From Sample to Result – Workflow Solutions for Genotyping and Pathogen Detection
•
1 gostou•1,277 visualizações
This document discusses pathogen detection methods using high-resolution melting (HRM) analysis and multiplex real-time PCR. It begins with an overview of challenges in pathogen detection workflows, including issues with sample collection/extraction and PCR inhibition. Methods for DNA extraction and real-time PCR detection assays are presented, including QIAGEN solutions. Applications discussed include developing assays for cannabis contamination detection and bovine respiratory/GI pathogen detection. Validation strategies for new assays include testing sensitivity/specificity using synthetic templates.
Recomendados
Recomendados
Mais conteúdo relacionado
Mais procurados
Mais procurados (20)
Destaque
Destaque (17)
Semelhante a From Sample to Result – Workflow Solutions for Genotyping and Pathogen Detection
Semelhante a From Sample to Result – Workflow Solutions for Genotyping and Pathogen Detection (20)
Mais de QIAGEN
Mais de QIAGEN (20)
Último
Último (20)
From Sample to Result – Workflow Solutions for Genotyping and Pathogen Detection
- 1. Sample to Insight Pathogen detection with HRM and multiplex real-time qPCR technology: Challenges, workflow and applications 1 James Qin, Senior Scientist, MDx Applications, QIAGEN
- 2. Sample to Insight Legal disclaimer Pathogen detection with HRM and multiplex RT-qPCR 2 • QIAGEN products shown here are intended for molecular biology applications. These products are not intended for the diagnosis, prevention or treatment of a disease. • For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.QIAGEN.com or can be requested from QIAGEN Technical Services or your local distributor.
- 3. Sample to Insight 4 Agenda Pathogen detection with HRM and multiplex RT-qPCR 3 Microbiome utilities and associated diseases Microbial DNA isolation challenges and solutions Pathogen detection methods • Challenges • Real-time PCR and Multiplex real-time PCR • Quantitative high-resolution melting (HRM) Applications 1 2 3
- 4. Sample to Insight 4 Agenda Pathogen detection with HRM and multiplex RT-qPCR 4 Microbiome utilities and associated diseases Microbial DNA isolation challenges and solutions Pathogen detection methods • Challenges • Real-time PCR and Multiplex real-time PCR • Quantitative high-resolution melting (HRM) Applications 1 2 3
- 5. Sample to Insight • Well-known microorganisms that cause GI infections • Adenovirus • Campylobacter • Clostridium difficile • Cryptosporidium • Entamoeba histolytica • Escherichia coli • Escherichia coli O157:H7 • Giardia • Helicobacter pylori • Rotavirus • Salmonella and Shigella • Staphylococcus aureus • Yersinia enterocolitica • Microbiome ecology: Suggests unique microbial residents are tuned to the environment of one’s body (genetics, diet, and developmental history) • Microbiome diversity: Hints immune and metabolic disorder might be related to a degraded microbiome and also because antibiotic resistance is limiting human ability to kill pathogens. • Microbiome composition: Associated with long-term distortions in the composition, function, and antibiotic resistance of the intestinal microbiota • Microbiome population profiling: Imbalance of the gut microbiota is linked with gastrointestinal conditions such as IBD, IBS, obesity, type 2 diabetes, and atopy • Microbiome transplants: New evidence linking changes in microbial diversity to a variety of diseases, including C. diff colitis; Helping to rationalize and validate the effectiveness of this controversial treatment strategy • Others – Monitoring disease progression and treatment … Microbiome and human diseases Pathogen detection with HRM and multiplex RT-qPCR 5
- 6. Sample to Insight 4 Agenda Pathogen detection with HRM and multiplex RT-qPCR 6 Microbiome utilities and associated diseases Microbial DNA isolation challenges and solutions Pathogen detection methods • Challenges • Real-time PCR and Multiplex real-time PCR • Quantitative high-resolution melting (HRM) Applications 1 2 3
- 7. Sample to Insight 7 Pathogen detection workflow: From Sample to Insight Overview of multiplex PCR assay workflow Sample isolation Multiplex amplification Detection and analysis Extract and purify microbial DNA 45 minutes, manually or automated Multiplex amplification of targeted pathogens 2 hour 1. Non-specific detection using DNA binding dyes 2. Specific detection using target specific probes Detection: 1 hr • What pathogens or microorganisms are present? • What is the relative abundance? • Are there any specific mutations associated with antimicrobial resistance? Sample collection & treatment Pretreat stool specimens to break down parasitic 30-45 minutes Pathogen detection with HRM and multiplex RT-qPCR
- 8. Sample to Insight General challenges of pathogen detection & ID workflow 8 Sample collection DNA or RNA extraction Detection Data analysis Different sample type represents challenges collecting samples: hair bulb, skin, dry blood, ear punch, FFPE, biopsy, saliva/ sputum, urine, stool… May contain agents that hamper DNA or RNA extraction Variable extraction efficiency Difficult to determine extraction efficiency Primer design: High diversity of microbes and majority unknown. It is difficult to design primers that capture all activity. Nucleases, enzyme inhibitors can interfere with qRT-PCR; sample abundancy may affect certain type of assays Normalization, quantitation methods: internal standards, housekeeping genes, Pathogen detection with HRM and multiplex RT-qPCR
- 9. Sample to Insight 9 Some considerations in sample extraction Microbial DNA extraction • PCR inhibitors presented in samples is one of big challenges • High abundance of host DNA can introduce bias of results Core questions to ask: • What samples are being analyzing? • Sample type: stool, soil, swabs, urine • How are samples collected and processed? • Lysis methods strongly depend on sample material (mechanical vs. enzymatic) • Additional pre-treatments might be necessary (e.g. deparaffinization) • How do you get rid of all the other stuff? • Most common biomolecules are relatively easy to remove (proteins, lipids, sugars) • Some metabolites are might be co-purified and inhibit your analysis • Deplete host DNA and enrich bacterial microbiome DNA • How much DNA can I possible get from my sample • Species may have low abundance • Stability of DNA Pathogen detection with HRM and multiplex RT-qPCR
- 10. Sample to Insight 10 QIAGEN Sample to Insight solutions for pathogen and microbial extraction Detection and analysis • QIAmp DNA Mini Kit • QIAmp UCP Pathogen Mini Kit • QIAmp DNA Stool Fast/ Mini Kit • QIAamp DNA Microbiome Kit • QIAamp 96 DNA QIAcube HT Kit • QIAsymphony mericon Bacteria Kit MO BIO Laboratories for NGS • PowerSoil DNA Isolation Kit • PowerFecal DNA Isolation Kit • … • QuantiFast Pathogen +IC Kits • QuantiTect Virus Kits • QuantiNova Probe PCR kits • Microbial DNA qPCR Arrays and Assays • Type-it HRM PCR kit for genotyping assays • Rotor-Gene Q • QIAxcel DNA Kits • Allprotect • InhibitEx • RNAlater Pathogen detection workflow: From Sample to Insight Sample isolation Sample collection & Stabilization qPCR or qRT-PCR Pathogen detection with HRM and multiplex RT-qPCR
- 11. Sample to Insight 11 Sample extraction: QIAamp 96 DNA QIAcube HT kit Automate extraction process: free up you time by automating DNA extraction Automatable on the QIAcube Fast and reliable 96-well DNA purification QIAamp silica-membrane technology Sample: genomic, mitochondrial, and pathogen DNA from blood, cells, and tissue samples • Number of samples: 24-96 samples • Elution volume: 200 ul • Duration: 24 samples in 45 mins, 96 samples in 96 mins • Cost/prep: $2.0 per sample Pathogen detection with HRM and multiplex RT-qPCR
- 12. Sample to Insight • Vortex 10 min • Heat de-activation 10 min • PK/ AL digestion 10 min • Quick spin; and transfer QIAamp 96 DNA QIAcube HT kit: Experiment data Move from manual process to fully automated process: • High yield • Simple and fast Pathogen detection with HRM and multiplex RT-qPCR 12
- 13. Sample to Insight 4 Agenda 13 Microbiome utilities and associated diseases Microbial DNA isolation challenges and solutions Pathogen detection methods • Challenges • Real-time PCR and Multiplex real-time PCR • Quantitative high-resolution melting (HRM) Applications 1 2 3 Pathogen detection with HRM and multiplex RT-qPCR
- 14. Sample to Insight 14 Current methods in pathogen detection and identification Each species of pathogens carries a unique DNA or RNA signature that differentiates it from other organisms • Culture • Gold standard for bacteria, such as Salmonella and Shigella • Utilized for pathogen identification, not directly characterize virulence factors • Laborious and time-consuming, and expensive • Rapid antigen • Simple and rapid • Can test a range of viruses and certain bacteria and toxins • Genomic assay: Singleplex and multiplex • High sensitivity and specificity • Genomic assay: NGS and Pyrosequencing • Allows quantitative detection and identification of all existing microbial • Proteomic assays • Rapid and allow function correlation Pathogen detection with HRM and multiplex RT-qPCR
- 15. Sample to Insight 15 Multiplex PCR-based pathogen detection • PCR based methods are the best for the specimens collected by non-invasive methods such as stool samples • Specific: only detects target sequence • Rapid: easy to set up, and run time within few hours • Sensitive: can detect low copy numbers • Standardized: can be automated and use stable chemical design • Can be used for the detection of bacteria as well as for characterization of pathogenic genes and specific mutations associated with antimicrobial resistance • Multiplex allows to focus either a panel of viruses or a panel of bacteria suspected in gastroenteritis with one test Challenge: The presence of PCR inhibitors in stool samples may cause low sensitivity； Assay designs. QIAamp DNA Kit can efficiently remove the inhibitors Pathogen detection with HRM and multiplex RT-qPCR
- 16. Sample to Insight 16 Multiplex real time PCR method SYBR Green-based detection method • Detection based on target specific primers only using non-specific DNA binding dyes • SYBR Green is the most widely used double-strand DNA-specific dye for real time PCR Target specific probe-based detection method • Specifically detect real time PCR with oligonucleotide probes labeled with both a reporter fluorescent dye and a quencher dye. • Hydrolysis probes such as TaqMan probes • Molecular Beacons • FRET Hybridization Probes • Scorpion Primers • Quantitative mRNA expression studies of cDNA • Microbial load or copy number measurements from microbial genomes • Allelic discrimination assays or SNP genotyping • Verification of microarray results Pathogen detection with HRM and multiplex RT-qPCR
- 17. Sample to Insight 17 QIAGEN Sample to Insight solutions for pathogen and microbial detection • QIAmp DNA Mini Kit • QIAmp UCP Pathogen Mini Kit • QIAmp DNA Stool Mini Kit • QIAamp 96 DNA QIAcube HT Kit • QIAsymphony mericon Bacteria Kit MO BIO Laboratories • PowerSoil DNA Isolation Kit • PowerFecal DNA Isolation Kit • … • QuantiNova Probe RT-PCR Kit • QuantiNova SYBR Green RT-PCR Kit • QuantiTect Multiplex PCR • QuantiFast Pathogen +IC Kits • Type-it HRM PCR Kit • Microbial DNA qPCR Arrays and Assay Kits • Rotor-Gene Q • QIAxcel DNA Kits • Allprotect • InhibitEx Pathogen detection workflow: From Sample to Insight Pathogen detection with HRM and multiplex RT-qPCR Detection and analysis Sample isolation Sample collection & Stabilization qPCR or qRT-PCR
- 18. Sample to Insight 18 QIAGEN RT-qPCR and multiplex PCR product lines • QuantiNova Probe RT-PCR Kit and QuantiNova SYBR® Green RT-PCR Kit • Novel QuantiNova two-phase hot-start mechanism • Reliable: Increased reliability of gene expression results using gDNA reduction • Convenient: Room-temperature reaction setup without compromising • Visual pipetting control: limit pipetting errors • Internal controls: positive in-process verification of successful RT-PCR and monitoring RT-PCR inhibition • Sensitive: High sensitivity for low-copy RNA targets Pathogen detection with HRM and multiplex RT-qPCR
- 19. Sample to Insight 19 QIAGEN RT-qPCR and multiplex PCR product lines • QuantiTect Multiplex PCR Kit and QuantiTect Probe PCR Kit • Real-time PCR and two-step RT-PCR • High PCR specificity with integrated hot start • Reliable quantification of low-abundance transcripts • Accurate quantification over several logs of template • Available with or without uracil-N-glycosylase (UNG) • No need to optimize reaction and cycling conditions Wide dynamic rangeHigh specificity Fast: Faster results with time savings of up to 50% Multiplex: Successful multiplex PCR without the need for optimization Sensitive: Detect up to 4 targets in 1 tube Reliable: Quantify low- and high-abundance targets Pathogen detection with HRM and multiplex RT-qPCR
- 20. Sample to Insight 20 QIAGEN RT-qPCR and multiplex PCR product lines • Type-it HRM PCR Kit • Fast and accurate detection of gene mutations and SNPs by High-Resolution Melting (HRM) analysis Scan for mutation Type mutation Pathogen detection with HRM and multiplex RT-qPCR
- 21. Sample to Insight 4 Agenda 21 Microbiome utilities and associated diseases Microbial DNA isolation challenges and solutions Pathogen detection methods • Challenges • Real-time PCR and Multiplex real-time PCR • Quantitative high-resolution melting (HRM) Applications • Cannabis contamination detection (Food Safety) • Bovine respiratory and GI track pathogen detection • HRM: Microbiome pattern recognition in clinical research and more 1 2 3 Pathogen detection with HRM and multiplex RT-qPCR
- 22. Sample to Insight 22 Star: Talaromyces stipitatus; Tree: Aspergillus nidulans Ornaments: Penicillium marneffei; Trunk: Aspergillus terreus Hat, Eyes, Mouth, Buttons: Aspergillus niger; Arms: Aspergillus nidulans; Nose: Aspergillus terreus with Penicillium marneffei; Body: Neosartorya fischeri • Purpose: Develop real-time pathogen detection assays food and agricultural products such as marijuana, organic drinks • Extraction chemistry: QIAamp DNA kit • Detection Kit: QuantiFast Pathogen + PCR Kit • Experimental Design and validation strategy • Result Development of Cannabis contamination assays in Cannabis quality control with QuantiFast Microbial Kit + IC Application 1: Cannabis contamination detection (Food Safety) Pathogen detection with HRM and multiplex RT-qPCR
- 23. Sample to Insight Application 1: Validation strategy • Assays were tested using synthetic templates • Microbial qPCR Mastermix, Microbial DNA-Free Water • Sensitivity • Standard curves from 0 to 1,000,000 copies were prepared • 1000 copy Ct<31 or Ct<34 (low end assays if NTC Ct=40) • Primer efficiency> 80%, R>0.995, LLOQ determined • Specificity • Human, mouse, rat gDNA • Microbial genomic DNA pools: • Each pool contains 10 genomic DNA at 2000 genome copy each or complete pool which contains all genomic DNA (110) • Genomic DNA from same genus put into separate pools • Staph/Strep species made into separate pool • Each specificity test performed in duplicate • Performance in complex background • Spiked in synthetic template in stool, sputum, sewage • dCt = Ct (background + synthetic template) – Ct (synthetic template) • dCt<3 23Pathogen detection with HRM and multiplex RT-qPCR
- 24. Sample to Insight Application 1: Standard curves using synthetic templates 24 y = -3.2871x + 40.07 20 25 30 35 40 0 1 2 3 4 5 6 CT Log target copy number Aspergillus niger y = -3.2356x + 41.04 20 25 30 35 40 0 1 2 3 4 5 6 CT Log target copy number Klebsiella pneumoniae y = -3.1423x + 41.649 20 25 30 35 40 0 1 2 3 4 5 6 CT Log target copy number Mucor/Rhizopus spp. y = -3.1608x + 41.204 20 25 30 35 40 0 1 2 3 4 5 6 CT Log target copy number Pan Aspergillus/Penicillium Pathogen detection with HRM and multiplex RT-qPCR
- 25. Sample to Insight Pool1 Pool2 Pool3 Pool4 Acinetobacter baumannii Aeromonas hydrophila Alcaligenes faecalis subsp. Faecalis Aspergillus fumigatus Bacillus licheniformis Bartonella henselae Bordetella pertussis Brevundimonas diminuta Campylobacter jejuni subsp. Jejuni Candida albicans Candida glabrata Candida parapsilosis Citrobacter freundii Clostridium difficile Clostridium perfringens Clostridium thermocellum Corynebacterium glutamicum Enterobacter aerogenes Enterococcus faecalis Enterococcus faecium Fusobacterium nucleatum subsp. Nucleatum Geobacillus stearothermophilus Haemophilus influenzae Helicobacter pylori Legionella pneumophila subsp. Pneumophila Listeria monocytogenes Mycobacterium tuberculosis Neisseria meningitidis Pantoea agglomerans Pediococcus pentosaceus Plesiomonas shigelloides Proteus mirabilis Rahnella aquatilis Ralstonia solanacearum Salmonella enterica subsp. enterica serovar Paratyphi A Serratia marcescens Vibrio cholerae Yersinia enterocolitica subsp. Enterocolitica Yersinia pestis Stenotrophomonas maltophilia Pool5 Pool6 Pool7 Pool8 Bacillus cereus-14579D-5 Aggregatibacter actinomycetemcomitans Akkermansia muciniphila Anaerococcus prevotii Burkholderia cenocepacia Bacteroides thetaiotaomicron Bacteroides ureolyticus Bacteroides vulgatus Candida tropicalis Burkholderia cepacia Campylobacter coli Campylobacter concisus Corynebacterium diphtheriae Capnocytophaga gingivalis Cryptobacterium curtum Cryptococcus gattii Escherichia coli-200928D-5 Enterobacter cloacae subsp. Cloacae Gardnerella vaginalis Lactobacillus jensenii Klebsiella pneumoniae Lactobacillus casei Lactobacillus gasseri Micrococcus luteus Ochrobactrum anthropi Methanobrevibacter smithii Mycoplasma pneumoniae Neisseria flava Pseudomonas aeruginosa Mycoplasma orale Porphyromonas gingivalis Prevotella intermedia Shigella flexneri Porphyromonas endodontalis Trichomonas vaginalis Ureaplasma parvum Yersinia pseudotuberculosis Treponema denticola Staphylococcus haemolyticus Streptococcus mitis Pool9 Pool10 Pool11 staph/strep Aspergillus flavus Atopobium rimae Bacillus subtilis Staphylococcus aureus subsp. Aureus- Bifidobacterium breve Bifidobacterium longum subsp. Infantis Bordetella parapertussis Staphylococcus epidermidis- Campylobacter gracilis Campylobacter rectus Campylobacter upsaliensis Staphylococcus saprophyticus subsp. Saprophyticus Cryptococcus neoformans Desulfovibrio desulfuricans Lactobacillus acidophilus Streptococcus gordonii Haemophilus ducreyi Klebsiella oxytoca Leptotrichia buccalis Streptococcus mutans Lactobacillus plantarum Lactobacillus reuteri Mycoplasma hominis Streptococcus pneumoniae Mycobacterium smegmatis Mycoplasma genitalium Peptostreptococcus anaerobius Streptococcus pyogenes Neisseria gonorrhoeae Parabacteroides distasonis Tannerella forsythia Streptococcus sanguinis Prevotella melaninogenica Proteus vulgaris Vibrio parahaemolyticus Veillonella parvula-#1 Vibrio harvey Streptococcus agalactiae Application 1: Specificity of bacterial fungal assays Title, Location, Date 25 20 22 24 26 28 30 32 34 36 38 40 NTC Template humangDNA mousegDNA ratgDNA pool1 pool2 pool3 pool4 pool5 pool6 pool7 pool8 pool9 pool10 pool11 staph/streppool Ct Thermoactinomyces spp. 2 20 22 24 26 28 30 32 34 36 38 40 NTC Template humangDNA mousegDNA ratgDNA pool1 pool2 pool3 pool4 pool5 pool6 pool7 pool8 pool9 pool10 pool11 staph/streppool Ct Thermoactinomyces spp. 1 20 22 24 26 28 30 32 34 36 38 40 NTC Template humangDNA mousegDNA ratgDNA pool1 pool2 pool3 pool4 pool5 pool6 pool7 pool8 pool9 pool10 pool11 staph/streppool Ct Klebsiella pneumoniae
- 26. Sample to Insight Application 1: Testing of assays with genomic DNA 26 y = 2.3205x + 28.295 20 25 30 35 40 0 1 2 3 4 5 6 CT Log Dilution Factor Aspergillus niger y = 3.1124x + 19.776 20 25 30 35 40 0 1 2 3 4 5 6 CT Log Dilution Factor Mucor/Rhizopus spp. y = 2.5678x + 26.308 20 25 30 35 40 0 1 2 3 4 5 6 CT Log Dilution Factor Pan Aspergillus/Penicillium y = 2.5111x + 18.293 20 25 30 35 40 0 1 2 3 4 5 6 CT Log Dilution Factor Thermoactinomyces spp. 1 y = -0.0137x + 39.881 20 25 30 35 40 0 1 2 3 4 5 6 CT Log Dilution Factor Thermoactinomyces spp. 2 Aspergillus brasiliensis gDNA Penicillium chrysogenum gDNA Mucor hiemalis gDNA Thermoactinomyces vulgaris gDNA Thermoactinomyces vulgaris gDNA Pathogen detection with HRM and multiplex RT-qPCR
- 27. Sample to Insight Performance and sensitivity (LLOQ) 27 Species slope E R LLOQ Aspergillus niger -3.53 92% 0.999 60 Klebsiella pneumoniae -3.24 104% 0.996 80 Mucor/Rhizopus spp. -3.51 93% 1.000 230 Pan Aspergillus/Penicillium -3.16 107% 0.995 100 Thermo spp. 1 -3.07 112% 0.999 30 Thermo spp. 2 -3.01 115% 1.000 60 -4 -3 -2 -1 0 1 2 3 4 Aspergillusniger Klebsiellapneumoniae MucorRhizo Pan Aspergillus/Penicillium Thermoactinomyces spp.1 Thermoactinomyces spp.2 dCt([metagenomicsample+synthetic template] –synthetictemplate) dCt (stool-template) dCt (sputum-template) dCt (sludge-template) Assay performance in metagenomic samples Application 1: Performance Pathogen detection with HRM and multiplex RT-qPCR
- 28. Sample to Insight • Research purpose • Develop real-time multiplex pathogen detection assays for Veterinary customers • T. foetus, Calf Diarrhea panel (K99, Sal. Crypto.) assays • Calf Scours 4-plex assay with InType internal control • Bovine Respiratory Disease (BRD) Panel 5-plex assay with InType internal control • Current challenges • Long process: Magnetic beads, cRNA, isopropanol, tissue disruption equipment/steps ... • Lost of sensitivity for calf diarrhea /Johne’s feces because of inhibitors, … • Cross reactivity and drop of signals: Singleplex assays multiplex assays • Products used • Extraction: QIAcube HighThroughput (QCHT) automation solution based on QIAamp DNA 96 kit (96-well plate, silicon columns) • Consistent and comparable performance with QuantiFast Pathogen PCR with IC kit as a multiplex PCR solution • Validation strategy and result Application 2: Bovine respiratory and GI pathogen detection Pathogen detection with HRM and multiplex RT-qPCR 28
- 29. Sample to Insight • The enzymes: • HotStarTaq Plus DNA Polymerase • Reactivation within 5 minutes • Unmatched specificity and sensitivity • The buffer: • Unique combination of K+ and NH4 + ions • High specificity • No optimization necessary • Q-BondTM: • Enables fast cycling by promoting annealing of Taq and primer/probe to the template • Synthetic Factor MP • Supports macromolecular crowding • Enables equal amplification of templates that differ in abundance Solving problems with fast real-time PCR High PCR specificity and macromolecular crowding Pathogen detection with HRM and multiplex RT-qPCR 29
- 30. Sample to Insight T. Foetus (Fam) Internal Control (Yellow) TH = 0.05 Tritrichomonas Foetus and InType IC: A clean multiplex assay Pathogen detection with HRM and multiplex RT-qPCR 30
- 31. Sample to Insight Negative NTCs; Good IC ranges; Sensitivity <= 10 copies InType IC Good correlation & assay efficiency TH = 0.05 InType Internal Control in Calf Scours 4-plex assay Pathogen detection with HRM and multiplex RT-qPCR 31
- 32. Sample to Insight A clean Crypto assay in 4-plex reaction with good dynamic range Cryptosporidium in 4-plexes assay: Good consistency and sensitivity of Crypto+ in the 4-plex TH = 0.20 Calf scours panel: E. Coli K99/ Sal./Crypto/ InType 4-Plex assay Pathogen detection with HRM and multiplex RT-qPCR 32
- 33. Sample to Insight InType internal control (yellow) 5-plexes TH = 0.05 Tight range of Internal control, no interference with the 4 targets vs. IC Bovine BRD Respiratory Panel M. haem/H. Somni/ M. bovis/ P. mult InType 5-Plex assay Pathogen detection with HRM and multiplex RT-qPCR 33
- 34. Sample to Insight Comparable target Cts. A good specificity on POS vs Negs; and a consistency of M. Haem in singleplex vs. 5-plex assays. Mannheimia Haemolytica in 5-plexes : Good consistency, specificity, and tighter range of M. Haem+ in the 4-plex TH = 0.05 Good signal and tight range in IC controls. InType ICM. Haem+ Bovine BRD Respiratory Panel: M. Haemolytica in 5-plexes Pathogen detection with HRM and multiplex RT-qPCR 34
- 35. Sample to Insight Comparable target Cts. Better specificity and great improvement on negative samples in 5-plexes vs. H. Somni singleplex Histophilus Somni in 5-plexes: Good consistency, specificity, and less cross-reactivity of M. Haem+ in the 4-plex TH = 0.05 A good signal and tight range in IC controls in 5-plexes. Bovine BRD Respiratory Panel: H. Somni in 5-plexes Pathogen detection with HRM and multiplex RT-qPCR 35
- 36. Sample to Insight Better specificity and good improvement on negative samples in 5-plexes vs. singleplex Mycoplasm bovis (Green, Fam) in 5-plexes: Good consistency, sensitivity, and less cross-reactivity in M.bovis+ in the 4-plex TH = 0.05 A good signal and tight range in IC controls in 5-plexes. Overcome primer-probe-target interactions in singleplex in IC. Bovine BRD Respiratory Panel: Myco. bovis in 5-Plex Assay Pathogen detection with HRM and multiplex RT-qPCR 36
- 37. Sample to Insight TH = 0.05 Better specificity and great improvement on negative samples in 5-plexes vs. H. Somni singleplex after optimization Consistent, good signal and tight range in IC controls. Pasteurella multocida in 5-plexes: Good consistency, specificity, and tighter range of P. mult + in the 5- plex Bovine BRD Respiratory Panel: P. mult + Optimization Pathogen detection with HRM and multiplex RT-qPCR 37
- 38. Sample to Insight • Purpose • To identify a real-time multiplex kit that is consistent, precise in real-time PCR, and is fast, easy-to-use and cost-effective • Extraction chemistry • QIAamp DNA kit • Extraction automation: QIAcube: silica column-based • Detection Kits: QuantiNova real-time probe PCR kit (recommended for duplex) • Validation strategy • Use DNA templates contain different copy numbers between target vs. reference • Result • Series of template dilutions for ∆∆Ct (relative quantitation) analysis Application 3: Copy number assay for allele discrimination analysis Pathogen detection with HRM and multiplex RT-qPCR 38
- 39. Sample to Insight Comparison study in copy number assay for allele discrimination analysis using QuantiNova and QuantiTect Probe real-time PCR Assays R2=0.8576 M=-3.361 Efficiency= 0.98 R2=0.9962 M=-3.275 Efficiency= 1.02 QuantiNova rt-PCR kit: Comparable result and faster QuantiTect R-PCR Kit: a multiplex alternative (4-plex) Result is highly comparable. Application 3: Copy number assay for allele discrimination analysis Pathogen detection with HRM and multiplex RT-qPCR 39
- 40. Sample to Insight Purpose & background Complex community microbiome profiling on healthy subjects: • Qiagen RotorGene Q 5-plex HRM real-time PCR with primers targeting V3 region of 16S rRNA gene (170bp amplicons) on PowerSoil and PowerFecal DNA kits extracted DNA samples from healthy subjects HRM Profiling for Microbiome Diversity on healthy subjects: • HRM melt on RGQ 5-plex HRM for possible profiling on the amplified samples above; no PMA-Rx’ed samples Qiagen kits and automation • Extraction: QIAcube HT with QIAamp DNA kit • Type-it HRM PCR kit • QIAgility for liquid handling & assay setup • Rotor-Gene Q PCR with 100-Disc Validation strategy • Look at the sample pretreatment options and protocols • Test samples on QIAcube HT instrument and protocol for an automation and full workflow solution (propose: total 48 – 64 combined fecal + soil samples) • Reference kit: Data will need to have full range of QC (Qxpert, Nanodrop 8000) • Will evaluate limited samples with real-time PCR Results Application 4: Microbiome profiling by high resolution melting (HRM) Pathogen detection with HRM and multiplex RT-qPCR 40
- 41. Sample to Insight Background Pathogen detection with HRM and multiplex RT-qPCR 41
- 42. Sample to Insight Primer Set NTC (5ul/20-ul PCR) Run-2 JE/Pro 33.76 V3 22.3 V5 20.83 V6 22.1 V7 19.45 Threshold = 0.01 Dynamic Tube Slope Correct! Results: Assay and primer sets testing Pathogen detection with HRM and multiplex RT-qPCR 42
- 43. Sample to Insight ºC 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 dF/dT 3.75 3.50 3.25 3.00 2.75 2.50 2.25 2.00 1.75 1.50 1.25 1.00 0.75 0.50 0.25 0.00 JE341/Pro805 V6 V5 V3 V7 RGQ real-time PCR melt curves Results: RotorGene melt curve analysis on five assays Pathogen detection with HRM and multiplex RT-qPCR 43
- 44. Sample to Insight JE/ Pro V3 V7V5 V6 XX X Results: Linearity and assay dynamic range check Pathogen detection with HRM and multiplex RT-qPCR 44
- 45. Sample to Insight Threshold = 0.18735 127 bps Threshold = 0.15662 460 bps JE341/ Pro805 V6 Ct = 10-12Ct = 22-25 Assay Condition-1 Assay Condition-2 Results: Assay optimization and dynamic range Pathogen detection with HRM and multiplex RT-qPCR 45
- 46. Sample to Insight ºC 71.5 72.0 72.5 73.0 73.5 74.0 74.5 75.0 75.5 76.0 76.5 77.0 77.5 78.0 78.5 79.0 79.5 80.0 80.5 81.0 81.5 82.0 82.5 83.0 83.5 84.0 84.5 85.0 85.5 86.0 86.5 87.0 87.5 88.0 88.5 55 50 45 40 35 30 25 20 15 10 5 0 -5 -10 JE341/Pro805 Normalized to V5: V6 V5V3 V7 ºC 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 0 -10 -20 -30 -40 -50 -60 Normalized to JE341/ Pro805: JE341/Pro805 V6 V3 V5 V7 Results: RotorGene HRM analysis on five assays Pathogen detection with HRM and multiplex RT-qPCR 46
- 47. Sample to Insight For many datasets, most of the eigenvalues “lambda” are negligible and can be discarded. The eigenvalue measures the variation In the direction e: Example: Principal Component Analysis (PCA) in ScreenClust Finding relationship from a large multi-layer sample set is always a challenge. One way to avoid the dimensionality is by projecting the data onto a lower-dimensional space. It rotates multivariate dataset into a new configuration which is easier to interpret. Techniques for dimension reduction: Principal Component Analysis (PCA) – most effective! Fisher’s Linear Discriminant Multi-dimensional Scaling. Independent Component Analysis. Pathogen detection with HRM and multiplex RT-qPCR 47
- 48. Sample to Insight Application of PCA in genomics Purposes • Simplify data – PCA is the most commonly used dimension reduction technique. • Look at relationships between variables – PCA is useful for finding new, more informative, uncorrelated features. • PCR reduces dimensionality by rejecting low variance features. • Look at patterns of units – i.e. new mutations, targets • Analysis of expression data • Analysis of metabolomics data (Ward et al., 2003) Example Run Sophisticated computing and bioinformatics Pathogen detection with HRM and multiplex RT-qPCR 48
- 49. Sample to Insight JE341/Pro805 V3 V5 V7 V6 HRM PCR Genotyping Kit/ RGQ: Offers distinct melt curves HRM analysis with ScreenClust software with RotorGene Pathogen detection with HRM and multiplex RT-qPCR 49
- 50. Sample to Insight ScreenClust: High predicting power in pattern recognition Example run report: HRM analysis with ScreenClust software Pathogen detection with HRM and multiplex RT-qPCR 50
- 51. Sample to Insight ºC 75 80 85 90 dF/dT 2.0 1.5 1.0 0.5 0.0 ºC 77 78 79 80 81 82 83 84 85 86 87 88 89 100 80 60 40 20 13003 13009 13000 13026 13031 13027 13003DS 13003DP13004 1300013029 HRM is able to pick up individual differences High Resolution Melt (V6 assay) Distinct melt curve patterns HRM and melt curve analysis by ScreenClust on all QCHT extracted Hu. samples Pathogen detection with HRM and multiplex RT-qPCR 51
- 52. Sample to Insight Result: Clustering Analysis by ScreenClust on All QCHT extracted samples Pathogen detection with HRM and multiplex RT-qPCR 52
- 53. Sample to Insight How Can I Qualify? • Have real funding, … • Consumable business that can be forecasted on a monthly basis w/ an annualized value of $50K or greater • $50K or more on automation for initial purchase or in the form of reagent rental • Combination of the above to meet $50K minimum requirement • Customer should agree to sign a Material Transfer Agreement (MTA) with the commitment to honor if requirements are met • Set expectations right with limited and achievable key deliverables • Investment is $10,000 with material/FTE cost for up to four weeks of time. It is free to qualified customers. If You Are… …A small startup that is for-profit and in growth mode, have funding & wants to accelerate their business; …A company that does not have the capacity or the time for development or portfolio expansion but is willing to spend when unique requirements are met. And … • Companies that have the following challenges: • Liquid cancers • Solid, difficult to extract tissues • Difficult plant, food, drink extraction • Look for assay solutions • Look for one-size-fits-all protocol • Customer also should have: • Clear, fixed objectives and short timeline • Has an established manual assay but wants to expand or improve • Currently in direct comparison w/ 3rd party offerings w/ a limited timeline to make a decision Contact QIAGEN: Local sales or application services Pathogen detection with HRM and multiplex RT-qPCR 53
- 54. Sample to Insight Questions? Thank you for attending Contact QIAGEN Technical Service Call: 1-800-426-8157 for US Call: +49 2103-29-12400 for EU Email: techservice-na@QIAGEN.com techservice-eu@QIAGEN.com QIAwebinars@QIAGEN.com Pathogen detection with HRM and multiplex RT-qPCR 54
Notas do Editor
- 1.) Proceedings of the National Academy of Sciences – reviewed that then microbial composition and gene content at these locations vary significantly from one person to the next. Consequently, it’s long been suspected that microbial communities found at certain body sites might have forensic utility. 2.) Sciences Advances today – westernization leads to reduction of diversity, to different compositions, and also to the activation of resistance genes in our bacteria. 3.) Nature Communications – found children who’ve taken certain antibiotics like azithromycin and clarithromycin experience long-lived shifts in their gut microbiome makeups, and metagenomic and other analysis of the intestinal microbiome of hundreds children review that macrolide use was linked with an increased risk of asthma and weight gain. 4.) Current Biology – gut microbiomes disproportionately affect some 11 Native American tribes. Microbiome population profiling – it matters when it comes to conditions such as obesity, type 2 diabetes, and autoimmune diseases. IBD – Inflammatory Bowel Disease, IBS – Irritable Bowel Sydrome。 5.) 2016 Personalized Medicine World Conference in Silicon Valley – show growing understanding of the interplay between the microbiome and human disease has opened up new opps. For theapeutic interventions aimed at modifying disrupted or dysfunctional microbiota
- QIAamp DNA Stool Mini Kit – Manual Silica membrane-based technology No organic extraction or alcohol precipitation Complete removal of contaminants and inhibitors Sample type: fresh or frozen human stool or others sample types with high concentrations of PCR inhibitors Yields: 10–30 µg Time: 50 minutes Elution volume: 200 µl
- Tritrichomonas foetus is a single celled flagellated protozoan parasite that is known to be a pathogen of the bovine reproductive tract as well as the intestinal tract of cats. In cattle, the organism is transmitted to the female vagina and uterus from the foreskin of the bull where the parasite is known to reside. It causes infertility, and, at times, has caused spontaneous abortions in the first trimester. Cases of infection in cattle have declined due to the use of artificial insemination. In the last ten years, there have been reports of Tritrichomonas foetus in the feces of young cats that have diarrhea[1] and live in households with multiple cats.
- Calf scours is caused by bacterial, viral and sometimes even by parasitic infections. Newborn dairy and beef animals are especially susceptible to calf scours because their immunity systems are not fully developed. Severe fluid loss due to calf scours results in dehydration and often leads to death. Unfortunately, even animals that survive calf scours often remain weak and perform poorly throughout their lives. Common Names Calf scours is also called calf diarrhea, or neo-natal diarrhea. Many consider calf scours as not a specific disease with a specific cause, but actually as a clinical sign of a complex disease. Whatever the truth may be, Calf scours is the primary cause of death in calves from 2 to 30 days of age. Causative Organism According to a leading research organization, the causative organisms of calf scours may be Coronavirus, Rotavirus, K99 E. coli bacteria or Clostridium perfringens Type C.
- Bovine respiratory disease (BRD) is the most common and costly disease affecting beef cattle in the world.[1] It is a complex, bacterial infection that causes pneumonia in calves and can possibly be fatal. The infection is usually a sum of three codependent factors: Stress, an underlying viral infection, and a new bacterial infection.[2] The diagnosis of the disease is complex since there are multiple possible causes.[3] The disease manifests itself most often in calves within four weeks of weaning, when calves are sorted and often sold to different farms. This gives it a common nickname, "Shipping Fever." It is not known whether the stress itself, commingling, or travel conditions are at most to blame, and while studies have identified general stressing factors like transport and cold weather conditions, there is still no conclusive evidence on more specific factors (e.g. distance, transport mode, temperature, or temperature volatility.
- P.Multocida are involved in most costly to North American cattle industry. So called: “shipping fever pneumonia”
- Current customer Workflow: Extraction: Mo Bio PowerSoil DNA Isolation Kit (manually) Standard culture method on agar to ID micro- organism population and profiling (manual) Platting method currently working to discriminate top 5 organisms… to what extend, able to meet FDA requirements? What is the method used in Trial II-B currently? MoBio PowerFecal, Powersoil DNA Isolation kits Agilent MX3005P for q-PCR (4 channels; manual). Samples: 16 DNA samples, 100ul @ greater than 15 ng/ul concentration. Samples will include reference samples from > three individuals extracted from Feces and soil with Powerfecal and Powersoil kits. 48- 64 human fecal and soil samples from multiple subjects. All of which will be QC’ed and a comparable potion of them will be assayed.
- RotorGene and HRM Type-it Chemistry: Quantification on PMA treatments -- Have created new protocols; demonstrated real-time PCR by Roter-GeneQ and HRM kit can detect down to ~1% Live PMA treated cells with good sensitivity, correlation (Slope= -3.22; Eff. = 100%; R2= 0.9983), comparable assay time, and linearity that is far better than the data created by “prepMan Ultra Kit” and Agilent MX3005P (Slope= -4.32; Eff. = 70.5%) HRM donor sample profiling-- Have demonstrated that the combination of ScreenClust software and Rotor-Gene Q instrument is capable of separating multiple sub-clusters from reference population of 12 healthy subjects. Full Automation – A “Walk away”, cost-saving solution: Have created customized protocols, and demonstrated QIAcube High Throughput (HT) with QIAamp DNA virus kit can extract human fecal sample from clinical treatments with better consistency. The nanodrop data have demonstrated good concentration ranges, yield, and purity and salt ratios. The real-time PCR data have shown good Ct range compare to previously extracted customer DNAs extracted from MO Bio “PowerFecal DNA Isolation kit”. QIAgility liquid handler/ assay setup instrument is ideal for fully automated assay setup on 72-Disc and 100-Disc rings. Data from previous customers (data not shown here) had demonstrated 28-32 min/ 100 samples capacity with greater consistency, time saving, and better %CV compare to manual setup.