points to consider for molecular detection for respiratory viruses

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

[email protected]

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

2Public and Environmental Health - Virology

Queensland4.8 million

Brisbane2.3 million

Workflow

• 3 rooms

• 5 rooms

• 1 direction

Public and Environmental Health - Virology 3

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


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


Oligonucleotides Primers & probes

• Have a protocol for design/use

• Adhere to it


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


Oligonucleotides Supplier variation


Amplicon structureShape & accessibility to oligonucleotides

Coxsackievirus A21 5’UTR

(A) amplicon @ 42C; (B) amplicon @ 55 C; (C) amplicon @ 72C;

(D) RNA @ 42 C


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


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


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?


QualityOngoing oversight


• 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…)


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


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


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..


Assessment panels

• External quality assessmentsoExternal to your lab

oExternal to your province

oExternal to your country


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



• 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

points to consider for molecular detection for respiratory viruses

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