catalogue - LED Techno

1
CATALOGUE
MATERIALS AND CONSUMM ABLES
January 2014
ADNUC LE IS S AS
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
TABLE of CONTENTS
I.
Conditions of a PCR lab implementation .......................................................................................3
1
2
3
4
II.
Necessary material .......................................................................................................................... 3
Training ........................................................................................................................................... 5
ADNucleis kits prices ..................................................................................................................... 6
ADNucleis’ kits advantages ........................................................................................................... 6
Methodology ....................................................................................................................................6
1
2
3
4
III.
Enrichment and culture media ....................................................................................................... 6
DNA extraction and purification .................................................................................................. 7
Amplification kits ........................................................................................................................... 7
External Point Control (EPC) & Internal Point Control (IPC) .................................................... 8
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
Real time RT PCR or QPCR
APPLIED BIOSYSTEMS
7000-7300-7500-7900
STRATAGENEAGILENT
QIAGEN
EPPENDORF
Rotorgene
Realplex
MX 3005P
COM PLEM ENTARY DEVIC ES
DRY BATH
MULTICHANNEL
MICROPIPETTE
1-10µL / 30-50µL
MICROBIOLOGICAL SAFETY CABINET
PLATE OR STRIPS
CENTRIFUGE
STOMACHER
REFRIGERATOR / FREEZER
(MSC-P2)
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
M aterial & Consummables necessary for the det ect ion of genet ic tar get s via real
time PCR
ENRICHMENT :
Bacterial concentration (optional)
Homogeneization
Graded tube with cap (1.5ml)
Stomacher
Centrifuge (rotor compatible with 1.5ml microtube)
Stomacher bag with filter
(precision : 400ml)
Incubation
DNA EXTRACTION
Pelleting reagents
Oven stabilised at 37°C or 30 °C
Plate or strips centrifuge
Transfer
Thermal and Chemical Lysis
Micropipettes : 25-200µl, 1 ml ….
Thermocycler or water bath or dry bath
Graded tube with cap (15ml)
Extraction buffer
Bacterial growth
Ethanol 96°
Culture media (PCRone, EPT, Half Fraser …)
Transfer
Deionised water
Micropipette 25-200µl
PURIFICATION using COLUMNS
PURIFICATION using MAGNETIC
MICROBEADS
Pelletting reagents
Pelletting reagents
Tubes, plates or strips centrifuge
Tubes, plates or strips centrifuge
Transfer
Transfer
Micropipette 25-200µl and 300µl (1 or 8
channels)
Micropipette 25-200µl and 300µl (1 or 8 channels)
Distripette with combitips
Reservoirs for buffers
Filtration
Magnetic separation
Filtration Columns
Deep-well plate, X-pierced film, aluminium foil
4 Collecting tubes
Magnetic separating device 96
Wash buffers 1 and 2
Wash buffers 1 and 2
Elution
Elution
Elution buffer
Elution buffer
Conic Tubes of 1.5ml volume
Conic Tubes of 1.5ml volume or smaller
DNA Conservation
DNA Conservation
200µl Microtubes
200µl Microtubes
AMPLIFICATION
Distripette with combitips
Reagents
Materials
Master mix PCR (1)
Thermocycler (sybr channel)
Master mix PCR Salmonella (AFNOR ADN 33/03 – 04/10) (1)
Centrifuge with plate rotor
Master mix PCR O157 (AFNOR ADN 33/01 – 02/10) (1)
Plate holder
Master mix PCR H7 (AFNOR ADN 33/01 – 02/10) (1)
Microtube (200µl) plate holder
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.
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
TRAINING activity registered under the
N° 82 69 09708 69 with Préfecture of Rhône-Alpes Region.
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
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
BPW (Buffered Peptone
Water) 500g
BLEB 500g
PCRone 400g
PCRone 400g
PCRone 2kg
Reference
Price € Tax excl.
BPW
45
BLEB
Mpcr_001_100g
Mpcr_001_400g
Mpcr_001_2kg
70
25
60
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
(food matrices,
complex water
matrices)
Simple water
matrices
HQS_ExtractP
Columns and Microbeads
HQS_Extract F
Microbeads
HQS_Extract E
Microbeads
DNA purification
kits
References
All matrices
For purification using Columns (included : extraction buffer,
wash buffers 1 and 2, elution buffer, columns and 4 collecting
tubes
For purification using magnetic microbeads (included : deepwell plate, X-pierced film, aluminium foil, wash buffers 1 and
2, elution buffer).
HQS_purif_Col
HQS_purif_BM
All matrices
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.
6600
6400
105
6200
100
6000
5800
95
5600
90
5400
85
5200
80
5000
75
4800
4600
70
4400
65
4200
60
55
3800
- dI / dT (%)
Fluorescence (norm)
4000
3600
3400
3200
3000
2800
50
45
40
35
30
2600
25
2400
2200
20
2000
15
1800
10
1600
5
1400
0
1200
1000
-5
800
-10
600
-15
400
-20
200
0
66
-200
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22 23
Cycle
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
Temperature [°C]
82
83
84
85
86
87
88
89
90
91
92
93
94
95
45
Threshold: 1%
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]
Kit HQS_salmo_sybr
5 successive dilutions of Salmonella spp DNA purified using magnetic beads
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.
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)
100
- dI / dT (%)
80
60
40
20
0
-20
76
78
80
82
84
86
88
Temperature [°C]
90
92
94
Kit HQS_BVD_sybr
Threshold: 15%
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).
External Point Control (EPC) & Internal Point Control (IPC)
Internal Point Control (IPC)
HQS_IPC (specify target)
External Point Control (IPC)
HQS_EPC
PCR M aster mix Buffer
Sybr Green PCR Master mix
HQS_AmpliS
TaqMan PCR Master mix
HQS_AmpliT
Standard DNA
DNA_target
ADNUCLEIS SAS
30 chemin des Mouilles, 69290 GREZIEU LA VARENNE
Email : [email protected]
Tél : + 33 (0) 478 571 640
9
T aq DNA Polymerase
Taq DNA Polymerase
HQS_Taq_Nucleis
Reverse T ranscriptase
Reverse transcriptase
HQS_RT_Nucleis
Food Safety
Cronobacter sakazakii spp
HQS_saka_sybr
(AFNOR Validation 12h-24h)
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
HQS_O157_sybr
(AFNOR Validation 12h-24h)
E. coli H7
HQS_H7_sybr
(AFNOR Validation 12h-24h)
E.coli O26
HQS_O26_sybr
E.coli O55
Listeria monocytogenes
HQS_O55_sybr
HQS_Listm_IPC_tqm
(validation in progress AFNOR 16h-24h)
Listeria spp
Salmonella spp
HQS_Listspp_sybr
HQS_Salm_sybr
(AFNOR Validation 12h-24h)
Anim al Health
Species
Canine
Feline
Birds
Fishes
Porcine
Reptiles
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
Rodents
Ruminants
10
Environnement
Listeria spp
HQS_listsp_sybr
Salmonella spp
HQS_salmo_sybr
Legionella spp
HQS_Legiospp_IPC_tqm
Legionella pneumophila
HQS_LegioP_tqm
Human Health
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 :
 Possibility of using 1
broth and 1
temperature for several
germs
CONCENTRATION (optional)
Optional step
Low speed centrifugation 5
minutes at 500g
Duration
 Optimal growth of
germs
10 to 20 minutes
+ pipetting 10s per sample
Speed
4500g
 Limited bacterial
competition
Capacity
6 to 52 sampling
Sampling
1mL
Detection
advantage
10 times higher when
compared to AFNOR
validated method
 Multiple germs growth
Enrichment broth
PCRone
(Salmonella, Cronobacter,
Bacillus, …)
 Limited PCR inhibition
Sampling
 25g of sample
Dilution factor
 10
ADNUCLEIS SAS
30 chemin des Mouilles, 69290 GREZIEU LA VARENNE
Email : [email protected]
Tél : + 33 (0) 478 571 640
11
DNA EXTRACTION
Extraction type
Enzymatic and/or chemical
+ thermal lysis
Duration
10 min to 60 min
Capacity
Packaging
Compatibility
Up to 96 simultaneous DNA
extractions
8 wells strips filled
beforehand with individual
caps
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
Reagents
conservation
Elution volume
45 min for 96 samples
1 year at Room
Temperature
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
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
Sensitivity
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
3 cfu/ml
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:
BM1824
BM2113
ETH10
ETH225
INRA023
SPS115
TGLA122
TGLA126
TGLA227
BM1818
ETH3
TGLA53
INRA Markers:
Samples:
Blood
Oral swab
HUJI177
ILSTS65
INRA72
INRA92
INRA135
INRA177
INRA222
Cartilage
FELINES:
Samples:
Blood
Oral swab
ISAG Markers:
FCA069
FCA075
FCA105
FCA149
FCA220
FCA229
FCA310
FCA441
FCA678
AMEL
ZFXY
ISAG Markers:
CANINES:
Samples:
Blood
Oral swab
AHTk253
AHT121
FH2054
CXX279
INRA21
AHTk211
REN54P11
REN162C04
AHTh260
FH2848
AHTh171
AHT137
REN105L03 REN247M23
AHTh130
INU005
REN169O18
INU030
AMELOGENIN
INU055
REN64E19
REN169D01
ADNUCLEIS SAS
30 chemin des Mouilles, 69290 GREZIEU LA VARENNE
Email : [email protected]
Tél : + 33 (0) 478 571 640