Serological methods for detection of viruses
- 1. Serological methods for detection of viruses D.INDRAJA
- 2. HAEMAGGLUTINATION ASSAY – VIRAL QUANTITATION • Agglutination • The interaction between antibody and a particulate(Insoluble) antigen results in visible clumping called agglutination. • Particulate antigen include: bacteria, white blood cells, red blood cells, latex particles • Agglutinin it is an antibody that interacts with antigen on the surface of particles such as erythrocytes, bacteria, or latex particles to cause their agglutination. • Agglutinogen it is an antigen on the surface of particles such as red blood cells that react with the antibody known as agglutinin to produce agglutination. • The most widely known agglutinogens are those of the ABO and related blood group systems.
- 3. • A)Qualitative agglutination tests Agglutination tests can be used in a qualitative manner to assay for the presence of an antigen or an antibody. The antibody is mixed with the particulate antigen and a positive test is indicated by the agglutination of the particulate antigen . • B) Quantitative Agglutination Test The Ab titre can be determined using serial dilution of the patient serum.
- 4. • Prozone phenomena • In an agglutination or precipitation reaction, the zone of relatively high antibody concentrations within which no reaction occurs. • As the antibody concentration is lowered below the prozone, the reaction occurs.(solve by dilution) • This phenomenon may be due simply to antibody excess or it may be due to blocking antibody or to nonspecific inhibitors in serum
- 5. HAEMAGGLUTINATION ASSAY – VIRAL QUANTITATION Discovery • Haemagglutination assay was developed by American virologist George Hirst in 1941- 1942. Defination The ability of certain viruses to bind with the red blood cells through their superficial glycoproteins and proteins had been utilised to quantitate these viruses and the assay is termed as haemagglutination assay. Principle • Ability of Certain Virus to agglutinate RBC due to the binding of Haemagglutinin- Neuraminidase (HN) Protein of virus to Receptors(sialic acid) on the surface of RBCS. • The Certain Virus that can agglutinate RBC are (Paramyxovirus, Orthomyxovirus, Reovirus, Adenovirus, togavirus) and Mycoplasma. • The basis of this assay is the ability of viral haemaglutinin to bind with the sialic acid present on the receptors of surface of the red blood cells causing haemagglutination.
- 6. • Formation of lattice depends on concentration of virus and RBC’S • The RBC used in HA or HAI are typically from chickens,turkeys,horses,guinea pigs or human depending on the selectivity of targeted viruses or bacteria and surface receptors of RBC
- 7. Procedure Serial dilution of viruses across the rows of U or V bottom shaped 96 well microtiter plate Standardized conc of RBC is added to each well and mix gently Incubated at 30 min at room temperature The first well have high conc of viruses and the remaining are diluted and the last well containing no virus (acts as negative control), each row has different viruses and same patterns of dilution Read the wells
- 9. Haemagglutination Inhibition test (HI) Definition • It is a serological test depending upon antigen antibody reaction in which inhibition of Haemagglutination occurs due to masking of virus receptors by specific Antibodies. Principle of Haemagglutination Inhibition test (HI) • Specific attachment of Antibody to Antigen sites (on HA molecule of virus) interfere with binding between the virus (Haemagglutinate) and receptors on the RBC Example: blood infected with measels virus • Hemagglutination occurs when measles viruses and red blood cells are mixed But, if the serum of a person infected with measles virus is mixed with RBC and measles virus, there won’t be any agglutination of RBC. This phenomenon is known as hemagglutination inhibition. • This arises because antibodies present in the serum of that infected person reacts with the measles viruses and neutralize them (positive result)
- 10. If the patient’s serum do not contain antibodies against surface proteins of test virus, there will be presence of hemagglutination as surface molecules are free to hemaaglutinate RBCs (negative result)
- 11. Patients antiserum is serially diluted and kept in rows of 96 well microtiter plate containing diff sample in each row A standard amount of virus is added to each well and allow to stand for 60 min at room temp A standard amount of RBC is added and incubate for 30 min Read the wells procedure
- 14. Advantages • Simple in use • Relatively inexpensive and available instrument • Provide result with in few hours • The assays are well established in many laboratories Disadvantages • Correct results require controlling several variables such as incubation time, RBC conc, type of blood cell • Non specific factors in sample can lead to interact and give incorrect titre values • No digital recording
- 15. Immunofluorescence Discovery • Coons et al developed the immunoflouroscence techniques for the frst time this discovery made possible to observe microscopically antigen and antibody and their related substances on tissue sections or cell smears
- 16. Principle • It mainly works on the principle of fluroscence • The property of certain dyes absorbing light rays at one particular wavelength (ultraviolet light) and emitting them at a different wavelength (visible light) is known as fluorescence. • In immunofluorescence test, fluorescent dye which illuminates in UV light are used to detect/show the specific combination of an antigen and antibody. • The dye usually used is fluorescein isothiocynate, which gives yellow-green fluorescence, tetramenthyl rhodamine isothiocyanate which gives red fluroscence • Immunofluorescence tests are also termed as fluorescent antibody test (FAT).
- 17. Types • Immunofluorescence tests have wide applications in research and diagnostics. These tests are broadly of two types: 1.Direct immunofluorescence test 2.Indirect immunofluorescence test Direct immunofluorescence test • Direct immunofluorescence test is used to detect unknown antigen in a cell or tissue by employing a known labeled antibody that interacts directly with unknown antigen • If antigen is present, it reacts with labeled antibody and the antibody coated antigen is observed under UV light of the fluorescence. It involves use of labeled antiviral antibody.
- 18. Method • The specimen is placed on slide; fluorescent labeled antibody is then added to it and allowed for some time for Antigen-Antibody reaction. The preparation is then washed which will allow the removal of other components except the complex of antigen and fluorescent labeled antibody. On microscopy (Fluorescence Microscopy), Antigen- Antibody complex are observed fluorescing due to the dye attached to antibody. • The need for preparation of separate labeled antibody for each pathogen is the major disadvantage of the direct immunofluorescence test.
- 19. Indirect immunofluorescence test • Indirect fluorescence is a double antibody technique. The unlabeled antibodies which have bound to the antigens are visualized by a fluorescent antiglobulin reagent directed at the unlabeled antibodies. The indirect immunofluorescence test is used for detection of specific antibodies in the serum and other body fluids for sero-diagnosis of many infectious diseases. Method • Indirect immunofluorescence is a two-stage process. • First stage: A known antigen is fixed on a slide. Then the patient’s serum to be tested is applied to the slide, followed by careful washing. If the patient’s serum contains antibody against the antigen, it will combine with antigen on the slide. • Second stage: The combination of antibody with antigen can be detected by addition of a fluorescent dye-labeled antibody to human IgG, which is examined by a fluorescence microscope.
- 20. • The indirect method has the advantage of using a single labeled antiglobulin (antibody to IgG) as a “universal reagent” to detect many different specific antigen–antibody reactions. The test is often more sensitive than the direct immunofluorescence test. • Amplification of signal and commercially available secondary antibody is the major advantage of this type
- 21. uses • Detect specific antibodies for serodiagnosis of syphilis, leptospirosis, amoebiasis, toxoplasmosis, and many other infectious diseases • Often used to detect autoantibodies Immunofluorescence may also be used to analyze the distribution of proteins, glycans, and small biological and non-biological molecules. Immunofluorescence has been widely used in biological research and medical research. The major limitation of immunofluorescence is that the technique requires 1.expensive fluorescence microscope and reagents, 2.trained personnel 3.have a factor of subjectivity that may result in erroneous results
- 22. ELISA Discovery and principle • ELISA is an antigen antibody reaction. In 1971, ELISA was introduced by Peter Perlmann and Eva Engvall at Stockholm University in Sweden. • ELISA stands for Enzyme-Linked Immunosorbent Assay. It comes under antigen and antibody reaction test and useful for identification of antigen or antibody of following specimens serum, urine, CSF, sputum, semen, supernatant of culture, stool etc. • It also applicable for qualitative as well as quantitative determination of antigen or antibody. • In qualitative test determines antigen or antibody is present or absent • in quantitative determines the quantity of the antibody in titer and titer is the highest dilution of the specimen usually serum which gives a positive reaction in the test
- 23. • ELISA is a plate based assay technique which is used for detecting and quantifying substances such as peptides, proteins, antibodies and hormones. • An enzyme conjugated with an antibody reacts with colorless substrate to generate a colored product. Such substrate is called chromogenic substrate. • A number of enzymes have been used for ELISA such as alkaline phosphatase, horse radish peroxidase and beta galactosidase. • Specific substrate such as ortho-phenyldiamine dihydrochloride (for peroxidase), paranitrophenyl phosphate (for alkaline phosphatase) are used which are hydrolysed by above enzymes to give colored end product.
- 24. Overall ELISA • ELISAs are typically performed in 96-well polystyrene plates. • The serum is incubated in a well, and each well contains a different serum. A positive control serum and a negative control serum would be included among the 96 samples being tested. • Antibodies or antigens present in serum are captured by corresponding antigen or antibody coated on to the solid surface. • After some time, the plate is washed to remove serum and unbound antibodies or antigens with a series of wash buffer. • To detect the bound antibodies or antigens, a secondary antibodies that are attached to an enzyme such as peroxidase or alkaline phosphatase are added to each well.
- 25. • After an incubation period, the unbound secondary antibodies are washed off. • When a suitable substrate is added, the enzyme reacts with it to produce a color. • This color produced is measurable as a function or quantity of antigens or antibodies present in the given sample. The intensity of color/ optical density is measured at 450nm. • The intensity of the color gives an indication of the amount of antigen or antibody.
- 26. ELISA Types • The four main types of ELISAs are • direct • Indirect • sandwich • competitive. • Each type of ELISA has its own advantages and disadvantages. Direct ELISA (antigen can be determined) • In a direct ELISA, an antigen or sample is immobilized directly on the plate and a conjugated detection antibody binds to the target protein. Substrate is then added, producing a signal that is proportional to the amount of analyte in the sample. Since only one antibody is used in a direct ELISA, they are less specific than a sandwich ELISA. Non-competitive.
- 27. Advantages: • Fast and simple protocol Disadvantages: • Less specific since you are only using 1 antibody. • Potential for high background if all proteins from a sample are immobilized in well
- 28. Indirect ELISA (antibody can be determined) • Antibody can be detected or quantitatively determined by indirect ELISA. • In this technique, antigen is coated on the microtiter well. Block all unbound sites to prevent false positive results. • Serum or some other sample containing primary antibody is added to the microtiter well and allowed to react with the coated antigen. and incubate the plate at 37°c. • Any free primary antibody is washed away and the bound antibody to the antigen is detected by adding an enzyme conjugated secondary antibody that binds to the primary antibody. • Unbound secondary antibody is then washed away and a specific substrate for the enzyme is added. • Enzyme hydrolyzes the substrate to form colored products. The amount of colored end product is measured by spectrophotometric plate readers that can measure the absorbance of all the wells of 96-well plate
- 29. Advantages: • Amplification using a secondary antibody • Increased sensitivity, since more than one labeled antibody is bound per primary antibody. • A wide variety of labeled secondary antibodies are available commercially. • Flexibility, since different primary detection antibodies can be used with a single labeled secondary antibody. • Cost savings, since fewer labeled antibodies are required. • Different visualization markers can be used with the same primary antibody. Disadvantages: • Potential for cross-reactivity caused by secondary antibody resulting in nonspecific signal.
- 30. Sandwich ELISA (antigen can be determined) • Sandwich ELISAs are the most common type of ELISA. • Antigen can be detected by sandwich ELISA. • In this technique, antibody is coated on the microtiter well. • A sample containing antigen is added to the well and allowed to react with the antibody attached to the well, forming antigen-antibody complex. and incubate the plate at 37°c • After the well is washed, a second enzyme-linked antibody specific for a different epitope on the antigen is added and allowed to react with the bound antigen. and then incubate at 37°c. • Then after unbound secondary antibody is removed by washing. Finally substrate is added to the plate which is hydrolyzed by enzyme to form colored products.
- 31. Advantages • High specificity, since two antibodies are used the antigen is specifically captured and detected. • Suitable for complex samples, since the antigen does not require purification prior to measurement. • Flexibility and sensitivity, since both direct and indirect detection methods can be used.
- 32. Competitive ELISA (antigen can be determined) • This test is used to measure the concentration of an antigen in a sample. • In this test, antibody is first incubated in solution with a sample containing antigen. • The antigen-antibody mixture is then added to the microtitre well which is coated with antigen. • The more the antigen present in the sample, the less free antibody will be available to bind to the antigen-coated well. • After the well is washed, enzyme conjugated secondary antibody specific for isotype of the primary antibody is added to determine the amount of primary antibody bound to the well. • The higher the concentration of antigen in the sample, the lower the absorbance.
- 33. Advantages • High specificity, since two antibodies are used. • High sensitivity, since both direct and indirect detection methods can be used. • Suitable for complex samples, since the antigen does not require purification prior to measurement
- 34. Application of ELISA 1.Presence of antigen or the presence of antibody in a sample can be evaluated. 2.Determination of serum antibody concentrations in a virus test. 3.Used in food industry when detecting potential food allergens. 4.Applied in disease outbreaks- tracking the spread of disease e.g. HIV, bird flu, common, colds, cholera, STD etc.
- 35. Polymerase chain reaction • PCR is a invitro DNA amplification technique used in the lab to make millions of copies of a particular section of DNA. It was first developed in the 1980s. • Sometimes called "molecular photocopying," the polymerase chain reaction (PCR) is a fast and inexpensive technique used to "amplify" - copy - small segments of DNA Discovery The polymerase chain reaction (PCR) was originally developed in 1983 by the American biochemist Kary Mullis. He was awarded the Nobel Prize in Chemistry in 1993 for his pioneering work. Principle • To amplify a segment of DNA using PCR, the sample is first heated so the DNA denatures, or separates into two pieces of single-stranded DNA. Next, an enzyme called "Taq polymerase" synthesizes - builds - two new strands of DNA, using the original strands as templates. This process results in the duplication of the original DNA, with each of the new molecules containing one old and one new strand of DNA. Then each of these strands can be used to create two new copies, and so on, and so on • The entire cycling process of PCR is automated and can be completed in just a few hours. It is directed by a machine called a thermocycler, which is programmed to alter the temperature of the reaction every few minutes to allow DNA denaturing and synthesis.
- 36. • A basic PCR set-up requires several components and reagents • Target • a DNA template that contains the DNA target region to amplify • Enzyme • a DNA polymerase; an enzyme that polymerizes new DNA strands • DNA polymerase I obtained from E. coli is used extensively for molecular biology research. However, the 5'→3' exonuclease activity makes it unsuitable for many applications. This undesirable enzymatic activity can be simply removed from the holoenzyme to leave a useful molecule called the Klenow fragment, widely used in molecular biology. In fact, the Klenow fragment was used during the first protocols of polymerase chain reaction (PCR) • Exposure of DNA polymerase I to the protease subtilisin cleaves the molecule into a smaller fragment, which retains only the DNA polymerase and proofreading activities.
- 37. • In the original PCR procedure, one problem was that the DNA polymerase had to be replenished after every cycle because it is not stable at the high temperatures needed for denaturation. This problem was solved in 1987 with the discovery of a heat-stable DNA polymerase called Taq, an enzyme isolated from the thermophilic bacterium Thermus aquaticus, which inhabits hot springs. • Primers • DNA polymerase can add a nucleotide only onto a preexisting 3'-OH group, it needs a primer to which it can add the first nucleotide • PCR primers are short pieces of single-stranded DNA, usually around 20 nucleotides in length. Two primers are used in each PCR reaction(forward primer and reverse primer), and they are designed so that they flank the target region (region that should be copied). • When the primers are bound to the template, they can be extended by the polymerase, and the region that lies between them will get copied.
- 38. Nucleotides (dNTPs or deoxynucleotide triphosphates) • single units of the bases A, T, G, and C, which are essentially "building blocks" for new DNA strands. buffer solution • a buffer solution providing a suitable chemical environment for optimum activity and stability of the DNA polymerase bivalent cations • bivalent cations, typically magnesium (Mg) or manganese (Mn) ions; Mg2+ is the most common, but Mn2+ can be used for PCR-mediated DNA mutagenesis, as a higher Mn2+ concentration increases the error rate during DNA synthesis,and monovalent cations, typically potassium (K) ions Thermal cycler • The reaction is commonly carried out in a volume of 10–200 μL in small reaction tubes (0.2–0.5 mL volumes) in a thermal cycler. • The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction . • Many modern thermal cyclers make use of the Peltier effect, which permits both heating and cooling of the block holding the PCR tubes simply by reversing the electric current. • Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibrium. • Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. Older thermal cyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.
- 39. Procedure: Extraction and Denaturation of Target Nucleic Acid For PCR, nucleic acid is first extracted (released) from the organism or a clinical sample potentially containing the target organism by heat, chemical, or enzymatic methods. Once extracted, target nucleic acid is added to the reaction mix containing all the necessary components for PCR (primers, nucleotides, covalent ions, buffer, and enzyme) and placed into a thermal cycler to undergo amplification THERMAL CYCLER
- 40. Denaturation(95°C ) The reaction mixture is heated to 95°C for a short time period (about 15–30 sec) to denature the target DNA into single strands that can act as templates for DNA synthesis Annealing (55 - 65C°) Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA. This annealing temperature is calculated carefully to ensure that the primers bind only to the desired DNA sequences (usually around 55oC). • One primer binds to each strand. The two parental strands do not re-anneal with each other because the primers are in large excess over parental DNA Extension(72°C) The temperature of the mixture is raised to 72°C (usually) and kept at this temperature for a pre-set period of time to allow DNA polymerase to elongate each primer by copying the single-stranded templates. Annealing of primers to target sequences provides the necessary template format that allows the DNA polymerase to add nucleotides to the 3’ terminus (end) of each primer and extend sequence complementary to the target template Taq polymerase is the enzyme commonly used for primer extension, which occurs at 72°C. This enzyme is used because of its ability to function efficiently at elevated temperatures and to withstand the denaturing temperature of 94°C through several cycles
- 41. • This cycle repeats 25 - 35 times in a typical PCR reaction, which generally takes 2-4 hours, depending on the length of the DNA region being copied. • If the reaction is efficient (works well), the target region can go from just one or a few copies to billions.
- 42. Using gel electrophoresis to visualize the results of PCR • The results of a PCR reaction are usually visualized (made visible) using gel electrophoresis. • Gel electrophoresis is a technique in which fragments of DNA are pulled through a gel matrix by an electric current, and it separates DNA fragments according to size. • A standard, or DNA ladder, is typically included so that the size of the fragments in the PCR sample can be determined. • DNA fragments of the same length form a "band" on the gel, which can be seen by eye if the gel is stained with a DNA-binding dye
- 43. Applications of PCR DNA sequencing has been greatly simplified using PCR, and this application is now common. By using suitable primers, it is possible to use PCR to create point mutations, deletions and insertions of target DNA which greatly facilitates the analysis of gene expression and function PCR is used in many research labs, and it also has practical applications in forensics, genetic testing, and diagnostics. For instance, PCR is used to amplify genes associated with genetic disorders from the DNA of patients (or from fetal DNA, in the case of prenatal testing). • PCR can also be used to test for a bacterium or DNA virus in a patient's body: if the pathogen is present, it may be possible to amplify regions of its DNA from a blood or tissue sample. • PCR in bioremediation to identify genetically modified organismsin the enviroinment • PCR in genetic engineering • Detection and diagnosis of infectious disease • Detection of mutations • Evolutionary studies • Site directed mutagenesis
- 44. Types of PCR : some of them are Colony PCR Colony PCR is a method in which, where identification of DNA of interest inserted into the plasmid is obtained by designing the inserted DNA specific primers. A bacterial colony is taken and added TO MICROFUGE TUBES and heated to extract the nucleic acids from the colonies and centrifuge and collect the supernatant and add into the master mix containing all other PCR reagents. The main application of colony PCR is in the identification of correct ligation and insertion of inserted DNA into bacteria as well as yeast plasmid. • Hot start PCR Hot start PCR is a novel form of conventional polymerase chain reaction (PCR) that reduces the occurrence of undesired products and formation of primer-dimers due to non-specific DNA amplification at room temperatures. The basic principle of hot-start PCR is the separation of one or more reagents from the reaction mix until the mixture reaches the denaturation temperature upon heating. Hot start PCR significantly reduces non-specific binding, the formation of primer-dimers, and often increases product yields. It also requires less effort and reduces the risk of contamination.
- 45. • Nested PCR Nested PCR is a useful modification of PCR technology where the specificity of the reaction is enhanced by preventing the non-specific binding with the help of the two sets of primer. The first set of primer binds outside of our target DNA and amplifies larger fragment while another set of primer binds specifically at the target site. In the second round of amplification, second set of primer amplifies only the target DNA. Nested PCR is a helpful method for the phylogenetic studies and detection of different pathogens. The technique has higher sensitivity; hence even if the sample contains lower DNA, it can be amplified which is not feasible in the conventional PCR technique
- 46. • Multiplex PCR Multiplex PCR is a common molecular biology technique used for the amplification of multiple targets(diff sequences and no similarities) in a single PCR test run. In Multiplex PCR, multiple primers and a temperature-mediated DNA polymerase are used for the amplification of DNA in a thermal cycler. All the primers pairs designed for Multiplex PCR have to be optimized so that the same annealing temperature is optimal for all the pairs during PCR. In diagnostic laboratories, multiplex PCR is useful to detect different microorganisms that cause the same types of diseases
- 47. • Inverse PCR Inverse polymerase chain reaction (Inverse PCR) is one of the many variants of the polymerase chain reaction that is used to amplify DNA when only one sequence is known. If We want to amplify the unknown sequence that is present at the ends of the sequence but when the know sequence is present at the middle we use the inverse pcr The inverse PCR involves a series of restriction digestion followed by ligation, which results in a looped fragment that can then be primed for PCR through a single section of known sequence. Then, like other polymerase chain reaction processes, the DNA is amplified by the temperature-sensitive DNA polymerase. Inverse PCR is especially useful for the determination of insert locations of various transposons and retroviruses in the host DNA
- 48. • Long-Range PCR Long-Range PCR is a method for the amplification of longer DNA lengths that cannot typically be amplified using routine PCR methods or reagents. Long-range PCR can be achieved by using modified high-efficiency polymerases with enhanced DNA binding, resulting in highly processive and accurate amplification of long fragments. This method allows the amplification of more extended targets within a shorter period and with efficient use of resources. • Reverse Transcriptase PCR (RT-PCR) Reverse transcription PCR (RT-PCR) is a modification of conventional PCR, whereby RNA molecules are first converted into complementary DNA (cDNA) molecules that can then be amplified by PCR. In RT-PCR, the RNA template is first converted into a complementary DNA (cDNA) using reverse transcriptase. The cDNA then acts as a template for exponential amplification using PCR. RT-PCR can be conducted either in a single tube or as two steps in different tubes. The one-step method is more effective with fewer chances of contamination and incorporation of variations. RT-PCR is used in research methods, gene insertion, genetic disease diagnosis and cancer detection.
- 49. • Real-Time PCR (Quantitative PCR (qPCR)) Quantitative PCR (qPCR), also called real-time PCR or quantitative real-time PCR, is a PCR-based technique that couples amplification of a target DNA sequence with quantification of the concentration of that DNA species in the reaction. Conventional PCR is a time-consuming process where the PCR products are analysed through gel electrophoresis. qPCR facilitates the analysis by providing real time detection of products during the exponential phase. The principle of real-time PCR depends on the use of fluorescent dye. The concentration of the nucleic acid present into the sample is quantified using the fluorescent dye or using the fluorescent labelled oligonucleotides. q-PCR is applied in genotyping and quantification of pathogens, microRNA analysis, cancer detection, microbial load testing and GMOs detection. • Reverse-Transcriptase Real-Time PCR (RT-qPCR) RT-PCR is commonly associated with q-PCR forming Reverse Transcriptase Real-Time PCR (RT- qPCR). This allows quantification of DNA in real-time after the amplification.
- 50. • Amplified fragment length polymorphism (AFLP) PCR It is a PCR-based technique that uses selective amplification of a section of digested DNA fragments to generate unique fingerprints for genomes of interest. AFLP PCR uses restriction enzymes to digest genomic DNA and allows attachment of adaptors to the sticky ends of the fragments. A part of the restriction fragments is then selected to be amplified by using primers that are complementary to the adaptor sequence. The amplified sequences are separated and visualized on denaturing on agarose gel electrophoresis. AFLP PCR is employed for a variety of applications, as to assess genetic diversity within species or among closely related species, to infer population-level phylogenies and biogeographic patterns, to generate genetic maps and to determine relatedness among cultivars. • Asymmetric PCR Asymmetric PCR is a variation of PCR used to preferentially amplify one strand of the original DNA more than the other. Asymmetric PCR differs from regular PCR by the excessive amount of primers for a chosen strand. Consequently, linear synthesis of the targeted single DNA strand from the excess primer is formed after depletion of the limiting primer. It is useful when amplification of only one of the two complementary strands is needed, such as in sequencing and hybridization probing
- 51. • Variable Number of Tandem Repeats (VNTR) PCR • They are important markers for the individualization in forensic science. • In VNTR PCR, fragments are amplified that showed little variation within a species, but did show differences between species. • It can successfully amplify from a very small amount of genomic deoxyribonucleic acid (DNA) by the polymerase chain reaction (PCR) • In-situ PCR •In-Situ Polymerase Chain Reaction(In-situ PCR) is an effective method that detects minute quantities of rare nucleic acid sequences in frozen or paraffin-embedded cells or tissue sections for the compartmentalization of those sequences within the cells. •This method involves tissue fixing that preserves the cell morphology, which is then followed by the treatment with proteolytic enzymes to provide an entry for the PCR reagents to act on the target DNA. •The target sequences are amplified by the reagents and then detected by standard immunocytochemical protocols. •In-situ PCR is applicable for the diagnosis of infectious diseases, quantification of DNA, detection of even small amount of DNA and is widely used in the study of organogenesis and embryogenesis.
- 52. RIA • Radioimmunoassay (RIA) is an in vitro technique used to measure concentrations of antigens (for example, hormone levels in the blood) without the need to use a bioassay History Developed in 1959 by Rosalyn Yalow and Solomon Berson for measurement of insulin in plasma. • It represented the first time that hormone levels in the blood could be detected by an in vitro assay. • In 1977 Yalow received the Nobel Prize for her and Berson’s development of RIA principle The basic principle of radioimmunoassay is competitive binding, where a radioactive antigen ("tracer") competes with a non-radioactive antigen for a fixed number of antibody or receptor binding sites
- 53. Most favoured Used when relatively small sample is to be determined
- 54. General Procedure for Performing a RIA Analysis • A known quantity of an antigen is made radioactive • This radiolabeled antigen is then mixed with a known amount of antibody for that antigen, and as a result, the two chemically bind to one another. • a sample of serum from a patient containing an unknown quantity of that same antigen is added • This causes the unlabeled (or "cold") antigen from the serum to compete with the radiolabeled antigen for antibody binding sites • As the concentration of "cold" antigen is increase, more of it binds to the antibody. • And by displacing the radio labelled variant and reduces the ratio of antibody- bound radio labelled antigen to free radio labelled antigen. • The bound antigens are then separated from the unbound ones • the radioactivity of the free antigen remaining in the supernatant is measured. • separating bound from unbound antigen is crucial • Initially, the method of separation employed was the use of a second "anti- antibody"
- 55. Measure radioactivity Measure radioactivity Decreased radioactivity Radioactive antigen Fixed antibody Fixed antibody with radioactive antigen Serum with antigen is added Competetive binding of radioactive antigen with normal antigen Interpretation Radioaactivity decreases more antigen in sample No change in radioactivity antigen is absent
- 56. Advantages Of RIA • Radio immuno assay is very sensitive technique used to measure minute concentrations of antigen without the need to use a bioassay. • It is structurally specific as antigen: antibody reaction are highly specific. • It is indirect method of analysis. • It is a saturation analysis as active reagent added in smaller quantity than that of analyte Disadvantages Of RIA • Prolonged reaction time (in days) as a consequence highly diluted reagent is used. • Radioactive Iodine used in is not a cheap reagent. • Possible health hazards due to handling of radioisotopes. • All the reagents must be added precisely. • Limited assay range. • Lack of direct linear relationship between analyte concentration and signal response. • Difficulty of automation.
- 57. Infectivity assay -plaque assay • One of the most important procedures in virology is measuring the virus titer – the concentration of viruses in a sample. • A widely used approach for determining the quantity of infectious virus is the plaque assay. • This technique was first developed to calculate the titers of bacteriophage stocks. • Renato Dulbecco modified this procedure in 1952 for use in animal virology, and it has since been used for reliable determination of the titers of many different viruses. • Plaque a clear area on an otherwise opaque field of bacteria that indicates the inhibition or dissolution of the bacterial cells by some agent, either a virus or an antibiotic.
- 58. Procedure • To perform a plaque assay, 10-fold dilutions of a virus stock are prepared, and 0.1 ml aliquots are inoculated onto susceptible cell monolayers. • After an incubation period, to allow virus to attach to cells, the monolayers are covered with a nutrient medium containing a substance, usually agar, that causes the formation of a gel. • When the plates are incubated, the original infected cells release viral progeny. The spread of the new viruses is restricted to neighboring cells by the gel. • each infectious particle produces a circular zone of infected cells called a plaque. • Eventually the plaque becomes large enough to be visible to the naked eye. • Dyes that stain living cells are often used to enhance the contrast between the living cells and the plaques. • Only viruses that cause visible damage of cells can be assayed in this way.
- 60. calculation • The titer of a virus stock can be calculated in plaque-forming units (PFU) per milliliter. (One virus is enough to form a plaque) • To determine the virus titer, the plaques are counted. • To minimize error, only plates containing between 10 and 100 plaques are counted, depending on the size of the cell culture plate that is used. • Statistical principles dictate that when 100 plaques are counted, the sample titer will vary by plus or minus 10%. • Each dilution is plated in duplicate to enhance accuracy Advantages • Plaque assay is an extremely sensitive • easy method • determines the number of infectious viruses by counting the number of plaques