Method of detection of food borne pathogen(methods).docx
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PCR and RT-PCR are commonly used molecular techniques for detecting foodborne pathogens through amplification of pathogen DNA or RNA. Multiplex PCR (mPCR) allows simultaneous detection of multiple pathogens. Real-time PCR monitors amplification in real-time without gel electrophoresis. Other methods like LAMP, NASBA, and microarrays provide isothermal amplification or detect multiple targets but require different primers or probes. Optical and electrochemical biosensors detect binding through surface plasmon resonance or changes in electrical signals. Mass-based sensors measure added mass through resonant frequency changes of piezoelectric crystals. ELISA is a common immunological technique that sandwiches the target antigen between immobilized and enzyme-conjugated antibodies for colorimetric detection.
Method of detection of food borne pathogen(methods).docx
- 1. Submitted by: Osama Alam Submitted to: Sir Sami Ullah Sb
- 2. Detection of Food Borne Pathogen PCR It is the common method which is used for detection of foodborne pathogens.Ithelps in detection of single strand of bacteria which is present in spoilage or food borne.while,the DNA sequence should be specific. Mechanism It is used for the detection of pathogens in thefood.It helpsin amplifying ofa specific target DNA sequence.It complete it’s own process in three steps (1) Denaturation of the template into single strands (2)Annealing of primers to each original strand for new strand synthesis (3)Extension of the new DNA strands from the primers. Firstly, the target double-stranded DNAis denatured into single stranded DNA at a temperature(760). Then, two single-stranded synthetic oligonucleotides or specific primers which are the forward and reverse primer will start annealing with each other.This is followed by the polymerization process whereby the primers complementary to the single-stranded DNA are extended with the presence of deoxyribonucleotides,Tag polymerase and a thermostable DNA polymerase.It is visualized on Agarose elecotrophoresis gel in bands form.PCR.
- 3. mPCR Multiplex PCR It helps fast rapid detection as compared to simple PCR through the automatic amplification of multiple target gene.The basic principle of mPCR is similar to conventional PCR. However,few sets of specific primers are used in mPCR technique.While only one strand of specific primers are used in conventional PCR technique. Difference PCR M PCR Only one DNA can be amplified. Upto 25 DNA it amplify. Required one primer. More then 5 primer needed. Detect one type of pathogen. Detect different pathogens. Mechanism It allows for simultaneousamplification ofmultiple targetsequences in a singletube using specific primer sets in combination with probeslabeled with spectrallydistinct fluorophores. Factors (1) Primer designing is very important for the development of mPCR technique.But there must be the primer have similar annealing,and it’s temperature in order to initiate a successeful mPCR process.
- 4. (2) With it, the concentration of primers is also important for mPCR, Because interacation may be occur between the multiple primer sets in mPCR results in primer dimers, thus, the concentration of primers mayneed to be adjusted to ensure the production of reliable PCR products. (3) Important factors for a successful mPCR assay include the PCR Buffer concentrations, the balance between magnesium chloride and deoxynucleotide concentrations, the quantitiesof DNA template, cycling temperatures and Taq DNA polymerase RT_PCR It stand for Real-time PCR and does not require agarose gel electrophoresis for the detection of food borne pathogen.This method is able to monitor the PCR products formation continuously in the entire reaction by measuring the fluorescent signal produced by specific duallabeled probes or intercalating dyes. The fluorescence intensity is proportional to the amount of PCR amplicons. Requirments: RT enzyme , Forward primer and Tag polymerase enzymes. Mechanism We can detect even single strand of RNA .It ContainsReverse Transcriptaseenzyme that help to convert RNA into DNA (double stranded).It is mostly used to amplify RNA targets,While COVID-19 is also detected by this technique . The RNA template is converted into complementary (c)DNA by the enzyme reverse transcriptase. The cDNA serves later as a template for exponential amplification using PCR. RT-PCR can be undertaken in one or two steps. One-step RT- PCR combines the RT reaction and PCR reaction in the same tube. Only sequence- specific primers may be used. During two-step RT-PCR, the synthesized cDNA is transferred into a second tube for PCR. Oligo (dT), random hexamer or gene- specific primers can be used. Oligo (dT) primers are generally preferred as they hybridize to the 3ʹ poly (A) tails in mRNAs (transcribed gene sequences), whereas random primers prime anything including ribosomal RNA
- 5. NASBA Nucleic acid sequence-based amplification It is developed in 1991 in the early 90 century and it helps in amplifying of nucleic acids underisothermal conditions, unlike PCR that requires thermocycling system. It is normally used for the amplification of RNA whereby the singlestranded RNA templateis converted into complementaryDNA(cDNA) by the reverse transcriptase during the reaction. Mechanism It performs at around 41◦C, that involves two specific primers and three enzymes: Avian, Myeloblastosis virus (AMV) and reverse transcriptase, T7 RNA polymerase and RNase H. The NASBA amplicons can be detected by agarose gel electrophoresis.It helps in amplification process that is able to amplify and detect RNA even in the presence of DNA.
- 6. LAMP Loop-mediated Isothermal Amplification. t is a very sensitive, easy and time efficient method.It is a novel nucleic acid amplification method which provides a rapid, sensitivity and specific detection of foodborne pathogens. Basis It is based on auto-cycling strand displacement DNA synthesis carried out by Bst DNA polymerase large fragment under isothermal conditions between 59◦C and 65◦C for 60 min. Mechanism There are four primers comprising two inner primers and two outer primers are used to target six specific regions of target DNA. Cauliflower-like DNA structures bearing multiple loops as well as stem-loop DNAs of different sizes are the final products of LAMP. Large amount of amplicons can be produced by LAMP within
- 7. 60 min which is usually 103-fold or higheras compared to simple PCR. The LAMP amplicons can be detected by agarose gel electrophoresis or SYBR Oligonucleate Microarray It is used to identify large numbers of specific DNA markers by molecular hybridization.The recent progress in multi-gene detection technology includes the microarray technology.Microarrays were originally used for the study of gene expression, but oligonucleotide DNA microarray has been widely used in the field of foodborne pathogen detection. Mechanism Microarrays are made up of glass slides or chips coated with up to hundreds of specific oligonucleotide probes and these probes are chemically synthesized short sequences range from 25 to 80 bp. Each oligonucleotide probe is able to target a specific part of a gene sequence. In this method, the samplenucleic acid fragments (DNA, mRNA or cDNA) are labeled with fluorescent dye, and then they are denaturated to generate single-stranded fragments. These fragments will hybridize
- 8. to the array through binding to their corresponding oligonucleotide probes. The results are obtained through the visualization of the fluorescence signal produced from the probe-sample complex. The fluorescence intensity is proportional to the concentration of each labeled nucleic acid fragment. OPTICAL BIOSENSORS It is devices thattransducethe biorecognition event (for example, a protein binding to antibodies, DNA hybridization, or a metabolite.Themost commonly used optical biosensor for the detection of foodborne pathogen is surface plasmon resonance (SPR) biosensor due to their sensitivity. SPR employs reflectance spectroscopy for the pathogen detection Mechanism In SPR, bioreceptors are immobilized on the surface of a thin metal. The electromagnetic radiation of a certain wavelength interacts with the electron cloud of the thin metal and produces a strong resonance. When the pathogen bindsto the metal surface, this interaction alters its refractive index which results in the change of wavelength required for electron resonance.
- 9. ELECTROCHEMICAL BIOSENSORS It is one of the typical sensing devices based on transducing the biochemical events to electrical signals.It is further classified into several types such as amperometric, impedimetric, potentiometric, and conductometric according to the measurementof changesin current, impedance, voltageand conductancerespectively, which caused by antigen-bioreceptor interactions .Many researchers had reported the successful detection of foodborne pathogens by electrochemical biosensors. Experiment Pal successfully detected Bacillus cereus present in alfalfa sprouts, strawberries, lettuce, tomatoes, fried rice and cooked corn by a direct-charge transfer conductometric biosensor. MASS-BASED BIOSENSORS
- 10. It involves measurement of mass change occurring as a result of biomolecular interaction. Piezoelectric crystals are employed to measure. Operate It operate on the base of detection of small changes in mass. Mechanism It involves the use of piezoelectric crystal which will vibrate at a certain frequency when induced by an electrical signalof a certain frequency. The bioreceptors (e.g., antibodies) for the detection of pathogens (e.g., antigens) are immobilized on this crystal. Once the target antigens bind to the antibodies immobilized on the crystal, this will causea measurablechangein thevibrationalfrequencyofthe crystal which correlates with the added masson the crystal surface. There are two major types of mass-based biosensors which are the bulk acoustic wave resonators (BAW) or quartz crystal microbalance (QCM) and surface acoustic wave resonator (ELISA) ENZYME-LINKED IMMUNOSORBENT ASSAY It helps to identify situations that lead your immune system to make antibodies. Certain diseases aren't easy to identify with other means like swab tests. In these cases, an ELISA blood test can help spot signs of infection or disease in your system.It is one of the most commonly used immunologicalmethodsfor the detection of foodborne pathogens. Sandwich ELISA is the most effective form of ELISA Mechanism It involves two antibodies.The primary antibody is usually immobilized onto the wallsof the microtiter platewells. Thetarget antigen like bacterial cells or bacterial
- 11. toxins from the food sample binds to the immobilized primary antibody and the remaining unbound antigens are removed. After that, an enzyme-conjugated secondary antibody is added and it will bind to the antigen and the remaining unbound antibodies are removed. The complex consisting antigen sandwiched between two antibodies is formed and it can be detected by adding a colorless substrate which will be converted into a colored form in the presence of the enzyme.Thereare different types of enzymes can be used in ELISA, some of the most commonly used enzymes include horseradish peroxidase (HRP), alkaline - phosphatase and beta-galactosidase