Global Array: DNA Oligonucleotide Microbial Diagnostic Microarray (MDM)

Tonelli Alfreda

13/09/2009

Introduction Pathogen isolation and identification considerations

enrichment and selective culture media appropriate incubation conditions

temperature atmosphere

various identification kits for confirmation interpretation of results is often subjective

qualified personnel essential Time to diagnosis a limiting factor

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Introduction A diagnostic test that will identify pathogens and zoonotic organisms, because of the great number of organisms, to this effect microarrays were chosen because they are; high throughput method easy to perform harmonizeable easy to interpret ? ? ?

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What is an MDM? An MDM is a Microbial Diagnostic

MicroarrayHigh throughput method for the detection of multiple genes belonging to one or more organismOligonucleotide microarray specific oligonucleotides, 35-70 nucleotides,

in length are chemically synthesized the oligonucleotides are spotted, at defined

positions, onto a glass slide to construct the array

binding of fluorescently labelled probes to the oligonucleotides is detected spectrophotometrically13/09/2009

What does an MDM look like ?

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Genes present on an MDM?

Probes to all genes of organisms of interest in veterinary and medical field Probes should be common within the genera inter and intra species specific

Probes are designed in such a fashion as to have few x-reactors Bacteria have different virulence mechanisms virulence genes are they unique to species?

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MDM Genes used in the design of microarray are; Housekeeping genes

16S rRNA, 16S-23S rRNA intergenic transcribed spacer

region (ITS), rpoB; RNA polymerase Beta Hsp60/groEL; Heat shock protein recA; Recombinase A gyrB; Gyrase Beta

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MDM Virulence genes

bacterial toxins adhesins important virulence factors

facilitate colonisation cause tissue damage

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Sample MDM: Pig zoonoses

The bacteria that will be detected will belong to the following genera

Campylobacter sp Staphylococcus sp Enterococcus sp Listeria sp Clostridium sp Salmonella sp Streptococcus sp Yersinia sp and others.

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Plan of the investigation1. Literature Search for pathogenic

organisms2. Identification in pathogens of

housekeeping genes virulence genes

3. Collection of all genes of interest in FASTA format

4. “Unique Sequence” search with OligoPicker2.3.2

5. “Unique Sequence” is checked for homology in BLAST (NCBI) and other BLAST engines 13/09/2009

Plan of the investigationaccording to species

ERIC PATRIC NMPDR Other BLAST engines particular to speciesbeing investigated

5. Choice of control sequences to be included in microarray

Negative and positive 6. Design of the MDM

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Plan of the investigation7. Choice reference strains to be used for

validation8. Purchase of reference strains9. Extraction of DNA from reference

strains10. Elaboration of Hybridization protocol:

Standard Operating Procedure for the MDM

11. Choice of software12. Hybridization of Reference strains13/09/2009

Plan of the investigation13. Analysis of Results ???14. Hybridization of field strains15. Analysis of Results ???16. Extraction of DNA from Test material17. Hybridization of Test material18. Analysis of Results ??? 19. Comparison of fluorescent profiles of

strains, HOW?

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Preliminary data Preliminary hybridization results are available at the BrIzsP-Db database

http: candida.bri.nrc,ca/Italy2/Microarray version V2.0 for strains of Listeria sp Campylobacter sp

Cluster analysis may be performed by using Four different methods Eight different algorithms

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Analysis of results Analysis of results is rather complex when 8096 fluorescent signals must be interpreted A interactive database of results was constructed which automatically transforms the fluorescent signals and performs cluster analysis with various mathematical algorithms

Organism Probes

Campylobacter 503

Yersinia 325

Salmonella 621

Listeria 142

Streptococcus 295

Clostridium 116

Others 22

Total 2024

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BrIzP-Db (database)

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Interpretation of data

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Plan of work for the next year

MIAME compliance Finish work with Reference Strains Application of Learning Algorithms to Data Hybridize to slides biological samples

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Timetable2008 2009 2010Sep-Dec Jan-Apr

May-Aug

Sep-Dec Jan-Apr

May-Nov

2nd set of farm samples1            

Storm samples2            

Species screening            

Multilocus sequence typing            

Survival studies            

Thesis preparation            1One week in October; 2Three storms to be sampled13/09/2009

Hybridization Protocol

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