qpcr diagnostics assay development for infectious disease working with industry - an academics...
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qPCR Diagnos-cs Assay Development for Infec-ous Disease Working with Industry – an Academics Perspec-ve
Dr Thomas Barry Nucleic Acid Diagnos-cs Research Laboratory (NADRL) Microbiology NUI, Galway Ireland
NADRL
Infec-ous Disease Clinical (Respiratory, Sepsis, STI,GI) Food Pathogen / Veterinary Environmental Monitoring Environmental Reservoir Contaminants Water contaminants Bioprocess Monitoring
Future Diagnos-cs CirculaEng RNA biomarkers / Small
RNA Sequencing
NADRL
NADRL Core Research Areas
NADRL
The NADRL has developed a range of diagnosEcs tests for mulEnaEonal industrial and SME partners including:
NADRL Commercial Partners/ Disclosures
Infec-ous Disease NAD Product Development Pipeline
‘From the Bench to the Bedside’
NADRL
Feasibility
NADRL Biological Resources
In‐house stocks of nucleic acid material from >1800 organisms including bacteria, fungi, and viruses
In-house stocks of cryopreserved cultures from >2000 species/strains representing >500 species of bacteria and >50 species of fungi
NucleoEde sequence database with ~ 3500 target sequences generated
NADRL
NADRL
NADRL‐ Diagnos-cs Assay Development Strategy
Target Biodiscovery
Identify novel nucleic acid
diagnostics targets
Bioinformatics In silico design / IP
landscape evaluation, filing
and prosecution
qPCR Assay Design
Species Specificity
Panel Assembly
Assay Performance Optimisation
Validation
Small scale Clinical / Application Trials
Technology Providers Licensing and R+D contracts with Industry
Translation
NADRL ‐ NAD Assay Development:
NADRL
Minimum InformaEon for PublicaEon of QuanEtaEve Real‐Time PCR Experiments (MIQE guidelines ‐hSp://miqe.info/ )
NADRL Proprietary Technologies – Intellectual Property
The NADRL has developed a range of nucleic acid diagnosEcs target technologies
Spacer Probe (ITS) ‐ Bacterial / Fungal markers
RiboSEQ* ‐ Bacterial markers
MycoSEQ - Fungal / Yeast markers
NucleoSEQ - Bacterial markers
11 Technologies Patented or Undergoing Office AcEons
NADRL
RioboSEQ IP – Bacterial Target Diagnos-cs Technology ssrA gene & tmRNA
NADRL
DNA / RNA target found in all bacterial species
Structure conserved in all bacteria
Nucleo-de sequence diversity allowing for assay specificity‐ Genus and species
Useful for syndromic infec-ous disease diagnos-cs applica-ons
Diagnos-cs patents granted worldwide
NADRL
Highly experienced research team ‐ staff have many years combined experience in both
academia and industry commercialisaEon
The laboratory endeavours to work to industry standard and has rigorous on site training, record keeping and SOPs etc.
in place.
Focuses on all aspects of technology transfer from
laboratory bench to industry se\ng
Staff members have experience in and have spent Eme working on site with various industrial partners, specifically at the technology
transfer stage
NADRL Staff Exper-se
NADRL
Water Monitoring for Pathogens – A qPCR NAD Assay Design and Development Case Study
Building Water Reservoir Contaminants / Water Contaminants /Bioprocess Monitoring
NADRL
• Water plays a crucial role in the industrial, manufacturing,
residenEal and clinical sectors. • Pathogenic microorganisms in building water distribuEon and
premise plumbing systems can pose a significant risk to human health parEcularly in hospital se\ngs.
Introduc-on
NADRL
Acquired Infec-ons Due to Contaminated Water Systems
NADRL
RouEne microbiological tesEng of water and water distribuEon systems are performed using century old culture techniques slow labour intensive
Microbial numbers can be significantly underesEmated
Limita-ons of Tradi-onal Culture Methodologies for Analysis of Water
NADRL
Gram –ve
• Legionella sp. • Pseudomonas aeruginosa • Burkholderia sp. • Serra5a marcescens • Stenotrophomonas maltophilia • Ralstonia sp. • Enterobacter aerogenes • Klebsiella pneumoniae • Proteus mirabilis • Acinetobacter baumannii
NAD Microbial Por_olio for Water Analysis
Gram +ve • Staphylococcus aureus • Bacillus sp. • Non Tuberculosis Mycobacteria
Fungal • Candida albicans
Amoeba (‘Trojan Horses’) Development stage
Amoeba ‘Trojan Horses’ – Indicators for Pathogens? Legionella ‐ Prey becoming the Predator
NADRL
Legionella are taken up into amoeba without being digested and replicate there within vacuoles. When the Legionella have reached a certain density, the vacuoles release them into the water system (hSp://www.pall.com/main/medical/medical‐cc5a1214.page)
NADRL
The Need for a Different Diagnos-c Approach: Quan-ta-ve Mul-plex qPCR ‐ a poten-al solu-on?
• Essen-al test criteria: Rapid‐ Culture independent QuanEtaEve Specific
• Desirable test criteria: MulEple microorganism idenEficaEon in a single test (mulEplexing) High throughput and simultaneous analysis of mulEple samples PotenEal for integrated / automated tesEng
The example: Legionella
NADRL
qPCR NAD assay to quan-ta-vely detect human pathogens from water, water distribu-on and premise plumbing systems
Legionella qPCR diagnos-cs assay requirements
NADRL
• Essen-al Assay Specificity Detec-on Requirements: • Must detect all clinically relevant Legionella species (n >35) – genus specific • Must specifically detect all L. pneumophila serogroups (n=15) – species specific • Must specifically detect L. pneumophila serogroup 1 – serogroup specific
• Essen-al Assay Sensi-vity Detec-on Requirements:
• Limit of DetecEon: 1‐10 genome equivalents – wet lab diagnosEcs • Limit of QuanEficaEon: 10 CFU / 100ml water – water sample diagnosEcs
Legionella genus assay: RiboSEQ*
• DetecEon of all clinically relevant Legionella species and strains tested (n=35)
• *This target is currently endorsed by the CDC for use in NAD for the detecEon of the Legionella genus (Current and Emerging Legionella DiagnosEcs for Laboratory and Outbreak SituaEons (2015) Clin Micro Rev. (28) 1).
Clinically relevant Tested Detected
Legionella anisa Legionella birminghamensis (n=2) Legionella bozemanii (n=2) Legionella cincinnatiensis Legionella dumoffii (n=2) Legionella erythra Legionella feeleii (n=2) Legionella gormanii (n=2) Legionella hackeliae (n=2) Legionella longbeachae Legionella micdadei Legionella oakridgensis (n=2) Legionella parisiensis Legionella wadsworthii Legionella pneumophila subsp. pneumophila‐1 (n=12) Legionella pneumophila subsp. pneumophila‐2‐16
Legionella pneumophila species (all serogroups): NucleoSEQ
• DetecEon of all clinically relevant Legionella pneumophila serogroups (n=15)
Clinically relevant Tested Detected
Legionella anisa Legionella birminghamensis (n=2) Legionella bozemanii (n=2) Legionella cincinnatiensis Legionella dumoffii (n=2) Legionella erythra Legionella feeleii (n=2) Legionella gormanii (n=2) Legionella hackeliae (n=2) Legionella longbeachae Legionella micdadei Legionella oakridgensis (n=2) Legionella parisiensis Legionella wadsworthii Legionella pneumophila subsp. pneumophila‐1 (n=12) Legionella pneumophila subsp. pneumophila‐2‐16
Biomarker Discovery ‐ Legionella pneumophila sg1 specific assay
• Requirement for a novel target for sg1:
15 serogroups associated with L. pneumophila species
• ComparaEve whole genome analysis
Comparison of L. pneumophila sg1 whole genome with L. pneumophila sg6 and sg12
• 95 nucleoEde sequence regions of difference idenEfied
• BioinformaEc analysis of these 95 regions
• Yielded 3 potenEal NAD targets in silico
Legionella pneumophila sg 1 specific NAD assay
AmplificaEon curves for Legionella pneumophila sg1 real‐Eme PCR diagnosEcs assay
• DetecEon of all L. pneumophila sg1 strains tested (n=12)
• Specific for L. pneumophila sg1 – no non sg 1 strains detected (n=23)
Legionella Mul-plex Assay: Current Status
• Incorporate all assays into mulEplex format in the presence of an Internal AmplificaEon Control‐ complete (4 –plex)
• Generate standard curves for quanEficaEon‐ complete
• OpEmise mulEplex assay – real water samples – Ongoing to improve LoQ (near compleEon)
Analysis of isolates from biocontaminated hot and c
• Test water samples (without culture) with our NAD mulEplex assay from a biocontaminated hot and cold water system
• Currently also working on a novel microbial nucleic acid enrichment process from water
• UlEmate goal is to develop an accurate, sensiEve and validated NAD test for Legionella with a rapid turnaround Eme (TAT)
Water Sample In to Result Out in <4 hours
Future Work
NADRL
Tips for Dealing with Industry
In general: • License negoEaEons ‐ bring in the professionals (University TTO / Legal / IP Managers) R+D Contracts with Industry: • MeeEng industry product specificaEons, milestones and deliverables deadlines – employ a
programme manager • “Moving goalposts” ‐ Chopping and changing of work programme / schedules – usually
influenced by markeEng component of industry partner (can be very frustraEng) • Physical separaEon of industry laboratory group from your day to day academic
laboratory group
Analysis of isolates from biocontaminated hot and c
• Significant nega-ve impact on your ability to publish research in peer reviewed journals
• ‘Le\ng your technology go’ – dependent on the nature of license structure
(exclusive / non‐ exclusive)
Downsides from Working with Industry
Analysis of isolates from biocontaminated hot and c qPCR NADRL Peer Reviewed Publica-ons 2015:
Cross Pla_orm Standardisa-on of an Experimental Pipeline for Use in the Iden-fica-on of Dysregulated Human Circula-ng MiRNAs. Kelly, H et al. (2015) PLoS ONE 10(9): e0137389. doi:10.1371/journal.pone.0137389. 100% (License Royalty Stream) v 0% A rapid culture independent methodology to quan-ta-vely detect and iden-fy common human bacterial pathogens associated with contaminated high purity water. Minogue, E et al (2015) BMC Biotechnol. 75% v 25% (SFI) Compara-ve genome analysis iden-fies novel nucleic acid diagnos-cs targets for use in the specific detec-on of Haemophilus influenzae. Coughlan, H. Et al. (2015) DiagnosJc Microbiology And InfecJous Disease. 50% v 50% (SFI) A comparison of established diagnos-c methodologies and a novel bacterial smpB real‐-me PCR assay for the specific detec-on of Haemophilus influenzae associated with respiratory tract infec-ons. Reddington, K et al. (2015) Journal Of Clinical Microbiology. 0% v 100% (EU)
Benefits from Working with Industry –
Non‐Repor-ng (%) v Repor-ng (%) Research Funding Contribu-on
Thank You PI: Dr Thomas Barry ([email protected]) To my core research team: Postdocs – Kate Reddington, Nina Tuite, Elizabeth Minogue Postgrads – Helena Coughlan, Helena Kelly, Shannon Fullbrook
CDC University College London / Royal Free Hospital NHS Hospitals London Public Health England UCHG
Our academic collaborators: