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ADNUCLEIS SAS 30 chemin des Mouilles, 69290 GREZIEU LA VARENNE
Email : [email protected]
Tél : + 33 (0) 478 571 640
1
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MMAATTEERRIIAALLSS AANNDD CCOONNSSUUMMMMAABBLLEESS
JJaannuuaarryy 22001144
AADDNNUUCCLLEEIISS SSAASS 1 001 000 € Capital
Head Offices : 30 chemin des Mouilles 69290 GREZIEU LA VARENNE
SIRET : 497 802 165 000 17 TVA intraCEE : FR 3649780216500017
Email : [email protected] Tel : + 33 (0) 478 571 640
Mob : + 33 (0) 660 960 141
ADNUCLEIS SAS 30 chemin des Mouilles, 69290 GREZIEU LA VARENNE
Email : [email protected]
Tél : + 33 (0) 478 571 640
2
TTAABBLLEE ooff CCOONNTTEENNTTSS
I. Conditions of a PCR lab implementation ....................................................................................... 3
1 Necessary material .......................................................................................................................... 3
2 Training ........................................................................................................................................... 5
3 ADNucleis kits prices ..................................................................................................................... 6
4 ADNucleis’ kits advantages ........................................................................................................... 6
II. Methodology .................................................................................................................................... 6
1 Enrichment and culture media ....................................................................................................... 6
2 DNA extraction and purification .................................................................................................. 7
3 Amplification kits ........................................................................................................................... 7
4 External Point Control (EPC) & Internal Point Control (IPC) .................................................... 8
III. Products ............................................................................................................................................ 8
IV. Technical features of ADNucleis real time PCR kits .................................................................. 10
V. Genotyping ..................................................................................................................................... 12
ADNUCLEIS SAS 30 chemin des Mouilles, 69290 GREZIEU LA VARENNE
Email : [email protected]
Tél : + 33 (0) 478 571 640
3
I. Conditions of a PCR lab implementation
1 Necessary material
RReeaall ttiimmee RRTT PPCCRR oorr QQPPCCRR
APPLIED BIOSYSTEMS 7000-7300-7500-7900
STRATAGENE-AGILENT MX 3005P
QIAGEN Rotorgene
EPPENDORF Realplex
CCOOMMPPLLEEMMEENNTTAARRYY DDEEVVIICCEESS
DRY BATH
MULTICHANNEL
MICROPIPETTE 1-10µL / 30-50µL
PLATE OR STRIPS
CENTRIFUGE
STOMACHER
MICROBIOLOGICAL SAFETY CABINET (MSC-P2)
REFRIGERATOR / FREEZER
Material installation + trials : 48h.
Level P1 laboratory : no specific sanitary requirements. Ventilation : air extraction from the amplification room /air entry inside the DNA extraction
room. Air-conditionning system recommended (room temperature inferior or equal to 25°C).
ADNUCLEIS SAS 30 chemin des Mouilles, 69290 GREZIEU LA VARENNE
Email : [email protected]
Tél : + 33 (0) 478 571 640
4
MMaatteerriiaall && CCoonnssuummmmaabblleess nneecceessssaarryy ffoorr tthhee ddeetteeccttiioonn ooff ggeenneettiicc ttaarrggeettss vviiaa rreeaall
ttiimmee PPCCRR
ENRICHMENT :
Homogeneization
Stomacher
Stomacher bag with filter (precision : 400ml)
Incubation
Oven stabilised at 37°C or 30 °C
Transfer
Micropipettes : 25-200µl, 1 ml ….
Graded tube with cap (15ml)
Bacterial growth
Culture media (PCRone, EPT, Half Fraser …)
Deionised water
Bacterial concentration (optional)
Graded tube with cap (1.5ml)
Centrifuge (rotor compatible with 1.5ml microtube)
DNA EXTRACTION Pelleting reagents
Plate or strips centrifuge
Thermal and Chemical Lysis
Thermocycler or water bath or dry bath
Extraction buffer
Ethanol 96°
Transfer
Micropipette 25-200µl
PURIFICATION using COLUMNS
Pelletting reagents
Tubes, plates or strips centrifuge
Transfer
Micropipette 25-200µl and 300µl (1 or 8
channels)
Distripette with combitips
Filtration
Filtration Columns
4 Collecting tubes
Wash buffers 1 and 2
Elution
Elution buffer
Conic Tubes of 1.5ml volume
DNA Conservation
200µl Microtubes
AMPLIFICATION
Materials
Thermocycler (sybr channel)
Centrifuge with plate rotor
Plate holder
Microtube (200µl) plate holder
Reagents
Master mix PCR (1)
Master mix PCR Salmonella (AFNOR ADN 33/03 – 04/10) (1)
Master mix PCR O157 (AFNOR ADN 33/01 – 02/10) (1)
Master mix PCR H7 (AFNOR ADN 33/01 – 02/10) (1)
Master mix PCR Ti (inhibition control) (AFNOR ADN 33/01 – 02/10)
Standard DNA for each target (3µl)
(1) Strip of 8 colored wells filled beforehand, breakable, with individual optic caps.
PURIFICATION using MAGNETIC MICROBEADS
Pelletting reagents
Tubes, plates or strips centrifuge
Transfer
Micropipette 25-200µl and 300µl (1 or 8 channels)
Distripette with combitips
Reservoirs for buffers
Magnetic separation
Deep-well plate, X-pierced film, aluminium foil
Magnetic separating device 96
Wash buffers 1 and 2
Elution
Elution buffer
Conic Tubes of 1.5ml volume or smaller
DNA Conservation
200µl Microtubes
ADNUCLEIS SAS 30 chemin des Mouilles, 69290 GREZIEU LA VARENNE
Email : [email protected]
Tél : + 33 (0) 478 571 640
5
2 Training
ADNucleis is registered for professionnal training :
SIREN number of the training organism: 497 802 165 000 17
TVA intraCEE : FR 3649780216500017
Fees and duration of training:
In accordance with the level of the initial education of the staff and the complexity of the demand. Trainers education level: Master degree or higher. Cost evaluation according to number of training days: - For 1 functionnal laboratory: 1 work day - For 1 laboratory in creation process: 5 work days
TRAINING activity registered under the N° 82 69 09708 69 with Préfecture of Rhône-Alpes Region.
ADNUCLEIS SAS 30 chemin des Mouilles, 69290 GREZIEU LA VARENNE
Email : [email protected]
Tél : + 33 (0) 478 571 640
6
3 ADNucleis kits prices
DNA extraction and PCR amplification kits prices vary in function of the target organisms and the number of kits purchased per year. For more information or for a quote, please contact us at the following address: [email protected]
4 ADNucleis’ kits advantages
Response in 3h, 19h if enrichment is needed. Optimised kits for use with complex matrices. Packaging in colored breakable strips or/ and in bulk. Different germs and targets with compatible thermal profile (allows for the determination of several
targets with the same DNA or in the same PCR run). No additionnal manipulation of DNA extraction or PCR material because strips are filled beforehand
(except in case of incompatibility with the thermocycler or in the case the kits are supplied in bulk). DNA purification using silica columns or magnetic microbeads for optimal purity of extracted DNA. Protocols compatible with large series (96) Compatibility with all qPCR thermocyclers (open system). Possibility of automation of the DNA purification step using the magnetic microbeads.
The following thermocyclers have been tested during the framework of the AFNOR Validation brand: Applied Biosystems : 7000, 7300, 7500, 7900 Qiagen : Rotorgene (Corbet) Stratagene-Agilent : MX 3005P Eppendorf : Realplex
II. Methodology
1 Enrichment and culture media
Dehydrated Media Reference Price € Tax excl.
BPW (Buffered Peptone Water) 500g
BPW 45
BLEB 500g BLEB 70
PCRone 400g Mpcr_001_100g 25
PCRone 400g Mpcr_001_400g 60
PCRone 2kg Mpcr_001_2kg 240
The PCRone® medium manufactured by ADNucleis is a multigerms
culture medium, specifically developed for the detection of germs by PCR after a short enrichment phase (12h to 24h). Its formula is finely designed and makes it particularily suitable for bacteria such as Listeria monocytogenes, Salmonella spp and Enterobacteriaceae (including E. coli O157:H7 and more widely all serotypes). Due to its conception, which is different from those customarily used in classical microbiology, it allows for the rapid growth of the germs present in samples while limiting the competition effect and without adding any PCR inhibitors. Advised enrichment temperature is of 37°C+/-1°C for all germs.
ADNUCLEIS SAS 30 chemin des Mouilles, 69290 GREZIEU LA VARENNE
Email : [email protected]
Tél : + 33 (0) 478 571 640
7
The development of the PCR as a routine method for the detection of pathogens allows for the analysis of several germs from one enrichment medium. Prepare the PCRone culture broth as indicated in the technical sheet of product (add 47 g/l of demineralized water, homogeneise, autoclave 15 minutes at 120°C).
2 DNA extraction and purification
ADNucleis offers several solutions in performing DNA extraction and purification. These solutions involve purification using either silica columns or magnetic microbeads. Buffers and DNA extraction protocols (bacterial cells lysis within the matrix) are dependent on the type and the complexity of the matrices. Once the bacterial cells are lysed, DNA purification protocols are the same whether it is done using columns or magnetic microbeads. Please consult us to obtain the solution most suitable for your activity and applications.
DNA extraction kits
References
Food matrices HQS_ExtractP Columns and Microbeads
(food matrices, complex water matrices)
HQS_Extract F Microbeads
Simple water matrices
HQS_Extract E Microbeads
DNA purification kits
References
All matrices HQS_purif_Col For purification using Columns (included : extraction buffer, wash buffers 1 and 2, elution buffer, columns and 4 collecting tubes
All matrices HQS_purif_BM For purification using magnetic microbeads (included : deep-
well plate, X-pierced film, aluminium foil, wash buffers 1 and 2, elution buffer).
3 Amplification kits
ADNucleis supplies bulk or strips filled beforehand for the amplification (qPCR) of the target(s) with Ti (inhibition) controls. The technology offered uses either hydrolysis probes (Taqman) or Sybr
® green.
Kit HQS_salmo_sybr
5 successive dilutions of Salmonella spp DNA purified using magnetic beads
Temperature [°C]
959493929190898887868584838281807978777675747372717069686766
- d
I / dT
(%
)
105
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
-5
-10
-15
-20
Threshold: 1%
Cycle
4544434241403938373635343332313029282726252423222120191817161514131211109876543210
Flu
ore
scence (
norm
)
6600
6400
6200
6000
5800
5600
5400
5200
5000
4800
4600
4400
4200
4000
3800
3600
3400
3200
3000
2800
2600
2400
2200
2000
1800
1600
1400
1200
1000
800
600
400
200
0
-200
Threshold: 134 (Noiseband)
Baseline settings: automatic, Drift correction OFF
Amplification and dissociation curves in sybr obtained with the amplification kit
HQS_salm_sybr,. ADNucleis on the PCR plateform Realplex Eppendrof
(Color-enhanced scanning electron
micrograph showing Salmonella typhimurium (red) invading cultured human cells Credit: Rocky Mountain Laboratories, NIAID, NIH Source: http://www2.niaid.nih.gov/biodefense/public/images.htm NIAID]
ADNUCLEIS SAS 30 chemin des Mouilles, 69290 GREZIEU LA VARENNE
Email : [email protected]
Tél : + 33 (0) 478 571 640
8
4 External Point Control (EPC) & Internal Point Control (IPC) The controls (IPC or EPC) supplied with each ADNucleis amplification kits correspond to a nulceic acid fragment. ADNucleis develops and makes EPCs and IPCs at the client demand and according to the target sought after.
Temperature [°C]
94929088868482807876
- dI / dT
(%
)
100
80
60
40
20
0
-20
Threshold: 15%
Kit HQS_BVD_sybr Sybr Dissociation curves obtained for the kit HQS_BVD_sybr
ADNucleis on PCR plateform Realplex Eppendrof
III. Products
ADNucleis products are supplied with 2 modes of packaging: - Breakable strips (8 reactions) - bulk (50 – 100 - 500 – 1000 – 5000 reactions)
ADNucleis standard Taq DNA polymerase is supplied with or without 10 X PCR Buffer, Mg2+ ions, dNTPs.
The Reverse Transcriptase is supplied with Oligo dT and random hexamers. Standard DNA packaging is fixed at 50µl (Ref: DNA_target).
EExxtteerrnnaall PPooiinntt CCoonnttrrooll ((EEPPCC)) && IInntteerrnnaall PPooiinntt CCoonnttrrooll ((IIPPCC))
Internal Point Control (IPC) HQS_IPC (specify target)
External Point Control (IPC) HQS_EPC
PPCCRR MMaasstteerr mmiixx BBuuffffeerr
Sybr Green PCR Master mix HQS_AmpliS
TaqMan PCR Master mix HQS_AmpliT
Standard DNA DNA_target
Sybr Dissociation curve obtained for the target BVD (Tm of 86.5°C +/-1°C)
Sybr Dissociation curve obtained for the IPC corresponding to the target BVD
(Tm of 90.75°C +/-1°C)
ADNUCLEIS SAS 30 chemin des Mouilles, 69290 GREZIEU LA VARENNE
Email : [email protected]
Tél : + 33 (0) 478 571 640
9
TTaaqq DDNNAA PPoollyymmeerraassee
Taq DNA Polymerase HQS_Taq_Nucleis
RReevveerrssee TTrraannssccrriippttaassee
Reverse transcriptase HQS_RT_Nucleis
FFoooodd SSaaffeettyy
Cronobacter sakazakii spp (AFNOR Validation 12h-24h)
HQS_saka_sybr
E.coli Stx1 HQS_stx1_sybr
E.coli Stx2 HQS_stx2_sybr
E.coli eaea HQS_eaea_sybr
E.coli O103 HQS_O103_sybr
E.coli O111 HQS_O111_sybr
E.coli O113 HQS_O113_sybr
E.coli O145 HQS_O145_sybr
E.coli O157 (AFNOR Validation 12h-24h)
HQS_O157_sybr
E. coli H7 (AFNOR Validation 12h-24h)
HQS_H7_sybr
E.coli O26 HQS_O26_sybr
E.coli O55 HQS_O55_sybr
Listeria monocytogenes
(validation in progress AFNOR 16h-24h) HQS_Listm_IPC_tqm
Listeria spp HQS_Listspp_sybr
Salmonella spp (AFNOR Validation 12h-24h)
HQS_Salm_sybr
AAnniimmaall HHeeaalltthh
Species Canine Feline Birds Fishes Porcine Reptiles Rodents Ruminants
BVD HQS_BVD_sybr
Coxiella burnetii HQS_FQ_IPC_tqmn
Listeria spp HQS_listsp_sybr
Mycobacterium avium paratuberculosis HQS_MAP_IPC_tqm
Salmonella spp HQS_salmo_sybr
ADNUCLEIS SAS 30 chemin des Mouilles, 69290 GREZIEU LA VARENNE
Email : [email protected]
Tél : + 33 (0) 478 571 640
10
EEnnvviirroonnnneemmeenntt
Listeria spp HQS_listsp_sybr
Salmonella spp HQS_salmo_sybr
Legionella spp HQS_Legiospp_IPC_tqm
Legionella pneumophila HQS_LegioP_tqm
HHuummaann HHeeaalltthh
Chlamydia trachomatis HQS_ChlamT_IPC_tqm
-haemolytic Streptococcus (S. agalactiae) HQS_GBS_IPC_tqm
Pseudomonas aeruginosa HQS_Paer_IPC_tqm
S. aureus meticillin sensitive HQS_SASM_IPC_tqm
Legionella spp HQS_Legiospp_IPC_tqm
Legionella pneumophila HQS_LegioP_tqm
E.coli eaea HQS_eaea_sybr
E.coli O157H7 HQS_O157_sybr // HQS_H7_sybr
E.coli Stx1 HQS_stx1_sybr
E.coli Stx2 HQS_stx2_sybr
Listeria monocytogenes HQS_listm_IPC_tqm
IV. Technical features of ADNucleis real time PCR kits
ENRICHMENT 12-24h :
Enrichment broth
PCRone
Possibility of using 1
broth and 1
temperature for several
germs
Multiple germs growth (Salmonella, Cronobacter,
Bacillus, …)
Optimal growth of
germs
Limited bacterial
competition
Limited PCR inhibition
Sampling 25g of sample
Dilution factor 10
CONCENTRATION (optional)
Optional step Low speed centrifugation 5
minutes at 500g
Duration 10 to 20 minutes
+ pipetting 10s per sample
Speed 4500g
Capacity 6 to 52 sampling
Sampling 1mL
Detection
advantage
10 times higher when
compared to AFNOR
validated method
ADNUCLEIS SAS 30 chemin des Mouilles, 69290 GREZIEU LA VARENNE
Email : [email protected]
Tél : + 33 (0) 478 571 640
11
AMPLIFICATION
Packaging Bulk or target-colored 8 wells strips with individual caps (vol 200µl) filled beforehand.
Other formats upon demand.
Reagents volume 20µl
PCR cycle number 40-50
Technology Sybr (AFNOR), Hydrolysis probes
Duration of a PCR run
Multiplexing
Specificity
Exclusivity
50 minutes to 1h30 depending on thermocycler
Depending on the tagets (non tested for ADNucleis kits AFNOR validated)
100%
100%
Capacity for a 96 block
* Sybr-Green
* Hydrolysis probes
*46 samples for 1 target, 30 samples for 2 targets (96-(nb targets+1))/(nb targets +1)
*94 samples for 1 target with the IPC, 44 samples for 2 targets
Conservation -20°C, 1 year
Material
ADNucleis PCR kits are compatible with the majority of real time thermocyclers available
on the market are compatible.
The following real-time thermocyclers have been tested within the framework of AFNOR
validation brand:
- Applied Biosystems : 7000, 7300, 7500, 7900,
- Qiagen : RotorGene
- Stratagen-Agilent : MX 3005P
- Eppendorf : Realplex
Automation YES
Detection limit in food
safety
With enrichment
* 30 to 100 cfu/ml of post-enrichment broth (depending on the germ sought after)
* <1 cfu/ 25ml of sample pre –enrichment
Without enrichment
* 30 to 100 cfu/ml depending on the germ sought after
Sensitivity 3 cfu/ml
DNA EXTRACTION
Extraction type Enzymatic and/or chemical
+ thermal lysis
Duration 10 min to 60 min
Capacity Up to 96 simultaneous DNA
extractions
Packaging
8 wells strips filled
beforehand with individual
caps
Compatibility All real time Thermocyclers,
dry bath
Conservation -20°C 1 year
Reagents volume 20µl
Sampling volume 100µl to 1 ml
DNA PURIFICATION
Duration and
capacity 45 min for 96 samples
Reagents
conservation
1 year at Room
Temperature
Elution volume 50 to 200µl
(16 to 65 PCR reactions)
Silica Magnetic
microbeads format
Individual tubes or 96 wells
plate
Silica Columns
format
Individual tubes or 8 wells
strips or 96 wells plates
Automation YES if Magnetic microbeads
ADNUCLEIS SAS 30 chemin des Mouilles, 69290 GREZIEU LA VARENNE
Email : [email protected]
Tél : + 33 (0) 478 571 640
12
V. Genotyping
ISAG Markers:
BOVINES:
INRA Markers:
Samples:
Blood
Oral swab
Cartilage
FELINES: ISAG Markers:
Samples:
Blood
Oral swab
ISAG Markers:
CANINES:
Samples:
Blood
Oral swab
BM1824 TGLA122
BM2113 TGLA126
ETH10 TGLA227
ETH225 BM1818
INRA023 ETH3
SPS115 TGLA53
HUJI177 INRA135
ILSTS65 INRA177
INRA72 INRA222
INRA92
FCA069 FCA310
FCA075 FCA441
FCA105 FCA678
FCA149 AMEL
FCA220 ZFXY
FCA229
AHTk253 AHTh260 FH2848
AHT121 AHTh171 AHT137
FH2054 REN105L03 REN247M23
CXX279 AHTh130 INU005
INRA21 REN169O18 INU030
AHTk211 AMELOGENIN INU055
REN54P11 REN64E19
REN162C04 REN169D01