2007. 9. 13. Food Microbiology Lab. Korea University

1. 2007. 9. 13. Food Microbiology Lab. Korea University Current and emerging molecular diagnostic technologies applicable to bacterial food safety Park Jwa-Haeng

2. Introduction

3. Introduction • Currently used FM testings • Traditional methods • The mainstay of FM testing • Involve the isolation and enumeration or multiple • enrichment and culturing steps • Take about 3-7days • Immuno-assays • Simple, rapid and automatable • Enzyme-Linked Immuno-sorbent Assays(ELISA) • Latex agglutination assays

4. Introduction • Nucleic acid-based diagnostic(NAD) assays • Have been successfully applied for the detection • and identification of food borne pathogenes in a • wide range of food types during last decade • Sensitive and specific detection of single or mul- • tiful pathogenes in foods • This review • Molecular technologies for detection of common • bacterial food borne pathogenes concretly

5. NADs for food borne pathogens

6. Nucleic Acid Diagnostics • General characteristics • Faster turnaround time and improved sensitivity and • specificity compared to conventional technologies • Available in a variety of formats ranging from simple • nucleic acid probe hybridization systems to tests in- • corporating amplification of a specific genomic target • Recently, real-time in-vitro amplification system, bio- • sensors, and microarray based platforms

7. Nucleic Acid Diagnostics • Molecular targets • The basis of any NAD assay is a specific nucleic acid • target sequence, unique to each species • To allow for differentiation of the pathogen at both • genus and species levels, • A candidate NAD target should be present in the • cell at relatively high copy number while being • sufficiently heterologous at the sequence level

8. Nucleic Acid Diagnostics • Popularly used target genes • Genomic DNA, multicopy rRNA • genes encoding toxins or virulence factors • and genes involved in cellular metabolism

9. Nucleic Acid Diagnostics

10. Nucleic Acid Diagnostics • RNA compared to DNA as target gene • A labile molecular that is quickly and easily degr- • aded once the organism is killed • Handling RNA is more difficult than DNA • The advantage of enabling viable organisms to • be distinguished from nonviable organisms

11. Nucleic Acid Diagnostics • Direct DNA probe based NADs • Applied for confirmationof the identity of organisms • following culture based isolation of the foodborne pa- • thogen of concern • Not require sophisticated equipment • Simple to perform • 104-105 bacterial cells of detection limit • Commercial NAD assays based on direct nucleic acid • probe technology • AccuprobeR (GenProbe, CA, USA) for • Campylobacter spp and L. monocytogenes • Gene-TrakR (Neogen, MI, USA) for • E. coli, Salmonella and Listeria spp

12. Nucleic Acid Diagnostics • NADs based on in vitro amplification technologies • General scope of Polymerase chain reaction(PCR) • Most popular platform applied in NAD assays for • food borne pathogens • Followed by gel electrophoresis, Southern blot • hybridization or a detecting method using spe- • cific nucleic acid probes • For fluorometric or colorimetric detection of PCR • products, labelled probes in membrane and • microwell are also used

13. Nucleic Acid Diagnostics • Food Microorganisms detected by Recently • developed PCR based assays • Campylobacter spp • - target genes : fla A, cadF, ceuE, cdt and 16S rRNA • Listeria and L.monocytogenes • - target genes : hly, hlyA and prfA • Salmonella spp and Salmonella typhimurium • - target genes : ogdH, invA • E.coli O174 , O177 and O157:H7 • - target genes : wzx, wzy, stx, vt1, vt2, eaeA, hlyA

14. Nucleic Acid Diagnostics • Multiplex PCR assays for simultaneous • detection of two or more FMOs • The simultaneous detection of • Salmonella spp., L. monocytogenes • and E. coli 0157:H7 • in a total assay time of 30h • The simultaneous detection of • E. coli 0157:H7, Salmonella and Shigella

15. Nucleic Acid Diagnostics • Real - time in vitro amplificationtechnologies • General scope of Real-time PCR • Refers to the collection of technologies and • chemistries that monitor the accumulation of • PCR product in a reaction while it is taking place • compared to endpoint detection of the PCR pro- • duct in conventional PCR • Provide sensitive, quantitative detection of PCR • products in a fast turnaround time in a closed • tube format, thereby significantly reducing the • risk of contamination

16. Nucleic Acid Diagnostics • A number of different fluorescent probes have • been employed in real-time PCR assays include- • ing SYBR greenⅠ, TaqMan(5’exonuclease), flu- • orescent resonance energy transfer(FRET), mol- • ecular beacons and scorpion probes

17. Nucleic Acid Diagnostics • Food Microorganisms detected by Recently • developed real-time PCR based assays • SYBR green real –time PCR assays • Salmonella, Campylobacter spp. • TaqMan real -time PCR assays • Salmonella(InvA, sefA), L.monocytogenes(hlyA) • FRET hybridization probe real –time PCR assays • Salmonella, Campylobacter spp.

18. Nucleic Acid Diagnostics • Multiplex real-time PCR assays for simultane- • ous detection of two or more FMOs • The simultaneous detection of • E. coli 0157:H7 , L. monocytogenes • and Salmonella strains • using SYBR green dye • The simultaneous detection of • L. monocytogenes and L.innocua • using TaqMan probe

19. Nucleic Acid Diagnostics

20. Nucleic Acid Diagnostics • Limitations and other considerations for • in vitro amplification NAD tests • Despite their demonstration as rapid, sensitive • and specific detection method, in-vitro amplifica- • tion NAD assays still await general acceptance • and official approvals as standard methods • because of the lack of universal validation and • standardization • The generation of false-positive results can be • originated from dead organisms’ presence in • the food sample • False-negative results can occur because of de- • gradation of target nucleic acid sequences and • the presence of substances inhibiting the PCR

21. Nucleic Acid Diagnostics • Calling for an international initiatives to focus on • the development of internal and external quality • assurance programs, suitable sample processing • guidelines and establishment of proficiency ring • trials • The problem of false positive results can be circu- • mvented by applying a culture enrichment step • prior to PCR analysis • The problem of false negative results can be over- • comed by filtration, centrifugation, use of deterge- • nt and organic solvent treatments, enzyme treat- • ment and sample dilution before PCR

22. Emerging NAD technologies for foodborne pathogen detection

23. Emerging NAD technologies • Biosensors • Definition : a group of devices and technologies that • use a biologically derived material immobilized on • a detection platform to measure the presence of one • or more analytes • Two kinds of Nucleic acid based biosensors • Quartz crystal microbalances(QCM) in combinat- • ion with PCR of the lac gene has been used to det- • ect 1-10 E. coli cells from 100ml of water • Optical based biosensors use surface plasmon • resonance(SPR) to monitor bio-molecular interact- • ions on a surface in real time

24. Emerging NAD technologies • Novel biosensors with advanced visualization and • signal amplification technologies have created the • possibility of monitoring single molecular interacti- • on in real time • The next generation of biosensors will have applic- • action in all sectors of the molecular diagnostics m- • arket

25. Emerging NAD technologies • Microarrays • Definition : consist of large numbers of probes(either • oligonucleotides or cDNAs) immobilized on a solid • surface such as specially treated glass • Have been demonstrated for the molecular identifica- • tion of E. coli O157:H7 and Campylobacter spp. Fro- • m cultures following PCR amplification of target genes • As microarray technologies matures, these planer ar- • rays are being supplemented by further evolutions • including microbead and suspension microarray formats

26. Emerging NAD technologies • Future studies • Current technologies are unable to detect such a • low bacterial load from a food matrix without either • sample amplification or extensive sample purification • techniques • so, these major hurdles have to be overcome before • biosensors and microarrays will provide ‘real time’ • detection of pathogens in food samples

27. Conclusion

28. Conclusion • The application of nucleic acid diagnostics tests for • foodborne pathogen identification is beginning to m- • ake an impact in this sector • Although costs remain high compared with tradition- • al methods, the reduced turnaround time to results is • becoming increasingly important for particular food • types • On going developments in molecular detection plat- • forms including biosensors and microarrays together • with the increasing awareness of the key criteria for • consideration in developing NAD assays provide po- • tential for new bioanalytical test methods that will en- • able multiparameter testing and at line monitoring for • microbial contaminants

29. Thank you !