•Two Real-Time PCR assays were selected for further analysis to investigate efficiency, sensitivity and specificity.

•Both assays demonstrated: ➢ Efficiency close to 100% and Sensitivity 1-10 genomic copies ➢ 100 % specificity (Figure 5C)

ConclusionThisinvestigationhasconfirmedthat C.sakazakiiisgeneticallydiverseandrequiresaccurateidentification.Wedevelopedhighlyspecific,sensitiveReal-TimePCRassaystodetectCronobactersakazakii.TheassayswerevalidatedusingasetofATCCC.sakazakiitargetstrainsandcarefullyselectedsetofcloselyrelatedspecies–anti-targets.Theassayswerealsovalidatedindifferentlaboratories.GoodcorrelationofC.sakazakiidetectionbyReal-TimePCRandstandardmethodswasdemonstrated.

Acknowledgements TheauthorswouldliketoacknowledgeDrs.ElenaBolchakovaandJamesBrucefor

scientificsupport

Legal Statements:For Research Use Only. Not for use in diagnostic procedures. ©Copyright 2008.Applied Biosystems. All rights reserved. AB (Design) and Applied Biosystems are registered trademarks of Applied Biosystems or its subsidiaries in the U.S. and/or certain other countries. TaqMan is a registered trademark of Roche Molecular Systems, Inc.

•The specific C.sakazakii genomic fragment –potential identification signature - was identified using whole genome comparison analysis

•A set of Real-time PCR assays were designed to probe the identified signature fragment for specific detection of the target C.sakazakii DNA. Twenty assays were designed and tested. Eight assays detect three target C.sakazakii strains (assays #2, 10, 11, 14, 15, 16, 19, 20) . One assay (#1) detected all four target strains.

•One out of four C.sakazakii strains demonstrated significantly different response when the strains were tested with Real-Time PCR assays

•The strain ATCC 51329 was further investigated using sequencing based MicoSeq system and phylogenetic analysis

•We compared all four C.sakazakii sequences and several closely related organisms (Figure 4A and 4B). ATCC 51329 strain was identified as Enterobacter hormaechei (Enterobacter hormaechei wasfirstidentifiedasauniquespeciesin1989)

Cronobacter sakazakii Real-Time PCR Detection: Sample Characterization, Assay Design and ValidationA. H. Wong, L. Wong, P. Brzoska, T. Schild, M. R. Furtado and O. Petrauskene. Applied Biosystems, Foster City CA 94404

Abstract

Background: Cronobacter sakazakiiisagram-negativebacteriumwithinthefamilyEnterobacteriaceae.Itisfoundthroughouttheenvironment.Powderedmilk-basedinfantformulashavebeenassociatedwithC.sakazakii-relatedoutbreaksinprematureorotherimmunocompromisedinfants.Thecase-fatalityratevariesfrom40-80%amongnewbornsdiagnosedwiththistypeofinfection.TheconventionalmethodapprovedbytheFDArequires5daysfordetection.Methods: Quantitativereal-timePCRisusedinawidevarietyofmolecularapplications,e.g.geneexpressionanalysisandaccuratedetectionofpathogenlevels.ToimprovesensitivityandshortendetectiontimewedesignedaTaqMan®PathogenDetectionassayforC.sakazakii.HavingextremelylimitedsequenceinformationforC.sakazakiiwedecidedtosequenceandanalyzeanumberofgenomicfragmentsaspotentialtargetsforTaqManassaydesigns.ThesetargetsincludedtheRNasePgeneandseveralregionsselectedbyourBioinfomaticsalgorithms.Results: UsingthenewsequenceinformationwedesignedtwoReal-TimePCRassaystodetectC.sakazakii.TheassaysweretestedwithfourATCCC.sakazakii strainswhichwerecharacterizedby16SribosomalDNAsequencingusingAppliedBiosystems’MicroSeqsystem.Specificityoftheassayswasconfirmedusingawidepanelofnon-C.sakazakiistrainsincludingSalmonella, Shigella, E.Coli, Citrobacter and Listeria species, aswellas Enterobacter species: E.nimipressuralis, E.dissolvens, E.cloacae and E.aerogenes.Herewedemonstratethattheassayshavenear100%PCRamplificationefficiencyandahighdegreeofspecificityfortheselectiveamplificationoftargetedgenesinthepresenceofcloselyrelatedtargets.Next step: Customervalidation–weperformedincollaborationwithdifferentlaboratories.Real-TimePCRresultswerecomparedwithISOMethod.Overallgoodcorrelationbetweenstandardmethodsandreal-timePCRresultswasdemonstrated.Conclusions: ThisinvestigationhasconfirmedthatC.sakazakiiisgeneticallydiverseandrequiresaccurateidentification.EnhanceddetectionassaysweredesignedtoenablespecificandhighlysensitivescreeningofCronobacter sakazakii.

IntroductionReal-timePCRdetectionofmicroorganismsinfoodandenvironmenthasbecomeanimportantpartofbiothreatmonitoringandepidemiologicalstudies.Traditionalculturebasedmethodsarelaborious,timeconsumingandoftenhamperedbyslowgrowthofmanyorganisms.WedesignedandtestedReal-timePCRspecificdetectionassaysfordetectionofCronobacter sakazakiiinscreeningenvironmentorfoodsamples.

Study Design ➢ WedesignedandtestedReal-timePCRspecificassaysfordetectionofCronobacter sakazakiibasedonspecificC.sakazakiigenomicfragment➢ TargetorganismswereselectedfromtheATCCpanel:ATCC12868,ATCC29004, ATCC29544andATCC51329➢ Todemonstratespecificityofthedesignedassayscloselyrelatedorganismswere selectedbasedontwocriteria:closeproximityonthephylogenetictreeandpotential presenceinthesamelocation

Assay #11 – target DNA

Assay #16 – target DNA

Assay #11 – target DNAAssay #11 – target DNA

Assay #16 – target DNA

Anti-target Panel Sample Name Copy no. Avg. Stdev Avg. Stdev FAM Ct VIC CtAssay11_citrobacter 2000000 45 0 29.25 0.25Assay11_E.aero 2000000 45 0 29.21 0.13Assay11_E.cloacae 2000000 45 0 29.19 0.02Assay11_E.ColiK12 2000000 45 0 29.20 0.21Assay11_E.diss 2000000 45 0 29.02 0.03Assay11_E.nimi 2000000 45 0 29.21 0.08Assay11_Listeria 2000000 45 0 29.10 0.01Assay11_NTC1 2000000 45 0 29.05 0.07Assay11_NTC2 2000000 45 0 29.16 0.10Assay11_NTC3 2000000 45 0 28.97 0.11Assay11_NTC4 2000000 45 0 29.03 0.13Assay11_Salmonella 2000000 45 0 29.09 0.08Assay11_Salmonella anatum 2000000 45 0 29.13 0.18Assay11_Salmonella dublin 2000000 45 0 29.16 0.04Assay11_Salmonella entr 2000000 45 0 29.36 0.10Assay11_Salmonella typh 2000000 45 0 29.03 0.05Assay11_Shingella 2000000 45 0 29.19 0.21Assay16_citrobacter 2000000 45 0 28.78 0.26Assay16_E.aero 2000000 45 0 29.03 0.12Assay16_E.cloacae 2000000 45 0 29.05 0.08Assay16_E.ColiK12 2000000 45 0 29.17 0.03Assay16_E.diss 2000000 45 0 29.00 0.11Assay16_E.nimi 2000000 45 0 29.08 0.11Assay16_Listeria 2000000 45 0 29.18 0.10Assay16_NTC1 2000000 45 0 29.08 0.20Assay16_NTC2 2000000 45 0 29.02 0.13Assay16_NTC3 2000000 45 0 28.97 0.06Assay16_NTC4 2000000 45 0 29.09 0.15Assay16_Salmonella 2000000 45 0 29.18 0.01Assay16_Salmonella anatum 2000000 45 0 29.10 0.11Assay16_Salmonella dublin 2000000 45 0 29.22 0.01Assay16_Salmonella entr 2000000 45 0 29.20 0.18Assay16_Salmonella typh 2000000 45 0 29.23 0.09Assay16_Shingella 2000000 45 0 29.25 0.21

Cronobacter sakazakii assays target test with 4 ATCC DNA strains

05

1015202530354045

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Assay ID

Ct

12868290042954451329

Figure 2Validation panel16S Phylogenetic tree

Figure 4A

Figure 4B

Figure 3

Figure 5A

Figure 5B

Figure 5C

100 % match to C. sakazakii

~100 % match to E. Hormachei

ATCC 29544 C. sakazakii

ATCC 51329

Figure 1