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Introduction to DiversiLab Knokke 11 oktober 2011 Dirk Jansen Application Specialist BioMerieux Benelux 1 Content Introduction DiversiLab Explanation E l ti System S t Libraries Questions 2 DiversiLab - 2006 BBCI Bacterial Barcodes Inc Ath Athens, G Georgia, i USA Developed DiversiLab System Small company focused on North American market (Distributor in Italy) To expand a partner was needed 2006 BBCI merged with BioMerieux Inc. BBCI still located in Athens 2007 DiversiLab launched as a BioMerieux product 2010 DiversiLab product line Transferred to Grenoble 3 Introduction t oduct o The DiversiLab System 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 distinguished at the subspecies and strain level 4 Introduction t oduct o DiversiLab is fragment based genotyping method DiversiLab can be uses for: Outbreak analysis Surveillance Enviromental monitoring Fields for application Clinical Food and Agriculture Pharmaceutical Biodefense Enviromental 5 Content Introduction DiversiLab Explanation p System y Libraries Questions Q 6 DiversiLab Description 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 b o é eu p provides o des the e equ equipment, p e , reagents eage s a and d so software a e for o microbial genotyping of bacteria and fungi. Packaged together, these components are referred to as the DiversiLab System. 7 DiversiLab Description 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 2. rep-PCR. PCR 3. DETECTION 3 C O o of the t ea amplified p ed material ate a by us using ge electrophoresis. ect op o es s 8 DiversiLab Description 1. ISOLATION of DNA from bacteria or fungi g with MoBio kit. A pure bacterial culture must be used to ensure correct fingerprinting. fingerprinting 9 DiversiLab Description 1. ISOLATION of DNA from bacteria or fungi with MoBio kit. 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 column based method result in a clean DNA sample perfect for use in the DiversiLab System. bioMérieux provides extraction kits in 50- and 250-test sizes. Accessories to aid this extraction method are also available. Use of EasyMAG still in R&D 10 DiversiLab Description 2 AMPLIFICATION of the isolated DNA with rep 2. rep-PCR. PCR The rep-PCR 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 DNA polymerase copies the DNA. Because numerous areas throughout the bacterial chromosome are amplified amplified, many potential genetic differences can be detected. 11 DiversiLab Description 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 2. Multiple fragments of various lengths are amplified rep-PCR primers genome 4. A unique rep-PCR DNA fingerprint profile is created with multiple bands of varying intensity 3. Fragments can be separated by mass and charge via electrophoresis (+) (-) Size of amplified fragments (time) 12 DiversiLab Description 3. DETECTION of the amplified p material by y using g chips. 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 electrokinetic forces. 13 DiversiLab Description The Diversilab System, what is needed? Isolation: MoBio UltraClean Microbial DNA isolation kit quantification equipment q p ((strongly g y advised)) DNA q Amplification: Diversilab web based software p DNA Fingerprinting g p g kit(s), ( ) species p specific p Rep-PCR Taq polymerase and accompanying buffer Thermal Cycler Detection: Agilent 2100 bioanalyzer PC with expert software, upload client and internet connection p and reagent g kit Micro fluidics chips Chip Priming Station General necessities: Vortex Centrifuge 14 DiversiLab Description The Diversilab System, instruments and reagents MoBio UltraClean Microbial DNA isolation kit rep PCR DNA rep-PCR fingerprinting kits Agilent 2100 bioanalyzer Micro fluidics chips and reagentt kits kit Web-based Software 15 DiversiLab Description The Diversilab Software S f is used ffor: Creation of worksheets for PCR (amp) and Chips (det). (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) (customer/BMX). 16 DiversiLab Description Diversilab, PCR Worksheet Assay information Including lot numbers Auto calculates reagentt volumes l Displays thermal cycling li program and parameters Provides PCR plate layout 17 DiversiLab Description Diversilab, Chip p Worksheet Brief loading protocol Assay information Chip number assigned Well number assigned 18 DiversiLab Description Chip QC: Validate raw data and normalized data send to Customer website Validation button Chip information Raw data Normalized data 19 DiversiLab Description Diversilab Report - Interactive tab 20 DiversiLab Description Interpretation report by Customer DiversiLab Analysis Report Problem 1 Report number #2 Analysis rules: Organism: Klebsiella Indistinguisahble 0 Bands differences Similar Different 1-2 3 or more Bands (denoted - - - - -) Bands (denoted ) Indistinguishable Similar * Indistinguishable to 1-3* Similar to 7 Similar to 6 Indistinguishable Similar* Indicates band difference See overlay following dendrogram 21 DiversiLab Description Interpretation report by Customer in Software 22 DiversiLab Libraries Typing Methods Comparative typing • Outbreak O tb k related l t d and d unrelated l t d isolates i l t are compared d • Sometimes sufficient for outbreak investigation Definitive (library) typing • 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) Monitoring outbreaks Compare to previous events p to events at other places p Compare Compare with global cases “Identification” – Illegal use, no prove to promote this 23 DiversiLab Libraries 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 (64 Harmony samples) Mold (179 samples) Library specifics see: Yeast (231 samples) htt //bi http://biomerieux-usa.com/diversilab/ i /di il b/ Mycobacterium (183 samples) C. Difficile (41 samples) Candida (241 samples) E. Coli (108 samples) Salmonella (313 samples, different libraries) G Gram Positive P iti Gram Negative 24 DiversiLab Description Genus Specific fingerprinting kits Acinetobacter Enterobacter Mycoplasma Archea Enterococcus Propionibacterium Aspergillus Escherichia Pseudomonas Bacillus Francisella Saccharomyces Bifidobacterium Klebsiella Salmonella C. Perfringens Lactobacillus Serratia Campylobacter Listeria Staphylococcus Candida M. Tuberculosis Streptococcus p Clostridium Mycobacterium General Fingerprinting kits Bacterial TCQC Fungal Training 25 DiversiLab Description p Genus missing in list Not for every genus a specific Kit Bacterial kit or other specific kit can be used in most cases Check on website – Libraries or Downloads Contact bioMerieux Kits size Kit size for fingerprinting kits: 24 tests Changed in 2011 To prevent reagent expiring at customer 26 Question Overwhelming ??? Always feel free to contact me. 27 Question 28