CCAATTAALLOOGGUUEE - Startpagina · ADNUCLEIS SAS 30 chemin des Mouilles, 69290 GREZIEU LA VARENNE Email : [email protected] Tél : + 33 (0) 478 571 640 1 CCAATTAALLOOGGUUEE

ADNUCLEIS SAS 30 chemin des Mouilles, 69290 GREZIEU LA VARENNE

Email : [email protected]

Tél : + 33 (0) 478 571 640

1

CCAATTAALLOOGGUUEE

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

mailto:[email protected]




Tél : + 33 (0) 478 571 640

2

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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




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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).




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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




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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.




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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.





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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]




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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)




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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 eaea HQS_eaea_sybr

E.coli O103 HQS_O103_sybr




E.coli O157 (AFNOR Validation 12h-24h)

HQS_O157_sybr

E. coli H7 (AFNOR Validation 12h-24h)

HQS_H7_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




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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



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




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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




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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


Documents

CCAATTAALLOOGGUUEE - Startpagina · ADNUCLEIS SAS 30 chemin des Mouilles, 69290 GREZIEU LA VARENNE Email : [email protected] Tél : + 33 (0) 478 571 640 1 CCAATTAALLOOGGUUEE