of analytical procedure-

• Types of Control
• Frequency and Number of Controls
• Evaluation of Controls and Calibrator

False Amplification Potential causes

Potential cause-

• Non-optimized assay conditions
• Unknown polymorphisms in target sites-

1. Gene duplications
2. Oligonucleotide mispriming at related sequences
3. Pseudogenes or gene families

• Oligonucleotide concentrations too high
• Nucleic acid cross-contamination

Quality Indicator

Measurement to monitor and record specific activities as part of the quality management system-

• Turnaround Time
• % of failed runs
• Population medium
• Calibrator parameters
• The graph to identify trend or shift
• Monitor frequency and acceptable range

Proficiency Testing

• Assessment of the Competence in Testing
• Performed twice a year
• If specimens are not commercially available alternative proficiency testing program has to be established (specimen exchange etc.)

Molecular Assay Proficiency Testing Material Sources

It may be of national or international origin.

Sample Acceptance and Tracking

1. Special specimen acceptance criteria?
2. Assign a unique code to each patient
3. Use two patient identifiers at every step of the procedure
4. Develop worksheets and document every step
5. Laboratory Information Management Systems (LIMS) interface and Positive ID

Reagents

• Labeling Reagents-

1. Content, quantity, concentration
2. Lot
3. Storage requirements (temperature etc.)
4. Expiration date
5. Date of use/disposal

• Know your critical reagents (enzymes, probes, digestion, and electrophoresis buffers) and perform QC checks as appropriate.

Critical Molecular Assay Components

• Nucleic Acids: Prepare aliquots appropriate to workflow to limit freeze-thaw cycles

1. Primers and probes
2. dNTPs
3. Genomic DNA
4. 4-8°C
5. -15 to -25°C

• Enzymes- Benchtop coolers recommended
• Fluorescent reporters-

1. Limit exposure to light
2. Amber storage tubes or wrap in shielding (foil)

Other QA/QC Considerations

1. Specimen storage
2. Laboratory Cleanliness, and Waste Disposal
3. Instrument Maintenance and Calibration
4. Instrument/Method Comparison
5.  Document Management
6. Personnel Training and Competency
7. Periodic Review of QA/QC
8. COOP Plan: A COOP plan addresses emergencies from an all-hazards approach. A continuity of operations plan establishes policy and guidance ensuring that critical functions continue and that personnel and resources are relocated to an alternate facility in case of emergencies.

Keynotes

• Potential sources of contamination are cross-contamination between specimens, amplification product contamination, laboratory surfaces, ventilation ducts, reagents/supplies, and hair, skin, saliva, and clothes of laboratory personnel.
• Contamination may cause-

1. Incorrect results
2. Require extensive cleanup
3. Loss of creditability
4. Impact on financial and performance

• Contamination can be controlled using proper-

1. Laboratory design
2. Laboratory practices
3. Chemical and enzymatic controls

Further Readings

• https://www.aphl.org/programs/newborn_screening/Documents/2015_Molecular Workshop/Molecular-Laboratory-Design-QAQC-Considerations.pdf
• https://www.scimmit.com/molecular-laboratory-design-and-its-contamination-safeguards/
• https://www.aacc.org/~/media/files/meetings-and-events/resources-from-past events/conferences/2014/molecular-testing/may-29-and 30/checklist_for_mdx_testing_slides_may_29_2014.pdf?la=en
• https://www.researchgate.net/publication/303176803_Introduction_To_Molecular_Diagnostics
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2797718/
• https://universe84a.com/molecular-laboratory-build/
• https://cdn.intechopen.com/pdfs/23728/InTechGood_clinical_laboratory_practice_gclp_for_molecular_based_tests_used_in_diagnostic_laboratories.pdf
• https://www.bu.edu/emd/emergencyplanning/coop/
• https://www.pomona.edu/academics/departments/molecular-biology/facilities
• https://academic.oup.com/femspd/article/49/2/184/493227