![Method Detection Li
mit
[cfu mL-
1 or g]
Time Before
Result
(hour)
Specificity
Bioluminesce
nce
104 0.5 No
Impedimetry 1 6-24 Moderate/go
od
Immunologica
l Method
104 1-2 Moderate/go
od
Nucleic Acid
Based As-
says
103 6-12 Excellent](https://image.slidesharecdn.com/rapidmethodsofdetectionoffoodbornepathogens-180116185154/85/Rapid-methods-of-detection-of-food-borne-pathogens-50-320.jpg)
Rapid methods of detection of food borne pathogens
- 1. RAPID METHODS OF DETECTION OF FOOD BORNE PATHOGENS Anchal Food technology
- 2. NEED FOR RAPID METHODS : -To reduce human errors -Validation -Automation and computerization -Simplicity -To save time -To save labor cost
- 3. TYPES OF RAPID METHOD: -Biosensors -Microscopic methods -Immunological Detection Methods -Molecular Detection Methods
- 4. Biosensors -Defined as the indicator of biological compound. -Biosensing methods for pathogen detection are centered on four basic physiological or genetic properties of microorganisms: 1. metabolic pattern of substrate utilization 2. phenotypic expression analysis of signature molecules by antibodies 3. Nucleic acids analysis 4. Analysis of the interaction of pathogens with eukaryotic cells
- 7. Types of biosensors: 1) Bacterial bioluminescence 2) Fiber optic biosensor 3) Electrical impedance biosensor 4) Piezoelectric biosensors 5) ATP Bioluminescence
- 8. 1. Bacterial bioluminescence: -Gene responsible for bacterial bioluminescence (lux gene) has been identified and cloned. -The DNA carrying this gene can be introduced into the host specific phages. -These phages do not posses the intracellular biochemistry necessary to express this gene, remain dark. -On transfer of lux gene to the host bacterium during infection results in light emission that can be easily detected by luminometers This technique can be used to detect 1 x 102 cells in 60 minutes Specificity of this assay depends on phage specificity Eg. Bacteriophage p22 is specific for salmonella typhimurium
- 11. Fiber optic biosensor -Basic principle of this is that when light propagates through the core of the optical fiber i.e. waveguide . -It generates an evanescent field outside the surface of the waveguide -When fluorescent labeled analytics such as pathogens or toxins bound to the surface of the waveguide ,are excited by evanescent wave generated by laser (635nm) and emit fluorescent signal -Signal travels back through the waveguide in high
- 13. Electrical impedance biosensor -Detects microbes directly due to production of ions from metabolic end products or indirectly from liberation co2 -Microbes metabolism results in an increase in both conductance and capacitance ,causing decrease in impedance. -A bridge circuit measures impedance . Method : -Population of microbes provided with nutrients (non-electrolyte) like lactose -Microbes utilize it ,and convert into lactic acid (ionic form),thus changing the impedance. -Impedance is measured over 20 hours after inoculation in specific media.
- 15. Advantages : -Simple to perform -faster than agar plate count -capable of analyzing hundreds of sample at the same time since the instrument (bactometer) is computer driven Disadvantages: -Applicable for samples with high number of microorganisms -food matrix may interfere with the analysis
- 16. Piezoelectric biosensors: -General principle is based on coating the surface of piezoelectric sensor with a selective binding substances . -e.g. antibodies to bacteria and then placing it in a solution containing bacteria -The bacteria will bind to the antibodies and the mass of the crystal will increase while the resonance will decrease proportionally.
- 17. ATP Bioluminescence -This technique measures the emission of light produced by an enzymatic reaction bet -between luciferin and luciferase that requires the presence of ATP . -The amount of light produced is proportional to the concentration of ATP and therefore the number of microorganisms in a sample. -Only applicable if number of microorganism is high ,more than 104 CFU/g. - Used to measure the cleanliness of surface that come into contact with food ,including the presence of organic residues and microbial contaminants.
- 18. -Lacks specificity -more specific way includes an IMS step for capturing target microorganism, which is then detected by bioluminescence.
- 19. Microscopic methods: 1)Direct Epifluorescent Filter Technique 2)Flow Cytometry 3)Solid-Phase Cytometry
- 20. Flow Cytometry -Flow cytometry quantitatively measures optical characteristics of cells when they are forced to pass individually through a beam of light. -Fluorescent dyes can be used to test the viability and metabolic state of microorganisms . -Samples are injected into a fluid (dye), which passes through a sensing medium in a flow cell. -The cells are carried by the laminar flow of water through a focus of light, each cell emits a pulse of fluorescence, and the scattered light is collected by lenses and directed onto selective detectors (photomultiplier tubes).
- 21. -This technique is fast, automatic, and potentially very specific, as long as appropriate dyes are available for selectively labeling specific types of microorganisms and appropriate methods for separating cells from food are utilized so as not to interfere with detection. -The sensitivity of flow Cytometry is low . -The detection limit with food samples is around 105–107 CFU/g . -Currently, there are various flow cytometry methods developed for foods, especially for liquid samples such as dairy products, water, and other beverages.
- 23. Solid-Phase Cytometry -Solid-phase cytometry (SPC) is a technique that combines aspects of flow cytometry and epifluorescence microscopy. -After filtration of the sample, the retained microorganisms are fluorescently labeled with argon laser excitable dyes on the membrane filter and automatically counted by a laser scanning device. Each fluorescent spot can be visually inspected with an epifluorescence microscope connected to a scanning device by a computer-driven moving stage. -Depending on the fluorogenic labels used, information on the identity and the physiological status of the microorganisms can be obtained within a few hours.
- 24. -only applicable if the number of bacteria present is high (103– 104 CFU/g). -Recommended for the determination of the total viable microbial count in liquid samples, and enumeration of pathogens in food samples.
- 25. Direct Epifluorescent Filter Technique -The direct epifluorescent filter technique (DEFT) is a microscopic method for the enumeration of viable cells in a sample. -Based on the binding properties of the fluoro- chrome acridine orange. -Once treated with detergents and proteolytic enzymes, the samples are filtered through a polycarbonate membrane. -The cells are stained on this same filter and examined under an epifluorescent microscope ,a process that
- 27. -DEFT is a very labor-intensive technique -Does not have the capability of processing a large number of samples -Only applicable if the number of bacteria present is high (103–104 CFU/g) -Additionally, fluo- rescent food material can be trapped on the filter, and the technique can only be used with raw food and usually for enumerating total viable microorganisms. -DEFT may be used for the detection and enumeration of specific bacteria in food samples
- 28. Immunological Detection Methods: -The antibody-based system has facilitated the design of a variety of assays and formats. -Incubation times are usually very short for methods such as agglutination reactions commonly used for the rapid identification of microorganisms . -Normally, the antibody is labeled with a fluorescent reagent or with an enzyme so that the antigen–antibody interaction may be visualized more easily when it occurs. -In some cases, the antigen–antibody complex formed is directly measurable or even visible.
- 30. Lateral Flow Devices -Lateral flow devices (LFD) are typically comprised of a simple dipstick made of a porous membrane that contains colored latex beads or colloidal gold particles coated with detection antibodies targeted toward a specific microorganism. -The particles are found on the base of the dipstick, which is put in contact with the enrichment medium. -If the target organism is present, then it will bind with the colored particles. -This conjugated cell/particle moves by capillary action until it finds the immobilized capture antibodies.
- 31. -Upon binding with these, it forms a colored line that is clearly visible in the device window, indicating a positive result. -As with other immunoassays, LFD also require previous enrichment. -The technique is extremely simple to use and easy to interpret, requires no washing or manipulation, and can be completed within 10 min after culture enrichment. -There are various LFD on the market that have been validated
- 33. Enzyme-Linked Immunosorbent Assay and Enzyme-Linked Fluorescence Assay -The enzyme-linked immunosorbent assay (ELISA) is a biochemical technique. -That combines an immunoassay with an enzymatic assay. As LFD, it is a “sandwich” assay. -An antibody bound to a solid matrix is used to capture the antigen from enrichment cultures and a second antibody conjugated to an enzyme is used for detection.
- 35. -The enzyme is capable of generating a product detectable by a change in color, or in the case of enzyme-linked fluorescence assay (ELFA) in fluorescence, which allows for indirect measurement using spectrophotometry of the antigen present in the sample. -Detection using automated and robotic ELISAs is widely used since they can reduce detection times after enrichment to as low as 1–3 h. -Thus, the results can be obtained in 2–3 days instead of the 3–5 days needed by conventional method.
- 36. Molecular Detection Methods: There has been an explosion in the past 15 years in the introduction of nucleic acid– based assays for the detection and identification of foodborne pathogens. There are many DNA-based assay formats, but only probes and nucleic acid amplification techniques have been developed commercially for detecting foodborne pathogens. TYPES: 1)Fluorescent In Situ Hybridization 2)Polymerase Chain Reaction
- 37. Fluorescent In Situ Hybridization -Fluorescent in situ hybridization (FISH) with oligonucleotide probes directed at rRNA is the most common method among molecular techniques not based on PCR. -The probes used by FISH tend to be 15–25 nucleotides in length, and are covalently labeled at their 5¢ end with fluorescent labels. -After hybridization, the specifically stained cells are detected using epifluorescence microscopy .
- 38. The detection limit of this technique is around 104 CFU/g. Following pre-enrichment to reach these detection levels, the results can be obtained quickly (in about 3 h). FISH in combination with flow cytometry has been used for rapid culture-independent detection of Salmonella spp. on the surfaces of tomatoes and other fresh produce.
- 40. Polymerase Chain Reaction There are three main processing steps in PCR: 1. Denaturation 2. Annealing 3. Extension In the first step of the PCR method, DNA is heated to 95- 98oC, at which point double stranded DNA separates into two single strands. In the annealing step , synthetic oligonucleotide primers are added and bind to the single strands at a temperature of 55oC.
- 41. Finally, in the last step of the process the primer exten ded by DNA polymerase in the presence of adenine, g uanine, cytosine,and thymine. The extension by polymerase occurs at a temperature near 72oC. These three steps result in new DNA strands that are complementary to the original strand that was separated in step 1 .
- 43. Polymerase chain reaction (PCR) is a method used for the In-vitro enzymatic synthesis of specific DNA sequences by Taq and other thermoresistant DNA polymerases. PCR uses oligonucleotide primers that are usually 20–30 nucleotides in length and whose sequence is homologous to the ends of the genomic DNA region to be amplified. The method is performed in repeated cycles, so that the products of one cycle serve as the DNA template for the next cycle, doubling the number of target DNA copies in each cycle The rapid increase in the number of copies of the target sequence that can be achieved with PCR-based methods makes them ideal candidates for the development of faster microbiological detection systems. Many PCR tests have been validated and commercialized to make PCR a standard tool used by food microbiology laboratories to detect pathogens in foods.
- 44. Conventional PCR relies on amplification of the target gene(s) in a thermocycler, separation of PCR products by gel electrophoresis, followed by visualization and analysis of the resulting electrophoretic patterns, a process that can take a number of hours. The specificity can be subsequently confirmed by sequencing the amplified fragment. PCR can be superior to culture for detecting the main pathogens in food samples. Real-time PCR It allows both the detection and quantification of a signal emitted by the amplified product by using the continuous measurement of a fluorescent label during the PCR reaction. The increase in fluorescence can be monitored in real time, which allows accurate quantification over several orders of magnitude of the DNA target sequence.
- 45. There are two general techniques used to obtain a fluorescent signal from the amplification of product in PCR. The first technique uses the inherent properties of fluorescent dyes such as SYBR Green I . As the dyes bind to dsDNA and undergo a change in shape, it increases their fluorescence
- 46. The second approach uses fluorescent resonance energy transfer (FRET). FRET relies on the presence of 2molecules that interact with one another, wh ere at least one of the molecules must have fluorescent properties. The fluorescent molecule is known as the donor, while The non-fluorescent molecule is known as the acceptor. During fluorescent resonance energy transfer, the donor molecule is excited by an external source. It emits light at a shifted, longer wavelength, which is then used to excite the acceptor molecule. It is not necessary for the acceptor molecule to emit light. The signal emanating from the acceptor molecule will be detected using the real-time instrument.
- 48. •Advantages: One of the main advantages over traditional PCR is that real- time PCR collects data in the exponential growth phase as opposed to the plateau phase. Results can be obtained in an hour or less, which is consider- ably faster than conventional PCR. Real-time PCR has greatly increased the speed and sensitivity of PCR- based detection methods . PCR requires only about 30–90 min. Aside from that, if a positive result for PCR is reached, this must be confirmed using cultures. The limit of quantification of real-time PCR with food samples is around 103– 104 CFU/g.
- 49. All currently available commercial real-time PCR into qualitative detection methods instead of quantitative. In some cases, multiple different microorganisms may be detected in a single PCR reaction by amplifying the corresponding loci simultaneously. In this type of multiplex PCR reaction, all necessary primers are combined in a single tube for detecting the presence of the main pathogens associated with a given food or the main subtypes within a given species.
- 50. Method Detection Li mit [cfu mL- 1 or g] Time Before Result (hour) Specificity Bioluminesce nce 104 0.5 No Impedimetry 1 6-24 Moderate/go od Immunologica l Method 104 1-2 Moderate/go od Nucleic Acid Based As- says 103 6-12 Excellent