points to consider for molecular detection for respiratory viruses
TRANSCRIPT
Points to considerfor molecular detection of
respiratory viruses
Ian M. Mackay, PhDSupervising Scientist, Development & Validation
Public and Environmental Health – Virology
Forensic & Scientific Services | Health Support Queensland
Department of Health | Queensland Government
Briefly
• Things to consider for respiratory virus diagnosticso Laboratory layout
oOligonucleotide (primer/probe) design, purchase & quality
oDevelopment and validation process
oAssay controls
oSampling site
oBuild capacity – be prepared
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Queensland4.8 million
Brisbane2.3 million
Workflow
• 3 rooms
• 5 rooms
• 1 direction
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Be organized
• Networked computerso Inside and outside laboratory areas
• Oligonucleotide databaseoAll oligonucleotides in use are listed
• Storage register (mixes, stocks, ivtRNA)oSearchable, up-to-date, simple
• Protocols for everythingoHow to make reagents/controls, receive specimens, use equipment, extract,
dilute, amplify, prepare an assay, order, store, calibrate…
• TrainingoNew staff
oCross-training and enrichment
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Plan aheadEasier to fit new thing into existing process
• Make base (kit) reagents up in relevant bulk and aliquoto –20C or -80C
oRT-rtPCR, rtPCR, RT-PCR, PCR
• Make oligonucleotide mixes in bulko –20C or -80C
• Make control stocks in bulko -80C
oAliquots for single use
• Aliquot water
• Use liquid handling robots
• Write everything down
• ProtocolsPublic and Environmental Health - Virology 5
OligonucleotidesPrimers & probes
HCoV-HKU1 N gene target showing BLASTn primer specificity.
(A) 20mers from Position #1; (B) 20mers from Position #5
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Oligonucleotides Primers & probes
• Have a protocol for design/use
• Adhere to it
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Oligonucleotides Quality and surge capacity
• QualityoMonitor performance of controls, curve height, shape, CT values
• Oligonucleotide stocksoHave a backup
oUse aliquots
oPlan ahead
• Commercial supplieroHave two
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Oligonucleotides Supplier variation
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Amplicon structureShape & accessibility to oligonucleotides
Coxsackievirus A21 5’UTR
(A) amplicon @ 42C; (B) amplicon @ 55 C; (C) amplicon @ 72C;
(D) RNA @ 42 C
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Assay controls
• PositiveoWild-type virus
– Hard to find
o “Synthetic” DNA/RNA– Just need a sequence
oContamination
• NegativeoWater
• No amplificationoNo enzyme – signal (probe) control
oDuring development
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Assay controls2 separate templates
• Primer & Probe controls to replace wild-typeoSynthetic DNA sequence incorporating virus primer or probe sequence
oCan be used to make ivtRNA
oTease out run problems more quickly
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Assay controls2 separate templates
• Primero If a run fails, was it due to primer problems?
• Probeo If a run fails, was it due to probe problems?
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QualityOngoing oversight
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• Batch change (kit, primer, probe, water)oCompare result using new reagent batch to result from previous reagent
batch, in same run
o Just one variable
oTakes up some extra cycler time/space
oSaves considerable use of cycler time/space troubleshooting later
Validation
• Every time we change a significant part of the assay
• For example…oExtraction method
oThermal cycler
oOligonucleotide concentration
oNew oligonucleotide sequence
oNew reagent kit/supplier
oNew type of positive control
• Not for…oNew user
oNew pipette
oNew batch of reagent (but…)
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ValidationSamples and assay sensitivity
• Store positive samples
• Assay sensitivity
• Other assay specificity
• -80C preferred over -20C
• May need to confirm amplifiable nucleic acids remainoStored for months to years
oUse the gold standard assay and re-test in parallel with new assay
oUse a host housekeeping gene to determine amount of amplifiable nucleic
acid left – will be an over-estimation
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ValidationWhat about co-detections?
• ≥20% of samples can have some other virus
• Co-detections (CoDes)
• Newly identified viruses will sometimes be part of a CoDe, sometimes not
• Validate your assay against a wide range of non-target viruses to be confident that your result is specific
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Sampling site
• Relevant to virus being sought
• MERS: studies/data mostly from
URT oViral loads higher in LRT
oURT has less receptor than LRT
o LRT recommended site
• Ebola virus diseaseoReminded us that we only find what we
look for, where we look for it
oBlood the gold standard
oCSF, semen, urine, aqueous humor..
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Assessment panels
• External quality assessmentsoExternal to your lab
oExternal to your province
oExternal to your country
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In house PCR
• It can be done welloPlans, preparations and protocols
oThink on a larger scale
• Be prepared, not caught out
• Communicate with your neighbours
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• University Hospital BonnoOrganizational team
• Central Veterinary Research Laboratory
• United Arab Emirates Ministry of Health
• World Health Organization–EMRO
• Public & Environmental Health | VirologyoHealth Support Qld | Department of Health