8/30/2016 Food Learning Objectives Microbiology 1.Identify the various categories of rapid identification methods Chapter 5 AN INTRODUCTION 2.Explain the basis of immunological,nucleic acid,and biochemical methods Rapid and 3.Recognize when rapid methods are Automated suitable to use Microbial Methods 4.Understand the advantages and disadvantages associated with the use of rapid methods Increased consumer awareness of Microbiological analyses of microbial food safety has increased foods are complex innovation among food microbiologist Food is complex structurally, Development of real-time sensors for physically,chemically detection of toxins in food Microbes may not be present Complexity of microbes associated with in high numbers food has increased Rapid microbiological Global marketplace methods have been Consumer food preference developed to identify Changes in food processing foodborne pathogens
8/30/2016 1 Chapter 5 Rapid and Automated Microbial Methods Learning Objectives 1. Identify the various categories of rapid identification methods 2. Explain the basis of immunological, nucleic acid, and biochemical methods 3. Recognize when rapid methods are suitable to use 4. Understand the advantages and disadvantages associated with the use of rapid methods • Microbiological analyses of foods are complex • Food is com p y, lex structurally, physically, chemically • Microbes may not be present in high numbers • Rapid microbiological methods have been developed to identify foodborne pathogens • Increased consumer awareness of microbial food safety has increased innovation among food microbiologist - Development of real-time sensors for detection of toxins in food • Complexity of microbes associated with food has increased - Global market place - Consumer food preference - Changes in food processing
8/30/2016 US Food Micro Market-Test Volume Testing for pathogens and spoilage microbes is essential to ensure safe and wholesome food Conventional test methods -sensitive cost effective labor intensive 00a require days to complete Products that are minimally processed have a short half-life conventional methods are not reasonable to use due to the short shelf life Rapid methods may be more appropriate Flowchart showing When considering whether an assay is processing steps and "rapid",all steps in the protocol must be relative time needed to considered detect a pathogen in a The rapid assay may only take 15 min food sample. A preenrichment and/or selective enrichment may be needed IMS-immune-magnetic separation Enrichment may take days before the 15 Particles with magnetic min rapid assay can be performed properties are modified Rapid tests have detection limits ranging with target-specific from 102 to 105 CFU/g or ml antibody to capture and purify the target using a magnetic field
8/30/2016 2 • Testing for pathogens and spoilage microbes is essential to ensure safe and wholesome food • Conventional test methods - sens t ve i i - cost effective - labor intensive - require days to complete • Products that are minimally processed have a short half-life - conventional methods are not reasonable to use due to the short shelf life • Rapid methods may be more appropriate • When considering whether an assay is ”rapid”, all steps in the protocol must be considered • The rapid assay may only take 15 min • A preenrichment and/or selective enrichment may be needed • Enrichment may take days before the 15 min rap y id assay can be performed • Rapid tests have detection limits ranging from 102 to 105 CFU/g or ml Flowchart showing processing steps and relative time needed to detect a pathogen in a food sample. IMS - immune-magnetic separation Particles with magnetic properties are modified with target-specific antibody to capture and purify the target using a magnetic field
8/30/2016 Sample Processing Sample processing Pathogens are generally found in low numbers in food Enrichment increases the probability Some type of processing is required of detecting low levels of target before use of a rapid method to detect microbes pathogen Duration of enrichment varies <12h- -Preenrichment-recovery of injured 24h,based on manufacturer's and VBNC cells directions,may even be days Enrichment-promotes pathogen A specific enrichment medium may be growth over other microbes specified or a standard growth Selective enrichment-promotes medium pathogen growth while inhibiting compefing microbes Requirements and Validation of Rapid Methods Reducing time required to obtain an accurate result is the intent of developing a rapid assay Identification of unique features of the target microbe-requires knowledge of the pathogen Ideally,want a near instantaneous result but not there yet ·Accuracy sensitivity-detect low numbers Conventional systems require several days specificity-differentiate target microbe from ·Rapid tests other microbes -provided results within 8 h False-negative-fails to detect the target microbe -require 6-24 h enrichment False-positive-fails to differentiate the target limit of detection 102 to 105 CFU/g or ml microbe from other microbe limitation on the number of samples tested Accuracy needed is <1 cell per 25 g of food
8/30/2016 3 Sample Processing • Pathogens are generally found in low numbers in food • Some type of processing is required before use of a rapid method to detect pathogen - Preenrichment – recovery of injured and VBNC cells - E ih nr c hment – promotes h pat hogen growth over other microbes - Selective enrichment - promotes pathogen growth while inhibiting competing microbes Sample processing • Enrichment increases the probability of detecting low levels of target microbes • Duration of enrichment varies <12h - 24h, based on manufacturer’s directions, may even be days • A specific enrichment medium may be specified or a standard growth medium Requirements and Validation of Rapid Methods • Identification of unique features of the target microbe – requires knowledge of the pathogen • Accuracy - sensitivity – detect low numbers - specificity – differentiate target microbe from other microbes • False-negative – fails to detect the target microbe • False-positive – fails to differentiate the target microbe from other microbe • Accuracy needed is <1 cell per 25 g of food • Reducing time required to obtain an accurate result is the intent of developing a rapid assay • Ideally, want a near instantaneous result but not there yet • Conventional systems require several days • Rapid tests - provided results within 8 h - require 6-24 h enrichment - limit of detection 102 to 105 CFU/g or ml - limitation on the number of samples tested
8/30/2016 Other Factors to Consider Other Factors to Consider Speed of sample processing All aspects of the assay should be easy small sample number-single diagnostic test -technically easy to perform equipment easy to operate results easy to interpret ·Accuracy ·Cost Suitability for the food matrix tested ·fit with company needs -Food constituents should not interfere with training of personnel assay performance ·special equipment Natural microflora should not interfere with service-maintenance contracts accuracy ·disposable supplies Debris should not interfere with accuracy reagents Accepted by industry or government agencies Organizations that Validate Testing Methods for foods AOAC-First or Final Action Status International Standards Organization AOAC International is the most widely rcognized International Dairy Federation and used service that provides testing for manufacturers of test kits AOAC International(formally Association of ·Two programs used Official Analytical Chemists) 、 Collaborative study -publishes FDA Bacteriological Analytical Manual >Rigorous testing >AOAC approved-first action status USDA and FDA do not validate but have manuals that outline the standard methods used in each -Peer-verified organization after 3 years of use-scientific community Food Safety Inspection Service(FSIS)publishes votes for final-action status the Microbiology Laboratory Guidebook Status by AOAC is listed on the kit package
8/30/2016 4 Other Factors to Consider • Speed of sample processing - small sample number - single diagnostic test - large sample large sample number - high-throughput system is needed though more costly • Accuracy • Cost - fit with company needs - training of personnel - special equipment - service – maintenance contracts - disposable supplies - reagents Other Factors to Consider • All aspects of the assay should be easy - technically easy to perform - equipment easy to operate - results easy to interpret • Suitability for the food matrix tested - Food constituents should not interfere with assa y performance - Natural microflora should not interfere with accuracy - Debris should not interfere with accuracy • Accepted by industry or government agencies Organizations that Validate Testing Methods for foods • International Standards Organization • International Dairy Federation • AOAC International (formally Association of Official Analytical Chemists) - publishes FDA Bacteriological Analytical Manual • USDA and FDA do not validate but have manuals that outline the standard methods used in each organization • Food Safety Inspection Service (FSIS) publishes the Microbiology Laboratory Guidebook AOAC - First or Final Action Status • AOAC International is the most widely rcognized and used service that provides testing for manufacturers of test kits • Two programs used - Collaborative study Rigorous testing AOAC approved – first action status - Peer-verified after 3 years of use- scientific community votes for final-action status • Status by AOAC is listed on the kit package
8/30/2016 Rapid Methods Based on Diluting the food sample Traditional Methods Traditional-manually pipetted Biochemical profiles differentiate and identify Rapid bacteria 1.manual diluters Bacterial isolation is required 2.automated plating systems Separation from food matrix that attain 1000-fold Traditional-sterile blender cups dilution on one plate Rapid-sterile bags and Stomacher Products that replace the Counting colonies standard agar plate Traditional method Petrifilm system >time consuming >rehydratable nutrients and a gelling agent embedded in >open to human error disposable cardboard -Rapid-colony counting >1 ml of diluted bacteria used systems to rehydrate the film >scanners and software >specialized for specific >images may be stored microbes E.coli=49(blue colonies with gas)beta- glucuronidase Total coliform=87(red and blue colonies with gas)
8/30/2016 5 Rapid Methods Based on Traditional Methods Biochemical profiles differentiate and identify bacteria Bacterial isolation is required • Separation from food matrix - Traditional – sterile blender cups - Rapid – sterile bags and Stomacher • Diluting the food sample - Traditional – manually pipetted - Rapid 1. manual diluters 2. automated plating systems that attain 1000-fold dilution on one plate • Counting colonies - Traditional method time consuming open to human error - Rapid – colony counting systems scanners and software images may be stored • Products that replace the standard agar plate - Petrifilm system rehydratable nutrients and a gell ing agent embedded in disposable cardboard 1 ml of diluted bacteria used to rehydrate the film specialized for specific microbes E. coli = 49 (blue colonies with gas) betaglucuronidase Total coliform = 87 (red and blue colonies with gas)
8/30/2016 Products that replace the standard agar plate Products that replace the standard agar plate -API 20E Media for detection,enumeration,and >A strip of wells that provide biochemical identification of specific bacteria.yeasts, profiles of the pathogen and mold >Wells are inoculated with target microbe to >CHROMagar as an example rehydrate the compounds Vibrio human pathogens >Incubated and color changes analyzed V.parahaemolyticus-mauve VBAAB1BRM01K09029102 V.vulnificus /V.cholerde Wells serve as 0000CEH-8 EERSS test tubes for green blue to turquoise blue ical V.alginolyticus-colourless F0sP6的8 WwwWwwwWo reactions Immunologically-Based Methods Immunologically-Based Methods Monoclonal antibodies -one specific cellular Most widely used rapid methods target Used to screen and identify specific bacteria Polyclonal antibodies-many cellular targets Limits of detection 103-105 CFU/ml Monoclonal antibodies eliminate cross- reactions with non-target bacteria 6
8/30/2016 6 • Products that replace the standard agar plate - API 20E A strip of wells that provide biochemical profiles of the pathogen Wells are inoculated with target microbe to rehydrate the compounds Incubated and color changes analyzed Wells serve as test tubes for various biochemical reactions • Products that replace the standard agar plate - Media for detection, enumeration, and identification of specific bacteria, yeasts, and mold CHROMagar as an example Vibrio human pathogens V. parahaemolyticus → mauve V l ifi / V h l V. vulnificus / V. cholerae → green blue to turquoise blue V. alginolyticus → colourless Immunologically-Based Methods • Most widely used rapid methods • Used to screen and identify specific bacteria • Limits of detection 103 – 105 CFU/ml Immunologically-Based Methods • Monoclonal antibodies – one specific cellular target • Polyclonal antibodies – many cellular targets • Monoclonal antibodies eliminate crossreactions with non-target bacteria
8/30/2016 Immunologically-Based Methods Immunologically-Based Methods Coat latex beads with antibodies against a specific Coat magnetic beads with antibodies to separate of bacterium specific bacterium from a sample Latex agglutination test Immunomagnetic separation Not sensitive -106 bacteria required for a Beads can be used to inoculate media or used in visible reaction a polymerase chain reaction or other tests Rapid and convenient Beads are available for a number of pathogens 龄 Immunologically-Based Methods Molecular Methods ELISA Enzyme-Linked Immunosorbent Assay- combine the specificity of antibodies with the sensitivity of Nucleic acid-based assays for differentiation simple enzyme assays,by using antibodies or antigens and identification of foodborne pathogens coupled to an easily-assayed enzyme One of many ·DNA methods types of ELISA Polymerase chain reaction PCR Antigen -Pulse-field gel electrophoresis 0。 -Ribotyping linked-enzyme Plasmid typing Restriction fragment length polymorphisms Substrate added Some of these methods are automated and positive wells change color Kits are available to purify DNA and RNA >
8/30/2016 7 Immunologically-Based Methods Coat latex beads with antibodies against a specific bacterium • Latex agglutination test - Not sensitive – 106 bacteria required for a visible reaction - Rapid and convenient Immunologically-Based Methods Coat magnetic beads with antibodies to separate of specific bacterium from a sample • Immunomagnetic separation - Beads can be used to inoculate media or used in a polymerase chain reaction or other tests - Beads are available for a number of pathogens Immunologically-Based Methods ELISA Enzyme-Linked Immunosorbent Assay – combine the specificity of antibodies with the sensitivity of simple enzyme assays, by using antibodies or antigens coupled to an easily-assayed enzyme One of many types of ELISA Antigen Secondary IgG antibody with a linked-enzyme Substrate added and positive wells change color Molecular Methods • Nucleic acid-based assays for differentiation and identification of foodborne pathogens • DNA m th ds DNA methods - Polymerase chain reaction PCR - Pulse-field gel electrophoresis - Ribotyping - Plasmid typing - Restriction fragment length polymorphisms • Some of these methods are automated • Kits are available to purify DNA and RNA
8/30/2016 g<,HiH亚 PCR-most widely used method Three-steps to amplify a specific DNA segment 1.Denaturation-template DNA is denatured into single strands 2.Annealing-primers are annealed to the template DNA 之 annealing temp provides stringency 3.Extension-polymerization of DNA from the primers using a thermostable polymerase .Within 2 h,a single copy of DNA may be amplified a million-fold DNA is visualized using an agarose gel stained PCR Ribotyping Few kits available for identification of Fingerprinting of genomic DNA restriction foodborne pathogens fragments that contain all or part of the genes ·Assay is coding for the 16S and 23S rRNA very sensitive -The genes are cut at specific sites using expensive to run -not technically simple-expertise needed restriction enzymes generating pieces of affected by food components DNA of different lengths -Results within 16 h Enrichment may be needed to dilute food components and increase cell numbers Used for characterizing and fingerprinting Alternatives to conventional PCR may be strains and for epidemiological useful investigations
8/30/2016 8 PCR - most widely used method Three-steps to amplify a specific DNA segment 1. Denaturation -template DNA is denatured into single strands 2. Annealing - primers are annealed to the template DNA annealing temp provides stringency 3. Extension -p y ol merization of DNA from the primers using a thermostable polymerase • Within 2 h, a single copy of DNA may be amplified a million-fold • DNA is visualized using an agarose gel stained PCR • Few kits available for identification of foodborne pathogens • A issay s - very sensitive - expensive to run - not technically simple – expertise needed - affected by p food components • Enrichment may be needed to dilute food components and increase cell numbers • Alternatives to conventional PCR may be useful Ribotyping Fingerprinting of genomic DNA restriction fragments that contain all or part of the genes codi f th 16S d 23S RNA ding for the 16S and 23S rRNA - The genes are cut at specific sites using restriction enzymes generating pieces of DNA of different lengths - R lt ithi 16 h Results within 16 h - Used for characterizing and fingerprinting strains and for epidemiological investigations
8/30/2016 ●●0 Ribotyping 3 different stains of bacteria Very labor intensive if done manually extract DNA Requires an automated system called 雳雳麗 the Riboprinter tasnanne 米 s23 DNA fragments separated by size transfer DNA to Potpourri of Rapid Direct epifluorescence filter technique Methods -Bacteria are illuminated Bioluminescence-based methods Membrane filtration of a food Provide a rapid estimate of sample to collect cells total microbial numbers Light production correlates Stain bacteria with fluorescent with the number of microbes antibodies Uni-Lite System:results <1 min Visualize using epifluorescent microscope DAPI stained bacterial cells from drinking water
8/30/2016 9 genes coding for the 16S d 23S RNA 16S and 23S rRNA Ribotyping Very labor intensive if done manually Requires an automated system called th e Rib i t Riboprinter Potpourri of Rapid Methods Bioluminescence-based methods - Provide a rapid estimate of total microbial numbers - Light production correlates with the number of microbes - Uni-Lite System: results <1 min Direct epifluorescence filter technique - Bacteria are illuminated - Membrane filtration of a food sample to collect cells - Stain bacteria with fluorescent antibodies - Visualize using epifluorescent microscope DAPI stained bacterial cells from drinking water
8/30/2016 More to Come Summary Bioterrorism inspired the development of rapid methods for foodborne pathogens Specialized media that target specific and other pathogens that may be used to contaminate the food supply biochemical reactions of the target microbe are available. Such as:handheld PCR units that may be Most widely used format for rapid used to fest foods at ports of entry and methods is immunologically based assays on farms An enrichment step is required for most The Next Target of assays to increase the number of bacteria to a detectable level Summary Most assays require 102 to 105 CFU per gram or ml PCR-based assays are extremely sensitive but are subject to interference from the presence of inhibitors in food. Future rapid assays will likely be based on nanotechnology. 10
8/30/2016 10 More to Come Bioterrorism inspired the development of rapid methods for foodborne pathogens and other pathogens that may be used to contaminate the food supply Such as: handheld PCR units that may be used to test foods at ports of entry and on farms Summary • Specialized media that target specific bi h i l ti f th t t biochemical reactions of the target microbe are available. • Most widely used format for rapid methods is immunologically based assays • An enrichment step is required for most assays to increase the number of bacteria to a detectable level Summary • Most assays require 102 to 105 CFU per gram l or m • PCR-based assays are extremely sensitive but are subject to interference from the presence of inh b tors n food. ibitors in food. • Future rapid assays will likely be based on nanotechnology
