Introduction to DiversiLab - wkbwv.be Introduction Knokke.pdf · Pharmaceutical Biodefense ......

Introduction to DiversiLab

Knokke 11 oktober 2011

Dirk JansenApplication Specialist BioMerieux Benelux

Knokke 11 oktober 2011

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Content

Introduction DiversiLabE l ti S tExplanation SystemLibraries

Questions

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DiversiLab

- 2006 BBCI Bacterial Barcodes Inc

Ath G i USAAthens, Georgia, USA Developed DiversiLab System Small company focused on North American market

(Distributor in Italy)(Distributor in Italy) To expand a partner was needed

2006 BBCI merged with BioMerieux Inc. BBCI still located in AthensBBCI still located in Athens

2007 DiversiLab launched as a BioMerieux product 2010 DiversiLab product line Transferred to Grenoble

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Introductiont oduct o

The DiversiLab System DNA fingerprinting and analysis DNA fingerprinting and analysis A powerful tool for tracking the spread and source of

microbial infection, contamination, or epidemics natural and man-made.

Based on patented rep-PCR technology, Microbial isolates can quickly and accurately be Microbial isolates can quickly and accurately be

distinguished at the subspecies and strain level

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Introductiont oduct o

DiversiLab is fragment based genotyping method DiversiLab is fragment based genotyping method

DiversiLab can be uses for: Outbreak analysis Surveillance Enviromental monitoring Enviromental monitoring

Fields for application Clinical Food and Agriculture Pharmaceutical Biodefense Enviromental

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Content

Introduction DiversiLabExplanation Systemp yLibraries

QuestionsQ

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DiversiLabDescriptionDescription

The DiversiLab System for DNA fingerprinting and analysis is a powerful tool for tracking the spread and source of microbial infection, contamination, or epidemics natural and man-made. Based on patented rep-PCR technology, microbial isolates can quickly and accurately be distinguished at the subspecies and strain level.

bioMérieux provides the equipment, reagents and software for b o é eu p o des e equ p e , eage s a d so a e omicrobial genotyping of bacteria and fungi. Packaged together, these components are referred to as the DiversiLab System.

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The DiversiLab technique consists of 3 parts:

1. ISOLATION of DNA from bacteria or fungi with MoBio kit.

2 AMPLIFICATION of the isolated DNA with rep PCR2. AMPLIFICATION of the isolated DNA with rep-PCR.

3. DETECTION of the amplified material by using electrophoresis.3 C O o t e a p ed ate a by us g e ect op o es s

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1. ISOLATION of DNA from bacteria or fungi with MoBio kit.g

A pure bacterial culture must be used to ensure correct fingerprintingfingerprinting.

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1. ISOLATION of DNA from bacteria or fungi with MoBio kit.

Use of the UltraClean kit is recommended for extraction of microbial Use of the UltraClean kit is recommended for extraction of microbial genomic DNA. This kit provides a standardized extraction procedure for all organisms including Mycobacterium and Fungi.

The mechanical lyses and column-based method result in a clean DNAThe mechanical lyses and column based method result in a clean DNA sample perfect for use in the DiversiLab System.

bioMérieux provides extractionkits in 50- and 250-test sizes. Accessories to aid this extractionmethod are also available.

Use of EasyMAG still in R&D

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2 AMPLIFICATION of the isolated DNA with rep PCR2. AMPLIFICATION of the isolated DNA with rep-PCR.

The rep PCR technology is based on the discovery that The rep-PCR technology is based on the discovery that repetitive sequences are interspersed throughout the genome of all bacteria studied to date.

The spacing between these repetitive sequences varies among bacterial isolates because their DNA is different.

During the amplification cycles of the PCR, the Taq DNADuring the amplification cycles of the PCR, the Taq DNA polymerase copies the DNA.

Because numerous areas throughout the bacterial chromosome are amplified many potential genetic differences can beare amplified, many potential genetic differences can be detected.

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2. AMPLIFICATION of the isolated DNA with rep-PCR.

Rep-PCR = repetitive Polymerase Chain Reaction

1. rep-PCR primers bind to many specific repetitive sequences interspersed throughout the genome

rep-PCR primers

2. Multiple fragments of various lengths are amplified

genome

3. Fragments can be separated by mass and charge via electrophoresis

4. A unique rep-PCR DNA fingerprint profile is created with multiple bands of varying intensity

electrophoresis

(+) (-)

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Size of amplified fragments (time)


3. DETECTION of the amplified material by using chips.p y g p

DNA analysis is done by on-chip electrophoresis. Agilent's Lab-on-a-Chip DNA assays allow you to automatically

separate and size PCR fragments and restriction digests accurately and reproducibly.

Samples are moved through a gel dye in the chips by electro-kinetic forces.

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The Diversilab System, what is needed?

Isolation: MoBio UltraClean Microbial DNA isolation kit DNA quantification equipment (strongly advised)q q p ( g y )Amplification: Diversilab web based software Rep-PCR DNA Fingerprinting kit(s), species specificp g p g ( ) p p Taq polymerase and accompanying buffer Thermal CyclerDetection: Agilent 2100 bioanalyzer PC with expert software, upload client and internet connection Micro fluidics chips and reagent kitp g Chip Priming StationGeneral necessities: Vortex

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Centrifuge


The Diversilab System, instruments and reagents

rep-PCR DNA Agilent 2100

MoBio UltraClean Microbial DNA isolation kit

rep PCR DNA fingerprinting kits

Micro fluidics chips and t kit

Agilent 2100 bioanalyzer

reagent kits

Web-based Software

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Software


S f fThe Diversilab Software is used for:

Creation of worksheets for PCR (amp) and Chips (det)Creation of worksheets for PCR (amp) and Chips (det). Transfer of data from/to the Agilent (det). Presentation of measurement data in readable format. Creation of reports. Interpretation and analysis of data, by the customer. Use and/or create libraries for comparison (customer/BMX) Use and/or create libraries for comparison (customer/BMX).

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Diversilab, PCR Worksheet

Assay informationIncluding lot numbers

Auto calculates t lreagent volumes

Displays thermal licycling program

and parameters

Provides PCR plate layout

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Diversilab, Chip Worksheetp

Brief loading protocol

Assay information

Chip number assigned

Well number assigned

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Chip QC: Validate raw data and normalized data send to Customer website

Chip information

Validation button

Raw data Normalized data

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Diversilab Report - Interactive tab

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Interpretation report by CustomerDiversiLab Analysis Report

Problem 1Problem 1Report number #2 Organism: Klebsiella

Analysis rules: Indistinguisahble 0 Bands differences

Similar 1-2 Bands (denoted - - - - -)Different 3 or more Bands (denoted )

Indistinguishable

Indistinguishable

Indistinguishable to 1-3*Similar *

Similar to 7Similar to 6

Indicates band difference

Indistinguishable

Similar*

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See overlay following dendrogram


Interpretation report by Customer in Software

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DiversiLabLibrariesLibraries

Typing Methods Comparative typing

O tb k l t d d l t d i l t d• Outbreak related and unrelated isolates are compared• Sometimes sufficient for outbreak investigation

Definitive (library) typing• Isolates from current outbreak and previous strains are compared• Isolates from current outbreak and previous strains are compared• Necessary to set up and maintain a collection• Robust and standardized method needed

Use of Libraries (niche identification)Use of Libraries (niche identification) Monitoring outbreaks Compare to previous events Compare to events at other placesp p Compare with global cases “Identification” – Illegal use, no prove to promote this

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DiversiLabLibrariesLibraries

Reference Libraries “Global” Libraries that can be used by different labs at different types

Customer Libraries Libraries generated by a customer with isolates from one location….

Diversilab reference libraries MRSA (70 samples) MRSA (70 samples) MRSA (64 Harmony samples) Mold (179 samples) Yeast (231 samples)

Library specifics see:htt //bi i /di il b/ Yeast (231 samples)

Mycobacterium (183 samples) C. Difficile (41 samples) Candida (241 samples)

http://biomerieux-usa.com/diversilab/

Candida (241 samples) E. Coli (108 samples) Salmonella (313 samples, different libraries) G P iti

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Gram Positive Gram Negative


Genus Specific fingerprinting kitsAcinetobacter Enterobacter MycoplasmaArchea Enterococcus PropionibacteriumAspergillus Escherichia PseudomonasAspergillus Escherichia PseudomonasBacillus Francisella SaccharomycesBifidobacterium Klebsiella SalmonellaC. Perfringens Lactobacillus SerratiaCampylobacter Listeria StaphylococcusCandida M. Tuberculosis StreptococcuspClostridium Mycobacterium

General Fingerprinting kitsBacterial TCQCFungal Training

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Fungal Training

DiversiLabDescriptionp

Genus missing in listNot for every genus a specific KitBacterial kit or other specific kit can be used in most casesBacterial kit or other specific kit can be used in most cases

Check on website – Libraries or Downloads Contact bioMerieux

Kits sizeKit size for fingerprinting kits: 24 testsKit size for fingerprinting kits: 24 tests

Changed in 2011 To prevent reagent expiring at customer

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Question

Overwhelming ???

Always feel free to contact me.

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Question

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Introduction to DiversiLab - wkbwv.be Introduction Knokke.pdf · Pharmaceutical Biodefense ......

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