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
Development of a fingerprinting method for Salmonella Requirements: o Capacity to fingerprint strains belonging to the most important serovars in Belgium o Universal ("one test fits all") o As discriminative as PFGE (if possible ...) o Applicable in routine (veterinary diagnostics, food control, ...) • Rapid (24h) • Robust, easy to perform • Cheap 27/06/2013 2
Serovars selection Animals humans (Human Salmonella frequency in 2011*) Typhimurium (62,8%) o Enteritidis (14,9%) o Kentucky (1,1%) o Derby (1,0%) o Infantis (1,0%) o Newport (0,8%) o ParatyphiB (0,6%) o Brandenburg (0,5%) o Virchow (0,4%) o Hadar (0,3%) o Ohio (0,1%) o Animal health Choleraesuis o Gallinarum biov. Pullorum/Gallinarum o *http://bacterio.wiv-isp.be/reporting/reportspdf/ 27/06/2013 3
SNPs Identification & Selection Source * * Database MLST (http://mlst.ucc.ie/mlst/dbs/Senterica): aroC, dnaN, hemD, hisD, purE, sucA, thrA (Kidgell et al. , 2002) CE Luminex 27/06/2013 4
SNPs Identification & Selection Source * * Database MLST (http://mlst.ucc.ie/mlst/dbs/Senterica): aroC, dnaN, hemD, hisD, purE, sucA, thrA (Kidgell et al. , 2002) SNP selection criteria CE Luminex • Highest discrimination potential at subserovar level • Discrimination potential for > 1 serovar • Matching technical requirements for probe design 21 SNPs selected + 1 Salmonella control ( invA ) 27/06/2013 5
Ligase Chain Reaction (LCR) - Principle Padlock Probe=PLP P2 P1 SNP Pi Tag T1 T2 Tag * P1 P2 T2 T1 = Cy5 (Beckman CE), Biotin (ClonDiag AT™, * Lx200™ high sensitivity) or Cy3 (Lx200™ low sensitivity) 27/06/2013 6
LCR Validation by CE o 22 PLPs targeting: 21 Intra-serovars SNPs • 1 Salmonella control • Mix 1 Mix 2 Mix 3 o Validation by CE (3 mix) N PLPs 9 9 4 Ctrl - (SE with T) Ctrl + (SE with G) fliC_165G o All probes were validated by CE 27/06/2013 7
LCR vs PFGE o Comparable discriminatory capacity for: - S. Hadar - S. Choleraesuis - S. Gallinarum 27/06/2013 8
S. Hadar LCR vs PFGE 27/06/2013 9
LCR vs PFGE o Satisfactory discriminatory power but lower than PFGE: - S. Virchow - S. Newport - S. Brandenburg - S. Paratyphi B - S. Kentucky - S. Ohio - S. Infantis 27/06/2013 10
S. Virchow LCR vs PFGE 27/06/2013 11
LCR vs PFGE or MLVA o Poor discriminatory capacity for: - S. Typhimurium - S. Enteritidis - S. Derby 27/06/2013 12
S. Typhimurium LCR vs PFGE 27/06/2013 13
Transfer to Lx200 instrument 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 • 27/06/2013 14
CE Luminex (3 mixes 1 mix) Mix Genomes Mix fliC Mix MLST BG1030 BG1031 BG1029 BG1033 BG1032 BG1034 BG1035 BG1036 BG1040 BG1041 BG1042 BG1093 BG1039 BG 935 BG 933 BG954 BG955 BG949 BG950 BG951 BG952 BG957 SGSC 1412 SGSC1412 Luminex 350 300 250 MFI 200 150 100 50 0 27/06/2013 15
Biotin – labeling, Purified DNA (MFI) o Biotin-labeled primer, streptavidin-PE detection, no background substraction, 30-min hybridization, 3 washings 14000 12000 MFI (Median) 10000 8000 MFI 6000 4000 2000 0 PLPs detected by Luminex Beads SGSC1412 Exp1 SGSC1412 Exp2 E.coli Exp1 E.coli Exp2 Good repeatibility: 2 PCR and Luminex experiments • Same qualitative results as by CE • Signal to noise Luminex < CE • Sensitivity Luminex < CE • 27/06/2013 16
Cy3 – labeling, purified DNA (MFI) o Cy3-labeling (direct detection), no background substraction, 30-min hybridization, 3 washings 27/06/2013 17
Sa LT2/ E coli , Biotin- vs Cy3-labeling, purified DNA (S/N ratio) o No background substraction, 30-min hybridization, 3 washings 50 45 40 35 SGSC1412/ E. coli 30 25 20 15 10 5 0 sgsc/Ecoli 0805 Cy3 sgsc/Ecoli 2105am Cy3 sgsc/Ecoli 2105 pm Cy3 sgsc/ecoli 25032013 biotin sgsc/ecoli 14032013 biotin 27/06/2013 18
Boiled extract vs purified template DNA (MFI) o Cy3-labeling (direct detection), no background substraction, 30-min hybridization, 3 washings 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 27/06/2013 19
Sa LT2/ E. coli, Boiled extract vs. purified template DNA (S/N ratio) o Cy3-labeling (direct detection), no background substraction, 30-min hybridization, 3 washings 27/06/2013 20
Washing effect (MFI) o Cy3-labeling (direct detection), no background substraction, 30-min hybridization, boiled extract, w or w/o washing 27/06/2013 21
Washing effect (S/N ratio) o Cy3-labeling (direct detection), no background substraction, 30-min hybridization, boiled extract, w or w/o washing 27/06/2013 22
Conclusion and perspectives Proof-of-concept OK, simplified procedure OK o (raw DNA extracts, Cy3-labeling) Competitive consumable price (9 € / 22-PLP LCR assay) o Intra-serovar variability of all SNPs o More SNPs required to better discriminate serovars Typhimurium Enteritidis CE platform : 3-step test conducted in 3 mixes o Luminex platform : 3-step, single-tube test ! o Multiplexing capacity of 22 markers in one assay o most probably amenable to 80 on Lx200 instrument ... 27/06/2013 23
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