MagPlex-TAG beads : application to Salmonella fingerprinting Pierre - - PowerPoint PPT Presentation

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Sep 28, 2023 625 likes •868 views

Development of a single-tube assay for multiple SNP typing using MagPlex-TAG beads : application to Salmonella fingerprinting Pierre WATTIAU Ccile BOLAND Veterinary & Agrochemical Research Centre (Brussels, BE) xSAMPLES 2013, Rotterdam

SLIDE 1

Development of a single-tube assay for multiple SNP typing using MagPlex-TAG beads : application to Salmonella fingerprinting

Pierre WATTIAU Cécile BOLAND Veterinary & Agrochemical Research Centre (Brussels, BE) xSAMPLES 2013, Rotterdam

SLIDE 2

Development of a fingerprinting method for Salmonella

Requirements:

Capacity to fingerprint strains belonging to the most

important serovars in Belgium

Universal ("one test fits all")
As discriminative as PFGE (if possible ...)
Applicable in routine (veterinary diagnostics, food control, ...)
Rapid (24h)
Robust, easy to perform
Cheap

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SLIDE 3

Serovars selection

Animals humans

(Human Salmonella frequency in 2011*)

Typhimurium (62,8%)
Enteritidis

(14,9%)

Kentucky

(1,1%)

Derby

(1,0%)

Infantis

(1,0%)

Newport

(0,8%)

ParatyphiB

(0,6%)

Brandenburg

(0,5%)

Virchow

(0,4%)

Hadar

(0,3%)

Ohio

(0,1%)

Animal health

Choleraesuis
Gallinarum biov. Pullorum/Gallinarum

*http://bacterio.wiv-isp.be/reporting/reportspdf/

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SLIDE 4

SNPs Identification & Selection

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Source *Database MLST (http://mlst.ucc.ie/mlst/dbs/Senterica):

aroC, dnaN, hemD, hisD, purE, sucA, thrA (Kidgell et al., 2002)

* CE Luminex

SLIDE 5

SNPs Identification & Selection

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Source *

Highest discrimination potential at subserovar level
Discrimination potential for > 1 serovar
Matching technical requirements for probe design

 21 SNPs selected + 1 Salmonella control (invA) SNP selection criteria

CE Luminex *Database MLST (http://mlst.ucc.ie/mlst/dbs/Senterica):

aroC, dnaN, hemD, hisD, purE, sucA, thrA (Kidgell et al., 2002)

SLIDE 6

Ligase Chain Reaction (LCR) - Principle

27/06/2013 6 T2 T1 P1 P2 Tag

Tag P1 P2 T1 T2 Pi SNP

*

Padlock Probe=PLP

*

= Cy5 (Beckman CE), Biotin (ClonDiag AT™, Lx200™ high sensitivity) or Cy3 (Lx200™ low sensitivity)

SLIDE 7

LCR Validation by CE

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Mix 1 Mix 2 Mix 3 N PLPs 9 9 4

Ctrl + (SE with G) Ctrl - (SE with T)

fliC_165G

22 PLPs targeting:
21 Intra-serovars SNPs
1 Salmonella control
Validation by CE (3 mix)
All probes were validated by CE

SLIDE 8

LCR vs PFGE

Comparable discriminatory capacity for:
S. Hadar
S. Choleraesuis
S. Gallinarum

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SLIDE 9

S. Hadar LCR vs PFGE

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SLIDE 10

LCR vs PFGE

Satisfactory discriminatory power but lower than PFGE:
S. Virchow
S. Newport
S. Brandenburg
S. Paratyphi B
S. Kentucky
S. Ohio
S. Infantis

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SLIDE 11

S. Virchow LCR vs PFGE

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SLIDE 12

LCR vs PFGE or MLVA

Poor discriminatory capacity for:
S. Typhimurium
S. Enteritidis
S. Derby

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SLIDE 13

S. Typhimurium LCR vs PFGE

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SLIDE 14

Transfer to Lx200 instrument

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Cy5-labeling -> Biotin-labeling (or Cy3)
3 LCR mixes -> 1 mix (single-tube)
90-min analysis time -> 30-min hybridization + 5-min analysis

(8 samples)

Comparable qualitative results
S/N ratio and sensitivity < CE

SLIDE 15

CE  Luminex (3 mixes  1 mix)

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BG1030 BG1031 BG1029 BG1033 BG1032 BG1034 BG1035 BG1036 BG1040 BG 935 BG1041 BG 933 BG1042 BG954 BG955 BG949 BG950 BG951 BG952 BG1093 BG1039 BG957 SGSC 1412 Mix Genomes Mix fliC Mix MLST

50 100 150 200 250 300 350 MFI

SGSC1412 Luminex

SLIDE 16

Biotin-labeled primer, streptavidin-PE detection, no

background substraction, 30-min hybridization, 3 washings

2000 4000 6000 8000 10000 12000 14000 MFI (Median) PLPs detected by Luminex Beads SGSC1412 Exp1 SGSC1412 Exp2 E.coli Exp1 E.coli Exp2

Biotin–labeling, Purified DNA (MFI)

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Good repeatibility: 2 PCR and Luminex experiments
Same qualitative results as by CE
Signal to noise Luminex < CE
Sensitivity Luminex < CE

MFI

SLIDE 17

Cy3–labeling, purified DNA (MFI)

Cy3-labeling (direct detection), no background substraction,

30-min hybridization, 3 washings

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SLIDE 18

No background substraction, 30-min hybridization,

3 washings

Sa LT2/E coli, Biotin- vs Cy3-labeling, purified DNA (S/N ratio)

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5 10 15 20 25 30 35 40 45 50 SGSC1412/ E. coli sgsc/Ecoli 0805 Cy3 sgsc/Ecoli 2105am Cy3 sgsc/Ecoli 2105 pm Cy3 sgsc/ecoli 25032013 biotin sgsc/ecoli 14032013 biotin

SLIDE 19

Cy3-labeling (direct detection), no background substraction,

30-min hybridization, 3 washings

Boiled extract vs purified template DNA (MFI)

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Boiled DNA N colonies DO SGSC1412 boiled 1 1 colony 0,151 SGSC1412 boiled 2 some colonies 0,349 SGSC1412 boiled 3 a lots of colonies 0,866

E. coli boiled 1

1 colony 0,137

E. coli boiled 2

some colonies 0,369

E. coli boiled 3

a lots of colonies 1,232

SLIDE 20

Cy3-labeling (direct detection), no background substraction,

30-min hybridization, 3 washings

Sa LT2/E. coli, Boiled extract vs. purified template DNA (S/N ratio)

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SLIDE 21

Cy3-labeling (direct detection), no background substraction,

30-min hybridization, boiled extract, w or w/o washing

Washing effect (MFI)

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SLIDE 22

Cy3-labeling (direct detection), no background substraction,

30-min hybridization, boiled extract, w or w/o washing

Washing effect (S/N ratio)

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SLIDE 23

Conclusion and perspectives

Proof-of-concept OK, simplified procedure OK

(raw DNA extracts, Cy3-labeling)

Competitive consumable price (9€ / 22-PLP LCR assay)
Intra-serovar variability of all SNPs

 More SNPs required to better discriminate serovars

 Typhimurium  Enteritidis

CE platform : 3-step test conducted in 3 mixes
Luminex platform : 3-step, single-tube test !
Multiplexing capacity of 22 markers in one assay

 most probably amenable to 80 on Lx200 instrument ...

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