From Sample to Result – Workflow Solutions for Genotyping and Pathogen Detection
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Transcript of From Sample to Result – Workflow Solutions for Genotyping and Pathogen Detection
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
Sample to Insight
2
Legal disclaimer
Pathogen detection with HRM and multiplex RT-qPCR
• 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.
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
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
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
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
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
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 extractionVariable extraction efficiencyDifficult 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 caninterfere 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
Sample to Insight
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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
Sample to Insight
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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
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
Lyse Bind Wash Elute
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
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
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
Sample to Insight
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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
Sample to Insight
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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
Sample to Insight
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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
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
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
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
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
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
Sample to Insight
22
Star: Talaromyces stipitatus; Tree: Aspergillus nidulans Ornaments: Penicillium marneffei; Trunk: Aspergillus terreusHat, 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
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
Sample to Insight
Application 1: Standard curves using synthetic templates
24
0 1 2 3 4 5 62022242628303234363840
f(x) = − 3.28705357142857 x + 40.0704464285714
Aspergillus niger
Log target copy number
CT
0 1 2 3 4 5 62022242628303234363840
f(x) = − 3.235625 x + 41.0404464285714
Klebsiella pneumoniae
Log target copy number
CT
0 1 2 3 4 5 62022242628303234363840
f(x) = − 3.14232142857143 x + 41.64875
Mucor/Rhizopus spp.
Log target copy number
CT
0 1 2 3 4 5 62022242628303234363840
f(x) = − 3.16080357142857 x + 41.2038392857143
Pan Aspergillus/Penicillium
Log target copy number
CT
Pathogen detection with HRM and multiplex RT-qPCR
Sample to Insight
Pool1 Pool2 Pool3 Pool4
Acinetobacter baumannii Aeromonas hydrophila Alcaligenes faecalis subsp. Faecalis Aspergillus fumigatusBacillus licheniformis Bartonella henselae Bordetella pertussis Brevundimonas diminutaCampylobacter jejuni subsp. Jejuni Candida albicans Candida glabrata Candida parapsilosisCitrobacter freundii Clostridium diffi cile Clostridium perfringens Clostridium thermocellumCorynebacterium glutamicum Enterobacter aerogenes Enterococcus faecalis Enterococcus faeciumFusobacterium nucleatum subsp. Nucleatum Geobacillus stearothermophilus Haemophilus influenzae Helicobacter pyloriLegionella pneumophila subsp. Pneumophila Listeria monocytogenes Mycobacterium tuberculosis Neisseria meningitidisPantoea agglomerans Pediococcus pentosaceus Plesiomonas shigelloides Proteus mirabilis
Rahnella aquatilis Ralstonia solanacearumSalmonella enterica subsp. enterica serovar Paratyphi A Serratia marcescens
Vibrio choleraeYersinia enterocolitica subsp. Enterocolitica Yersinia pestis Stenotrophomonas maltophilia
Pool5 Pool6 Pool7 Pool8
Bacillus cereus-14579D-5Aggregatibacter actinomycetemcomitans Akkermansia muciniphila Anaerococcus prevotii
Burkholderia cenocepacia Bacteroides thetaiotaomicron Bacteroides ureolyticus Bacteroides vulgatusCandida tropicalis Burkholderia cepacia Campylobacter coli Campylobacter concisusCorynebacterium diphtheriae Capnocytophaga gingivalis Cryptobacterium curtum Cryptococcus gattii
Escherichia coli-200928D-5Enterobacter cloacae subsp. Cloacae Gardnerella vaginalis Lactobacillus jensenii
Klebsiella pneumoniae Lactobacillus casei Lactobacillus gasseri Micrococcus luteusOchrobactrum anthropi Methanobrevibacter smithii Mycoplasma pneumoniae Neisseria flavaPseudomonas aeruginosa Mycoplasma orale Porphyromonas gingivalis Prevotella intermediaShigella flexneri Porphyromonas endodontalis Trichomonas vaginalis Ureaplasma parvumYersinia pseudotuberculosis Treponema denticola Staphylococcus haemolyticus Streptococcus mitis
Pool9 Pool10 Pool11 staph/strep
Aspergillus flavus Atopobium rimae Bacillus subtilisStaphylococcus aureus subsp. Aureus-
Bifidobacterium breveBifidobacterium longum subsp. Infantis Bordetella parapertussis
Staphylococcus epidermidis-
Campylobacter gracilis Campylobacter rectus Campylobacter upsaliensisStaphylococcus saprophyticus subsp. Saprophyticus
Cryptococcus neoformans Desulfovibrio desulfuricans Lactobacillus acidophilus Streptococcus gordoniiHaemophilus ducreyi Klebsiella oxytoca Leptotrichia buccalis Streptococcus mutansLactobacillus plantarum Lactobacillus reuteri Mycoplasma hominis Streptococcus pneumoniaeMycobacterium smegmatis Mycoplasma genitalium Peptostreptococcus anaerobius Streptococcus pyogenesNeisseria gonorrhoeae Parabacteroides distasonis Tannerella forsythia Streptococcus sanguinisPrevotella melaninogenica Proteus vulgaris Vibrio parahaemolyticusVeillonella parvula-#1 Vibrio harvey Streptococcus agalactiae
Application 1: Specificity of bacterial fungal assays
Title, Location, Date 25N
TCTe
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ate
hum
an g
DN
Am
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NA
rat g
DN
Apo
ol 1
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8po
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10
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11
stap
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rep
pool
2022242628303234363840
Thermoactinomyces spp. 2Ct
NTC
Tem
plat
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man
gDN
Am
ouse
gD
NA
rat g
DN
Apo
ol 1
pool
2po
ol 3
pool
4po
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pool
6po
ol 7
pool
8po
ol 9
pool
10
pool
11
stap
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rep
pool
2022242628303234363840
Thermoactinomyces spp. 1Ct
NTC
Tem
plat
ehu
man
gDN
Am
ouse
gD
NA
rat g
DN
Apo
ol 1
pool
2po
ol 3
pool
4po
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pool
6po
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pool
8po
ol 9
pool
10
pool
11
stap
h/st
rep
pool
2022242628303234363840
Klebsiella pneumoniaeCt
Sample to Insight
Application 1: Testing of assays with genomic DNA
26
0 1 2 3 4 5 62022242628303234363840
f(x) = 2.32053571428571 x + 28.2950025
Aspergillus niger
Log Dilution Factor
CT
0 1 2 3 4 5 62022242628303234363840
f(x) = 3.112405 x + 19.776405
Mucor/Rhizopus spp.
Log Dilution Factor
CT
0 1 2 3 4 5 62022242628303234363840
f(x) = 2.56781080357143 x + 26.307851875
Pan Aspergillus/Penicillium
Log Dilution Factor
CT
0 1 2 3 4 5 62022242628303234363840
f(x) = 2.51111133928571 x + 18.2925016964286
Thermoactinomyces spp. 1
Log Dilution Factor
CT
0 1 2 3 4 5 62022242628303234363840
f(x) = − 0.0137010714285716 x + 39.8811464285714
Thermoactinomyces spp. 2
Log Dilution Factor
CT
Aspergillus brasiliensis gDNA Penicillium chrysogenum gDNA Mucor hiemalis gDNA
Thermoactinomyces vulgaris gDNA Thermoactinomyces vulgaris gDNA
Pathogen detection with HRM and multiplex RT-qPCR
Sample to Insight
Performance and sensitivity (LLOQ)
27
Species slope E R LLOQAspergillus niger -3.53 92% 0.999 60Klebsiella pneumoniae -3.24 104% 0.996 80Mucor/Rhizopus spp. -3.51 93% 1.000 230Pan Aspergillus/Penicillium -3.16 107% 0.995 100Thermo spp. 1 -3.07 112% 0.999 30Thermo spp. 2 -3.01 115% 1.000 60
Aspe
rgill
us n
iger
Kleb
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umon
iae
Muc
orRh
izo
Pan
Aspe
rgill
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enici
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Ther
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Ther
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es sp
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-3
-2
-1
0
1
2
3
4
dCt (stool-template) dCt (sputum-template) dCt (sludge-template)
dCt (
[met
agen
omic
sam
ple+
synt
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te
mpl
ate]
–
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Assay performance in metagenomic samples
Application 1: Performance
Pathogen detection with HRM and multiplex RT-qPCR
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
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
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
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
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
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
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
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
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
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
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
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=-3.361Efficiency=0.98
R2=0.9962
M=-3.275Efficiency=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
Sample to Insight
Purpose & backgroundComplex 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
Sample to Insight
Background
Pathogen detection with HRM and multiplex RT-qPCR 41
Sample to Insight
Primer Set NTC (5ul/20-ul PCR) Run-2
JE/Pro 33.76V3 22.3V5 20.83V6 22.1V7 19.45
Threshold = 0.01Dynamic TubeSlope Correct!
Results: Assay and primer sets testing
Pathogen detection with HRM and multiplex RT-qPCR 42
Sample to Insight
ºC71 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
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
Sample to Insight
Threshold = 0.18735127 bps
Threshold = 0.15662460 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
Sample to Insight
ºC71.5 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
Nor
mal
ised
min
us V
5
55
50
45
40
35
30
25
20
15
10
5
0
-5
-10
JE341/Pro805Normalized to V5:
V6 V5V3
V7
ºC72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88
Nor
mal
ised
min
us J
E341
/Pro
805
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
Sample to Insight
For many datasets, most of the eigenvalues “lambda” are negligible and can be discarded.
The eigenvalue measures the variationIn 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
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 RunSophisticated computing and bioinformatics
Pathogen detection with HRM and multiplex RT-qPCR 48
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
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
Sample to Insight
ºC75 80 85 90
dF/d
T2.0
1.5
1.0
0.5
0.0
ºC77 78 79 80 81 82 83 84 85 86 87 88 89
Nor
mal
ised
Flu
ores
cenc
e
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
Sample to Insight
Result: Clustering Analysis by ScreenClust on All QCHT extracted samples
Pathogen detection with HRM and multiplex RT-qPCR 52
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
Sample to Insight
Questions?
Thank you for attending
Contact QIAGEN Technical ServiceCall: 1-800-426-8157 for US
Call: +49 2103-29-12400 for EU
Email: [email protected]
Pathogen detection with HRM and multiplex RT-qPCR 54