Download Introduction to DiversiLab

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