The world leader in serving science
November, 7th 2016 - Prague
Diagnostic solutions for mycobacterial animal pathogens
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• 50,000 employees
• 50 countries
• $700 million spent on R&D
• 5 premier brands
The World Leader in Serving Science
3
We enable our customers to make the world
healthier, cleaner, and safer.
We help customers monitor and
control economically significant
farm animal diseases.
We help maintain herd health,
which may lead to improved
efficiencies and better use of
resources.
We help prevent the spread of
zoonotic disease at the farm and
slaughterhouse.
Healthier
Cleaner
Safer
Passion. Applied.
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Diagnostic For Animal Productivity and Zoonotic Diseases
- Offering advanced
diagnostic solutions for farm
animal designed to meet your
challenges of today and
tomorrow
- Workflow optimization from
sampling to result
- Sample prep solutions
- ELISA, qPCR kits and
machines
- Next-generation sequencing
(NGS) and genotyping by
sequencing (GBS) solutions
for the livestock and animal
health markets.
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Let’s work today on Mycobacteria
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Speakers
Dr Vet Med Jean Louis Moyen
Director
Laboratoire Départemental
d’Analyse et de Recherche,
Coulounieix-Chamiers, France
Dr Björn Schröder
Global Product Manager
Thermo Fisher Scientific,
Schlieren Zurich, Switzerland
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Mycobacterial infections – Impact on animal
health and innovative solutions of their
pathogens
Dr Björn Schröder
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Mycobacterial infections - Content
• Mycobacterial animal pathogens and diagnostic solutions
− Mycobacterium avium subsp. avium,
− Mycobacterium avium subsp. Paratuberculosis
− Mycobacterium tuberculosis
• Applied Biosystems™VetMAX™ M. tuberculosis Complex PCR
kit
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Mycobacterial infections - Overview
• Mycobacterium is a genus of Actinobacteria given its own
family, the Mycobacteriaceae. The genus includes
pathogens known to cause serious diseases in mammals,
including tuberculosis and leprosy.
• Robert Koch identified and described on March 24th 1882 the
first time the Mycobacterium tuberculosis.
• The distinguishing characteristic of all Mycobacterium species is
that the cell wall is thicker than in many other bacteria, being
hydrophoci, waxy, and rich in myclic acids/mycolates.
http://medicineworld.org/images/blogs/11-
2007/mycobacterium-tuberculosis-299290.jpg
Robert Koch
(1843* – 1910†)
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Mycobacterial infections - Overview
• Mycobacterium avium subsp. Avium
- M. avium avium belongs to the M. avium complex members
which do not necessarily clinical symptoms in pigs but can cause
zoonotic diseases in man
• Mycobacterium avium subsp. Paratuberculosis
- M. paratuberculosis is the cause of Johne’s disease
• Mycobacterium bovis
- M. bovis belongs to the M. tuberculosis complex (MTBC)
members that are causative agents of human and animal
tuberculosis.
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Mycobacterium avium
subsp. avium
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Mycobacterium avium - Infection with zoonotic potential
• Mycobacterium avium (MA) belongs to the M. avium complex (MAC)
• It is an important pathogen in both animals and humans.
• 28 MAC serotypes have been identified of which to M. avium subsp. avium (MAA) and to M. avium subsp. hominissuis (MAH)
• Zoonotic transmissions from pigs to humans due occur, and can induce a broad range of symptoms.
• Especially immunocompromised people are at higher risk.
• An infection with MA in pigs develops mostly subclinically, only seen as granulomatous lesions in the submaxillary and mesenteric lymph nodes.
• The current diagnosis, based on palpation and the incision of lymph nodes, is not specific and not sensitive
Preferred alternative to pathological examination:
Applied Biosystems™ PrioCHECK™ M. avium Antibody
ELISA kit, porcine
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PrioCHECK M. avium Antibody ELISA kit, porcine
• Risk-based surveillance schemes - only a fraction of pigs are tested
• Helps to save time and resources compared to pathological examination
• Allows efficient implementation of appropriate control measures
Convenient
• No false positive results due to lesions caused by Rhodococcus equi or other infective agents •More sensitive than the routine pathological examination
Preferred alternative
• Fast and robust test, follows a short 4 step protocol
• Allows efficient testing of large numbers of samples
• Uses the same sample in other PrioCHECK tests for swine diseases
• Allows efficient addressing of the herd health status
Efficient
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Mycobacterium avium
subsp.
Paratuberculosis
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MAP - Paratuberculosis
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Unique MAP offering
• Antibody ELISA tests*
• Applied Biosystems™ LSIVet™ Ruminant
Paratuberculosis Advanced Serum ELISA kit
• Applied Biosystems™ PARACHEK 2 Kit
• Real-time PCRs*
• Applied Biosystems™ VetMAX™ MAP Real
Time PCR screening Kit
• Applied Biosystems™LSI VetMAX™
M.paratuberculosis advanced kit
• Applied Biosystems™ LSI VetMAX™ Triplex
M. paratuberculosis kit
*For Veterinary Use Only. For In Vitro Use Only. Regulatory requirements vary by
country; products may not be available in your geographic area.
• Sample extraction (including
instruments)
• Applied Biosystems™ MagMAXTM Total
Nucleic Acid Isolation Kit
• Thermo Scientifc™
KingFisher™ instrument
• Real-time PCR Instruments
• e.g:QuantStudio 5 Real-Time PCR System
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MAP - Samples and testing purposes
Faeces
Milk
•qPCR for screening and
confirmation
•ELISA and qPCR
Serum
•ELISA - Screening
Environmental samples
•qPCR
Milk powder, cheese
•qPCR
Organs
•qPCR
Bacterial colonies /
broth culture
•qPCR
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Mycobacterium bovis
and other members of
the Mycobacteria
Tuberculosis Complex
(MTBC)
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Time after infection
CFT CCT SCT
BOVIGAM Serology
PCR Culture
Products – The bTb Disease
The interferon-gamma field trial: background, principles and progress M. Vordermeier, A. Goodchild, R. Clifton-Hadley, R. de la Rua Veterinary Record (2004) 155, 37-38
World Free of Bovine Tuberculosis World Free of Bovine Tuberculosis
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Bovine Tuberculosis Solution
Lelystad Tuberculin
PPD for Skintest
CCT, CFT or SCT
(PPDB or PPDB+PPDA)
Primary Test (PPD tuberculin for Skin test)
To be
slaughtered
+++
Necropsy
(Lymph nodes)
-
Auxiliary Test (IFN-y Release Assay High prevalence)
(Antibody ELISA very low prevalence)
BOVIGAM ELISA +
Stimulant
Confirmatory Test (PCR or Cell culture)
Lelystad Tuberculin PPD
and/or Peptide Cocktails
and/or Pokeweed
To be
slaughtered
+++ - +
Staying
alive
+ -
+
A generalized TB program decision tree – Serial testing
VetMAX M. tuberculosis
Complex PCR
Herd
Closed
Herd
Open
Testing regime
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Introducing our new VetMAX M.
Tuberculosis Complex
PCR kit (MTBC)
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VetMAX M. tuberculosis Complex
A new tuberculosis PCR – Why ? • The Applied Biosystems™ VetMAX M. tuberculosis Complex PCR kit will be
used as a confirmatory test to determine the TB status of a suspicious
animal.
• The VetMAX M. tuberculosis Complex PCR completes the tuberculosis
solution portfolio for Thermo Fisher Scientific.
• The new VetMAX M. tuberculosis Complex PCR is an improved version of
the current TUB2W100 PCR kit.
Duplex, single well instead of a double well for customer convenience
and cost optimization.
Multiple species approach
Increased robustness against inhibitors
Increased sensitivity by having an excellent specificity
Detects all TB strains belonging to the MTBC including M.bovis,
M.tuberculosis, M.microti, M.caprae, M.pinnipedi, M.africanum, M.canetti
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Workflow from sample to result
Three different workflows have been investigated running the LDMETHOD (UG/µL per spiked
matrix) for Applied Biosystems™ LSI MagVet™ Universal Isolation kit 1 UG/ µL and QIAamp®
DNA Mini Kit 1 UG/ µL
Individual sample :
• Lymph nodes
Sample in
validation :
• Other tissues
• Milk
Automated purification system :
Thermo ScientificTM KingFisherTM mL
or Flex Magnetic Particle instruments
Magnetic bead based kit:
MagVet Universal Isolation kit
PCR instruments :
Applied Biosystems™ 7500, 7500 Fast
and QuantStudioTM 5 Real-Time PCR
System
Real-time PCR kit :
VetMAX M. tuberculosis Complex
• Single real-time PCR
• Duplex: Tb & internal positive control
• Ready-to-use master mix
~3 hrs
Samples Nucleic acid extraction Amplification
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VetMAX M. tuberculosis Complex
• Kit Format
Duplex kit instead of two wells kit. Single well approach, no separated
IPC
• Robustness
Less sensitive to inhibitors than original kit. Change of MMx; Change
of probe IPC Ct values between 26-34; Rate of re-test <5%.
• Stability
Less sensitive to inhibitors than original kit. Change of MMx; Change
of probe. IPC Ct values between 26-34; Rate of re-test <5%.
• IPC
Duplex kit instead of two wells kit. Change of MMx; Change of probe
IPC Ct values between 26-34; Rate of re-test <5%.
• Other
Fast mode.
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VetMAX M. tuberculosis Complex
• Efficiency The PCR efficiency is close to 100% (99.14%).
• Repeatability
PCR repeatability shows a coefficient of variation (CV) comprised
between 0.70 % and 1.55 % (3x same technician)
• Intermediate Precision TUB PCR intermediate precision shows a coefficient of variation (CV)
comprised between 0.74 % and 1.52 % (different operators, days and
thermal cyclers)
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VetMAX M. tuberculosis Complex
• Analytical Sensitivity
The detection limit it is 16 copies of nucleic acid per PCR.
• Analytical Specificity
Analytical Specificity (PCR exclusivity) was evaluated on 22
Mycobacterium strains not belonging to the complex and on 20
pathogens typically found in the same ecological niches, or
phylogenetically close, or because they have the same clinical
symptoms in the target species. Analytical Specificity has been
assessed - based on the tested sample panel – to be 100%
Campylobacter fetus fetus, Campylobacter fetus venerealis, Coxiella burnetii, Mycobacterium avium hominisuis, Mycobacterium avium
subsp avium, Mycobacterium avium subsp paratuberculosis, Mycobacterium bourgelatii, Mycobacterium chelonae subs chelonae,
Mycobacterium flavescens, Mycobacterium fortuitum fortuitum, Mycobacterium gastri, Mycobacterium gordonae, Mycobacterium
intermedium, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium malmoense, Mycobacterium marinum, Mycobacterium
non chromogenicum, Mycobacterium paraffinicum, Mycobacterium scrofulaceum, Mycobacterium simiae, Mycobacterium smegmatis,
Mycobacterium terrae, Mycobacterium vaccae, Mycobacterium xenopi, Mycobacterium xenopi, Mycoplasma bovis, Mycoplasma
hyopneumoniae, Mycoplasma hyosynoviae, Mycoplasma meleagridis, Mycoplasma synoviae, Pasteurella multocida toxinogène,
Salmonella enteritidis, Salmonella typhimurium, Streptococcus agalactiae
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Diagnostic Sensitivity and Specificity
Species H2SO4/NaOH PBS1X Total
Bovine 445 148 593
Badger 82 3 85
Wild boar 2 2 4
Total 529 153 682
Global Results
(Both sample preparation)
TUB2W100 (PCR currently
used in France)
POS NEG
MTBC POS 307 6
NEG 8 361
Kappa correlation coefficient 0.96
Sensitivity (Se)=VP/(VP+FN) 97.5% [95.1 – 98.9%]
Specificity (Sp)=VN/(VN+FP) 98.4% [96.5 – 99.4%]
Field validation study & Field trial study
French samples tested : lymph
nodes and surrounding tissues
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World field test plan
France; Jean Louis Moyen Laboratoire
Départemental D`Analyses Agriculture et
Vétérinaire:
Testing on lymph node and
surrounding tissues
UK; APHA, New Haw
Addlestone, Surrey KT15 3NB, UK
Testing on lymph node and
surrounding tissues
Uruguay; DILAVE,
Montevideo
Testing on lymph node and
surrounding tissues
Philippines;
INFINNOMED
ENTERPRISE's
Quezon City; Manila
Philippines
Testing on lymph node and
milk
New Zealand; Farina Khan Laboratory
Manager (TB Diagnostics) Infectious Diseases,
Animal Science, Agriresearch
Testing on lymph node and surrounding
tissues
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Take home message
• VetMAX M. tuberculosis Complex PCR kit is a reliable
diagnostic tool to confirm suspect cases of TB infected
animals of different species - derived from mycobacteria
belonging to the MTB Complex.
• The duplex single well approach is customer convenient in
terms of number of tests per plate and analysis speed.
• VetMAX M. tuberculosis Complex PCR kit is embedded
into three well defined workflows
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Timelines
• VetMAX M. tuberculosis Complex PCR kit will be available
for purchase at 15.12.2016.
• If country specific batch release procedure applies in you
country the PCR will be available at a country specific time
point.
• Kits for validation purposes are available free of charge
with immediate effect.
• Please contact Dr. Björn Schröder for further information.
© 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise
specified.
Validation of the MTC PCR kit Jean-Louis Moyen EAVLD congress 7th November 2016
Diagnostic of Mycobacterium
tuberculosis complex—back over 9 years of PCR use
Field evaluation of a new qPCR kit from
Thermo Fisher Scientific
J-l. Moyen, L. Brugère, H. Gares
Laboratoire départemental d’Analyse et de Recherche de la Dordogne
Validation of the MTC PCR kit Jean-Louis Moyen EAVLD congress 7th November 2016
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PCR use for bTB diagnostic: French diagnostic scheme
Mycobacterium tuberculosis complex
First Real Time PCR with IS6110
Extraction
Se/Sp evaluation
M. microti
New master mix
Decontamination process
Field evaluation of a new PCR kit from Thermo Fisher Scientific: Comparison with first generation test (current kit)
Comparison with normal or fast protocol
Effect of bacteriological decontamination on PCR
Evolution of PCR test since 2006: SCHEDULE
Validation of the MTC PCR kit Jean-Louis Moyen EAVLD congress 7th November 2016
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PCR use for bTB diagnostic: French diagnostic scheme
Mycobacterium tuberculosis complex
First RT PCR with IS6110
Extraction
Se/Sp evaluation
M. microti
New mix
Decontamination process
Field evaluation of a new PCR kit from Thermo Fisher Scientific: Comparison with first generation test (current kit)
Comparison with normal or fast protocol
Effect of bacteriological decontamination on PCR
Evolution of PCR test since 2006: SCHEDULE
Validation of the MTC PCR kit Jean-Louis Moyen EAVLD congress 7th November 2016
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bTB diagnosis in France
Network of authorized laboratories for diagnostic tests
Trained and managed by the NRL (Anses Maisons Alfort)
Challenge for the network: a rapid direct diagnosis
PCR is used in parallel with bacteriology in compliance with European regulation
LDAR24: is agreed for these tests (about 10.000 bTB pcr, 10.000
bacteriology and 20.000 γ-IFN /year)
Collaboration with ANSES on bTB diagnosis (PHD students)
Validation of the MTC PCR kit Jean-Louis Moyen EAVLD congress 7th November 2016
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how we perform first line PCR: the Network
= PCR / bact (17)
= NRL
First-line diagnosis (official) by network of authorised Regional labs:
= 2012 = 2010- 2011 = 2006-2009
Breakdowns:
Appropriate premises (L3)
Staff trained by the NRL
Follow NRL’s SOPs
Participate to NRL’s Ring Trials
Be accredited (ISO 17025) *
Submit strains / DNA / samples to NRL
(identification/genotyping/expertise) AFNOR NF U 47-104 (bact) & NF U 47-600 (PCR)
*
Validation of the MTC PCR kit Jean-Louis Moyen EAVLD congress 7th November 2016
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NRL molecular diagnostic methods allow: • confirming PCR positive as MTBC (99%)
• confirming PCR first-line positive samples as belonging to a concrete
MTBC entity (95%)
• find the spoligopatterns
This molecular diagnostic scheme provides rapid direct reliable results
Field confidence Gain in the new diagnostic scheme
since 2011, quicker management of infection and avoid further transmission provides very quick info for ameliorating investigation of breakdown origin up to 10% outbreak confirmation by molecular results alone Confirmation of presence of other tuberculous non notifiable agents that confound diagnosis
Confirmation and molecular typing by NRL
Validation of the MTC PCR kit Jean-Louis Moyen EAVLD congress 7th November 2016
Current Techniques of diagnosis
Animal
sampling
Bacterial isolation (Gold
standard): 3 months of culture for
diagnosis
Real-time PCR: 2 days Histology: 8 days
Validation of the MTC PCR kit Jean-Louis Moyen EAVLD congress 7th November 2016
~ 6 months
8 days
8-10 days
3 months 1 month
CCID HISTOLOGY CULTURE IDENTIFICATION
& TYPING
Loss of qualification
6-8 weeks
SID
Diagnostic
cull
PCR IFN-g
(PPD+EC)
Diagnostic scheme
2/3 days 2/3 days 1 month
Validation of the MTC PCR kit Jean-Louis Moyen EAVLD congress 7th November 2016
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PCR use for bTB diagnostic: French diagnostic scheme
First RT PCR with IS6110
Mycobacterium tuberculosis complex
Extraction
Se/Sp evaluation
M. microti
New mix
Decontamination process
Field evaluation of a new PCR kit from Thermo Fisher Scientific: Comparison with first generation test (current kit)
Comparison with normal or fast protocol
Effect of bacteriological decontamination on PCR
Evolution of PCR test since 2006: SCHEDULE
Validation of the MTC PCR kit Jean-Louis Moyen EAVLD congress 7th November 2016
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REMINDER
EAVLD 2010: presentation of the results with LSI 2 wells* kit
Target: IS6110
Inclusion list: all members of the MTBC (M. bovis, M. tuberculosis,
M. caprae, M. microti, M. africanum, M. Canetii, M. pinipedii)
Extraction spin columns or magnetic beads (for hightroughput process)
A better sensitivity than official culture (Se: 95% of the detected samples instead of 90%)
*LSI VetMAX Mycobacterium tuberculosis real time PCR kit complex
(TUB2W100)
Validation of the MTC PCR kit Jean-Louis Moyen EAVLD congress 7th November 2016
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Progress from 2010
• About 70.000 PCR tests realized since 6 years on bovine and wildlife lymph nodes and tissues
• NRL has developed optimized molecular tools for identification, confirmation, typing and epidemiology
• A larger study (2008/2012) to assess Se and Sp • Sensitivity of the PCR 87.7% [82.5 - 92.3%] versus bacteriology 78.1% [72.9 - 82.8%]
Validation of the MTC PCR kit Jean-Louis Moyen EAVLD congress 7th November 2016
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Parameter Mean 95% credible interval
Seculture 78.1 [72.9 – 82.8]
SePCR 87.7 [82.5 – 92.3]
Sehistology 93.6 [89.9 – 96.9]
Spculture 99.1 [97.1 – 100]
SpPCR 97.0 [94.3 – 99.0]
Sphistology 83.3 [78.7 – 87.6]
2008-2012
How well does our PCR method work?
Latent classe analysis
Validation of the MTC PCR kit Jean-Louis Moyen EAVLD congress 7th November 2016
43
PCR use for bTB diagnostic: French diagnostic scheme
First RT PCR with IS6110
Mycobacterium tuberculosis complex
Extraction
Se/Sp evaluation
M. microti
New mix
Decontamination process
Field evaluation of a new PCR kit from Thermo Fisher Scientific: Comparison with first generation test (current kit)
Comparison with normal or fast protocol
Effect of bacteriological decontamination on PCR
Evolution of PCR test since 2006: SCHEDULE
• Detected as member of MTBC group
• Non notifiable agent
• Zoonotic (immunocompromised people)
• Wild life /domestic animals
• Better detection by PCR
Validation of the MTC PCR kit Jean-Louis Moyen EAVLD congress 7th November 2016
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0
2
4
6
8
10
12
No
mb
re d
e ca
s
Faune sauvage Animaux domestiques
Other applications PCR+ve samples:
widespread distribution of M. microti
• Michelet et al JCM, 2015 • Michelet et al Vet Rec, 2015 • Michelet et al EID, 2016
Validation of the MTC PCR kit Jean-Louis Moyen EAVLD congress 7th November 2016
45
PCR use for bTB diagnostic: French diagnostic scheme
First RT PCR with IS6110
Mycobacterium tuberculosis complex
Extraction
Se/Sp evaluation
M. microti
New master mix
Decontamination process
Field evaluation of a new PCR kit from Thermo Fisher Scientific: Comparison with first generation test (current kit)
Comparison with normal or fast protocol
Effect of bacteriological decontamination on PCR
Evolution of PCR test since 2006: SCHEDULE
Validation of the MTC PCR kit Jean-Louis Moyen EAVLD congress 7th November 2016
46
Progress since 2010
New master mix (in 2 wells current version):
A new master mix was introduced in mid 2014 by Thermo Fisher Scientific to reduce inhibition.
Today inhibition are unusual (less than 2% analysis with more than 2 CT later)
1/10 Dilution of the extracts is rare. Allows more concentrate extracts (the use of a small volume of PBS
is possible)
Evaluation and comparison of sample preparation with PBS or H2SO4/NaOH Sample preparation (dissection, crushing…) is a critical point H2SO4/NaOH was used in the french standard for bacteriology
(NF U 47-104)
Crushed material was used for culture and PCR (reproducibility) PBS allows more concentrate samples and gives a better Se and
higher DNA concentration for typing All decontamination process have the same effect
Validation of the MTC PCR kit Jean-Louis Moyen EAVLD congress 7th November 2016
48
PCR use for bTB diagnostic: French diagnostic scheme
First RT PCR with IS6110
Mycobacterium tuberculosis complex
Extraction
Se/Sp evaluation
M. microti
New mix
Decontamination process
Field evaluation of a new PCR kit from Thermo Fisher Scientific: Comparison with first generation test (current kit)
Comparison with normal or fast protocol
Effect of bacteriological decontamination on PCR
Evolution of PCR test since 2006: SCHEDULE
Validation of the MTC PCR kit Jean-Louis Moyen EAVLD congress 7th November 2016
49
Field evaluation of the new PCR kit
MTBC PCR Single well internal positive control (IPC) + target
Material Bovine & badgers (+ a few wild boars): - Infected lymph nodes (confirmation by
spolygotyping) - Non infected lymph nodes ( already tested with the
kit TUB2W100) - We have extract (DNA) and crushed material
Method
- Crushed material was thawned and extracted again - Extraction with KF 96 and magnetic beads - Extracts were amplified with the three methods:
Current kit (TUB2W100) New kit (MTBC), New kit with fast method (MTBC fast)
- Amplified with the thermocycler, Applied Biosystems™ 7500 and 7500 fast Real-Time PCR System
Validation of the MTC PCR kit Jean-Louis Moyen EAVLD congress 7th November 2016
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First trial - Experimental kit
78 extracts from infected animals:
• 3 Wild Boars
• 19 Badgers
• 56 Cattle
• 60 extracts from PBS and H2SO4/NaOH crushed material
• 18 extracts from H2SO4/NaOH only
214 crushed material from:
• Badgers: 12 (1 infected)
• Bovine: 202 (10 infected)
• All but 9 were tested after H2SO4/NaOH
Validation of the MTC PCR kit Jean-Louis Moyen EAVLD congress 7th November 2016
51
First trial - experimental kit
Extracts (magnetic beads): • Target
- No difference between two methods - High difference between crushing methods (H2SO4/ NaOH versus PBS)
• IPC (internal positive control): - Later Ct with new kit and both methods => better inhibition detection - Same IPC Ct values reproducibility (about 3%)
Crushed material (H2SO4/ NaOH) : 11 infected and 203 non infected • Lower Ct with PBS • Later IPC Ct with new methods
H2SO4/NaOH PBS
target IPC target IPC
TUB2W100 31.3 26.6 26.7 26.7
MTBC 31.0 29.0 26.3 28.7
MTBC fast 31.3 28.8 26.8 29.3
Validation of the MTC PCR kit Jean-Louis Moyen EAVLD congress 7th November 2016
52
Second Trial - Commercial kit
166 animal tested :
95 positive (34 badgers & 61 cattle)
71 negative
77 positive were tested with H2SO4/NaOH and PBS preparation
All samples were tested with the three amplification methods
Validation of the MTC PCR kit Jean-Louis Moyen EAVLD congress 7th November 2016
53
Commercial kit results
Negative samples:
• Same results for all methods: no false positive
Positive samples:
• comparison between the 77 samples with all tests
• Very good correlation between results
• More false negative results with H2SO4/NaOH
H2SO4/NaOH PBS
target IPC target IPC
LSI 2w 32.5 26.4 28.6 26.4
MTBC 31.6 27.8 27.8 27.7
MTBC fast 32.0 27.9 28.2 28.4
Validation of the MTC PCR kit Jean-Louis Moyen EAVLD congress 7th November 2016
54
bTB target Ct distribution
No difference between different amplification protocols and kits Large difference between different preparation methods
Validation of the MTC PCR kit Jean-Louis Moyen EAVLD congress 7th November 2016
55
IPC Ct distribution
With the new kit (MTBC): • Same value for both protocols • Higher Ct for MTBC and MTBC fast
Validation of the MTC PCR kit Jean-Louis Moyen EAVLD congress 7th November 2016
56
Summary
Datas from all analysis with PBS protocol for both trials (experimental batch and commercial) :
Sensitivity: (88 infected lymph nodes) *
87/88 positive (86/88 with fast method, not significant)
*in diagnosis animals were tested with at least 3 PCR ( 3 lymph nodes pools/animal)
Specificity: 0 false positive / 274 lymph nodes tested
Validation of the MTC PCR kit Jean-Louis Moyen EAVLD congress 7th November 2016
57
Conclusions
For bTB diagnosis with PCR: PBS preparation should be prefered
→ but decontamination is necessary for bacteriology wich is mandatory in EU regulation
New mix (TUB2W100 – 2014) allows a sharp decrease of inhibitions and a
better sensitivity
Field evaluation of the new kit (2016 MTBC and MTBC fast) No difference of sensitivity between three protocols (TUB2W100/
MTBC/MTBC fast) Rare inhibitions (master mix) Higher detection of inhibition thanks to higher IPC Ct values with new
kit (MTBC or MTBC fast) Easy to use kit :
→ Single well → Ready to use reagents → Same kit for fast protocol
Validation of the MTC PCR kit Jean-Louis Moyen EAVLD congress 7th November 2016
Thank you for your attention
Acknowledgement:
ANSES Maisons Alfort (bTB NRL):
Laura Boschiroli, Sylvie Henault
Technicians of LDAR24
Thermo fisher team
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Questions?
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For Veterinary Use Only. For In Vitro Use Only. Regulatory requirements vary by country; products may not be available in your geographic area. © 2016 Thermo Fisher
Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. QiaAMP is a trademark of Qiagen
Information and materials presented or provided by third parties are provided as-is and without warranty of any kind, including regarding intellectual property rights and
reported results. Parties presenting images, text and material represent they have the rights to do so.
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