This document discusses techniques for detecting and identifying plant viruses in quarantine. It covers:
1. An overview of plant viruses, their characteristics and modes of transmission.
2. Various detection techniques including biological, physical, biochemical, serological and molecular methods. Serological techniques include ELISA and molecular methods include PCR, RT-PCR and real-time PCR.
3. Details on techniques like mechanical inoculation, electron microscopy, and nucleic acid-based methods.
4. The use of these techniques to detect and identify important quarantine viruses.
Effective detection of MLN pathogens in leaf tissues and seed lotsCIMMYT
The document describes and assesses various detection methods for plant viruses, including Maize Lethal Necrosis virus. It finds that immuno-capture reverse transcription PCR (IC-RT-PCR) combines the specificity of ELISA-based detection with PCR-based detection. A study demonstrated IC-RT-PCR can successfully detect Maize Chlorotic Mottle Virus in infected plant leaves and seeds, with results available in hours and at low labor cost once the technique is established. However, IC-RT-PCR requires specialized equipment and antibodies may not always be available.
Detection of plant pathogens using non pcr based techniquesPuja41124
This document discusses various molecular techniques for detecting and identifying plant pathogens, beginning with conventional techniques like visual observation, culture-based methods, and microscopy. It then covers several antibody-based techniques like serological methods, ELISA, immunofluorescence, and immunoelectron microscopy. Hybridization methods like Southern blotting, Northern blotting, and DNA microarrays are also introduced. Biochemical approaches involving fatty acid analysis, Biolog identification systems, and volatile compound analysis are briefly covered. The document concludes by discussing the surface plasmon resonance technique for pathogen detection. In summary, it provides an overview of conventional, antibody-based, hybridization-based, and biochemical molecular techniques for plant pathogen detection and identification.
Electron microscopy and production of antisera can be used to detect and identify plant viruses.
1) Electron microscopy can detect virus particles in infected plant tissue and identify unknown viruses. It is often combined with antisera in a technique called immuno electron microscopy.
2) Antisera are produced by injecting purified antigens into rabbits to stimulate an immune response and antibody production. Test bleedings are taken from the rabbits and the resulting antisera are evaluated for avidity, specificity, and titer against the target antigen.
disease diagnosis, types of diagnostic kits, nucleic acid based that include pcr, rt pcr, microarray, protein based which include ELISA, types of elisa, comparision among all types of diagnostic kits
This document summarizes diagnostic technologies for differentiating bacterial and viral infections. It discusses using host biomarkers in a diagnostic test developed by ILRI and SLU. Rapid nucleic acid tests and point-of-care tests are mentioned. Methods like lateral flow assays, DNA/RNA amplification, and technologies currently on the market are briefly described. The document also notes challenges like RNA isolation and potential solutions like using paper-based platforms.
MLN Workshop: Maize lethal necrosis diagnostic methods -- A WanguiCIMMYT
This document discusses diagnostic methods for detecting plant viruses, specifically focusing on methods used to detect cassava mosaic viruses (SCMV and MCMV). It describes that correct identification of causal agents is important for disease management and safe trade. Detection methods discussed include symptoms, indicator plants, electron microscopy, serological methods like ELISA, and molecular methods like PCR. For SCMV and MCMV, ELISA and RT-PCR are commonly used due to their high specificity. The document provides details on how to perform DAS-ELISA and one-step RT-PCR, including required materials and steps of the procedures. It also discusses factors that can influence ELISA and PCR results.
Current methods for plant disease diagnosisSHIVANI PATHAK
Shivani presents an overview of current and prospective methods for plant disease detection. Several techniques are discussed including serological based methods like ELISA and lateral flow devices, nucleic acid based methods like PCR and real-time PCR, optical sensor based methods like fluorescence imaging and hyperspectral imaging, and digital imaging based methods. Examples of each technique being used to detect various plant viruses, bacteria, fungi, and nematodes are provided. The techniques allow for rapid, sensitive, and accurate detection of pathogens before symptom development.
Effective detection of MLN pathogens in leaf tissues and seed lotsCIMMYT
The document describes and assesses various detection methods for plant viruses, including Maize Lethal Necrosis virus. It finds that immuno-capture reverse transcription PCR (IC-RT-PCR) combines the specificity of ELISA-based detection with PCR-based detection. A study demonstrated IC-RT-PCR can successfully detect Maize Chlorotic Mottle Virus in infected plant leaves and seeds, with results available in hours and at low labor cost once the technique is established. However, IC-RT-PCR requires specialized equipment and antibodies may not always be available.
Detection of plant pathogens using non pcr based techniquesPuja41124
This document discusses various molecular techniques for detecting and identifying plant pathogens, beginning with conventional techniques like visual observation, culture-based methods, and microscopy. It then covers several antibody-based techniques like serological methods, ELISA, immunofluorescence, and immunoelectron microscopy. Hybridization methods like Southern blotting, Northern blotting, and DNA microarrays are also introduced. Biochemical approaches involving fatty acid analysis, Biolog identification systems, and volatile compound analysis are briefly covered. The document concludes by discussing the surface plasmon resonance technique for pathogen detection. In summary, it provides an overview of conventional, antibody-based, hybridization-based, and biochemical molecular techniques for plant pathogen detection and identification.
Electron microscopy and production of antisera can be used to detect and identify plant viruses.
1) Electron microscopy can detect virus particles in infected plant tissue and identify unknown viruses. It is often combined with antisera in a technique called immuno electron microscopy.
2) Antisera are produced by injecting purified antigens into rabbits to stimulate an immune response and antibody production. Test bleedings are taken from the rabbits and the resulting antisera are evaluated for avidity, specificity, and titer against the target antigen.
disease diagnosis, types of diagnostic kits, nucleic acid based that include pcr, rt pcr, microarray, protein based which include ELISA, types of elisa, comparision among all types of diagnostic kits
This document summarizes diagnostic technologies for differentiating bacterial and viral infections. It discusses using host biomarkers in a diagnostic test developed by ILRI and SLU. Rapid nucleic acid tests and point-of-care tests are mentioned. Methods like lateral flow assays, DNA/RNA amplification, and technologies currently on the market are briefly described. The document also notes challenges like RNA isolation and potential solutions like using paper-based platforms.
MLN Workshop: Maize lethal necrosis diagnostic methods -- A WanguiCIMMYT
This document discusses diagnostic methods for detecting plant viruses, specifically focusing on methods used to detect cassava mosaic viruses (SCMV and MCMV). It describes that correct identification of causal agents is important for disease management and safe trade. Detection methods discussed include symptoms, indicator plants, electron microscopy, serological methods like ELISA, and molecular methods like PCR. For SCMV and MCMV, ELISA and RT-PCR are commonly used due to their high specificity. The document provides details on how to perform DAS-ELISA and one-step RT-PCR, including required materials and steps of the procedures. It also discusses factors that can influence ELISA and PCR results.
Current methods for plant disease diagnosisSHIVANI PATHAK
Shivani presents an overview of current and prospective methods for plant disease detection. Several techniques are discussed including serological based methods like ELISA and lateral flow devices, nucleic acid based methods like PCR and real-time PCR, optical sensor based methods like fluorescence imaging and hyperspectral imaging, and digital imaging based methods. Examples of each technique being used to detect various plant viruses, bacteria, fungi, and nematodes are provided. The techniques allow for rapid, sensitive, and accurate detection of pathogens before symptom development.
DNA microarrays can be used to diagnose plant diseases by detecting pathogens. Microarrays work by hybridizing DNA samples to probes on a chip or slide. They allow researchers to simultaneously analyze thousands of genes. Studies show microarrays can identify fungi, bacteria, viruses, and phytoplasmas faster and more efficiently than existing methods like PCR and ELISA. Microarrays have also been used to study gene expression, toxicology, comparative genomics, and for applications in drug discovery and disease classification. They are a powerful tool but further development of portable biosensors may make disease detection even more accessible.
SEROLOGICAL METHODS FOR DETECTION OF PLANT PATHOGENSHARISH J
This document discusses serological methods for detecting plant pathogens. It explains that serodiagnosis involves inducing an immune response in an animal to produce antibodies against a pathogen's antigens. These antibodies can then be used to detect the presence of the pathogen. The document describes several serological testing methods including ring interface tests, microprecipitin tests, double diffusion tests, ELISA, immunosorbent electron microscopy, and immunofluorescent staining. It concludes that serodiagnosis is a sensitive tool for identifying pathogens, detecting infections, and quantifying crop diseases.
Avs molecular diagnostic techniques for detection of plant pathogensAMOL SHITOLE
PCR is a technique used to detect plant pathogens through amplification of DNA. It involves denaturing DNA, annealing primers, and polymerizing new strands of DNA. This process is repeated to exponentially increase the amount of target DNA. Nested PCR improves sensitivity by adding a second round of amplification. Other techniques like RT-PCR, IC-PCR, bio-PCR, and co-operational PCR have also been used to detect pathogens through nucleic acid amplification and analysis. PCR provides an efficient way to diagnose and study plant pathogens.
This document discusses techniques for serologically detecting plant viruses. It begins by defining serology and its use in agriculture for detecting pathogens with variable or latent symptoms. It then describes the basics of antigen-antibody reactions and the types of antigens, antibodies, and reactions. The rest of the document focuses on specific serological tests used in plant virology, including liquid phase tests like precipitation, agglutination, and immunodiffusion assays as well as solid phase tests like ELISA, SDS-PAGE, ISEM, western blotting, and dot/tissue immunobinding assays. These tests allow detection of plant viruses through the reaction of viral coat proteins or antigens with specific antibodies.
1. Advanced diagnostic techniques such as nucleic acid-based methods like PCR and hybridization methods have overcome limitations of conventional diagnostic methods by being more sensitive, specific, and rapid.
2. PCR exponentially amplifies DNA or RNA sequences, allowing single molecule detection, but cannot differentiate live from dead organisms. Hybridization methods pair complementary nucleic acid strands to detect specific sequences.
3. Other techniques discussed include flow cytometry for analyzing cell properties, Southern blotting for detecting DNA fragments, and ELISA for detecting antibodies or antigens. These techniques have various applications in microbiology, human genetics, forensics, and more.
Culture independent methods for detection & enumeration of gut microfloraAmna Jalil
This document discusses various culture-independent methods for detecting and enumerating gut microflora, including: 1) Design of PCR primers and hybridization probes to target specific species or groups; 2) PCR-ELISA to combine PCR amplification with ELISA detection; 3) Sequence analysis of randomly amplified 16S rRNA genes; 4) Temperature gradient gel electrophoresis (TGGE) and denaturing high performance liquid chromatography (DHPLC) to separate amplified DNA fragments by size; 5) Terminal restriction fragment length polymorphism (T-RFLP) to generate terminal restriction fragments for profiling; 6) Oligonucleotide arrays and quantitative real-time PCR for rapid, accurate detection of thousands of bacteria; 7) Fluorescence in
This document discusses using a bioinformatic approach for DNA signature-based diagnostics. It describes three main molecular diagnostic methods - biochemical, antibody, and DNA-based. DNA-based methods such as PCR and DNA probes are highlighted as being highly sensitive and specific. The use of DNA barcoding to identify species via unique DNA sequences is discussed. Developing DNA signatures for pathogens like Ug99 stem rust fungus is presented as an important application of bioinformatics.
Moleecular mechanism of disease diagnosisjeeva raj
This document discusses molecular techniques for disease diagnosis, including antibody-based and nucleic acid-based methods. Antibody-based methods include using polyclonal and monoclonal antibodies in techniques like ELISA and lateral flow. PCR and RAPD are described as nucleic acid-based techniques that use primers and DNA amplification to detect pathogens. DNA microarrays are also mentioned as a diagnostic tool that screens for multiple pathogens by probing arrays of known DNA sequences.
The document discusses general principles for diagnosing infectious diseases, including:
1. Physical examination, clinical diagnosis, and epidemiological assessment help identify possible pathogens.
2. Laboratory tests are needed to confirm the causative agent, including microscopic examination, culture-based methods, and immunological or molecular detection techniques.
3. Proper specimen collection, transport, and timing are important for accurate diagnostic results.
The document discusses molecular diagnostics and genetic testing techniques. It provides an overview of molecular diagnostics, their significance in medicine, and how they are transforming fields like prenatal testing, disease detection, and drug selection. It then covers various immunological diagnostic methods like ELISA, radioimmunoassay, western blotting, and their characteristics. The document also discusses molecular genetic tests, genetic alterations detected, and techniques for DNA-based diagnosis of diseases. It focuses on the principles and procedures of molecular diagnostic methods like hybridization assays and PCR and their applications in detecting pathogens and genetic disorders.
This document discusses laboratory diagnosis of viral infections. It describes River's postulates which are modified Koch's postulates used to identify viruses as the cause of disease. It also discusses indications for laboratory diagnosis such as managing diseases with available antiviral treatment. General approaches for diagnosis include direct demonstration of viruses and components, virus isolation, and detection of specific antibodies. Common methods described are microscopy, cell and tissue culture, serology including ELISA and PCR, and detection of cytopathic effects.
This document provides an overview of the history and methods of microbial identification. It discusses how identification methods have evolved from using tubed and plated media in the 1960s to now using miniaturized biochemical reactions and system-dependent approaches comparing reaction patterns to databases. Modern rapid identification approaches include varying conventional testing, unique substrates that detect activity without growth, antigen-antibody reactions, and molecular detection methods. Specific techniques like colorimetry, fluorescence, and turbidity are used to detect metabolic activity. Rapid tests for identifying common bacteria like Staphylococcus aureus and Streptococcus pyogenes using agglutination, chromogenic media, DNA probes, PCR, and immunochromatographic assays are also overviewed.
This document provides an overview of molecular pathology and various molecular techniques. It discusses the use of nucleic acid testing for clinical purposes such as diagnosis, prognosis, and pharmacogenetics/pharmacogenomics. It describes several molecular techniques including DNA sequencing, PCR, real-time PCR, FISH, microarrays, and their applications. It also discusses DNA/RNA structure, gene expression, mutations, molecular pathology specimen handling, and instrumentation.
This document discusses molecular diagnosis of tuberculosis through nucleic acid amplification (NAA) methods like polymerase chain reaction (PCR) and transcription mediated amplification (TMA). NAA allows direct detection of mycobacterial DNA or RNA from specimens before culture results. PCR amplifies a specific DNA sequence using primers and DNA polymerase, resulting in exponential multiplication of the target sequence. TMA is an isothermal method that uses RNA transcription and DNA synthesis to amplify nucleic acids. Commercial NAA tests for tuberculosis detection include Amplicor and Enhanced Mycobacterium Tuberculosis Direct test, which have reported sensitivities of 79.4-95.2% and specificities of 98.8-100% compared to culture.
Techniques for identification of bacterial and viral pathogensAmbica Bora
This document discusses various techniques for identifying bacterial and viral pathogens. It covers microscopic, histological, microbiological, immunological, and molecular techniques.
Microscopic techniques include basic staining methods to identify organisms under a microscope. Histological techniques examine stained tissue sections to detect pathogens. Microbiological techniques involve culturing samples and conducting biochemical tests on isolates.
Immunological techniques like ELISA, agglutination, and immunofluorescence use antigen-antibody reactions for detection. Molecular techniques like PCR, multiplex PCR, and DNA microarrays allow sensitive detection and differentiation of pathogens through nucleic acid analysis.
After detection, antibiotics are usually administered but alternative remedies like photoinactivation of pathogens using blue light and bacteri
Viruses. Methods of Indication & Identification. Diagnosis of Viral diseasesEneutron
This document discusses methods for indicating, identifying, and diagnosing viral diseases in the laboratory. It describes several methods for indicating viruses including observing cytopathic effects in cell cultures, hemadsorption, hemagglutination, metabolic inhibition assays, and plaque or pock formation. Identification methods covered include serological techniques like neutralization testing, complement fixation testing, hemagglutination inhibition, and enzyme-linked immunosorbent assays (ELISA). Molecular identification using PCR to detect viral nucleic acids is also mentioned. The document outlines the main stages of viral diagnosis as detection of viruses in samples, viral cultivation and identification, and serological diagnostics including various serological tests.
India is one of 12 megabiodiverse countries containing around 7-8% of the world's species. It has high biodiversity at genetic, species, and ecosystem levels due to its varied climate, geography and 10 biogeographic zones. India hosts numerous threatened species and is home to 4 of the world's 36 biodiversity hotspots. It has over 100 national parks and 500 wildlife sanctuaries covering 4.7% of its land area and has designated numerous biosphere reserves, Ramsar sites, and UNESCO World Heritage sites to protect its natural heritage.
This document outlines standard operating procedures for collecting, handling, and storing data from plant quarantine samples tested for mycotoxins. It describes filling out forms with sample information, processing samples in the lab including recording results on control logs, and using data worksheets and software to analyze results and calculate mycotoxin concentrations. All electronic records are stored securely with backups in a separate location from the original data.
This document describes a study assessing the genetic stability of taro (Colocasia esculenta) germplasm conserved in vitro. The study aims to develop an efficient micropropagation system for taro varieties, evaluate the field performance of tissue-cultured and conventionally propagated taro, and establish an in vitro germplasm collection of taro. Shoot tips from taro corms were sterilized and cultured on Z10 medium, then subcultured and transferred to the field. Genetic analysis using DNA quantification and gel electrophoresis showed the tissue-cultured plants were genetically stable. The project will produce disease-free planting materials for distribution to farmers and for genetic resource conservation.
DNA microarrays can be used to diagnose plant diseases by detecting pathogens. Microarrays work by hybridizing DNA samples to probes on a chip or slide. They allow researchers to simultaneously analyze thousands of genes. Studies show microarrays can identify fungi, bacteria, viruses, and phytoplasmas faster and more efficiently than existing methods like PCR and ELISA. Microarrays have also been used to study gene expression, toxicology, comparative genomics, and for applications in drug discovery and disease classification. They are a powerful tool but further development of portable biosensors may make disease detection even more accessible.
SEROLOGICAL METHODS FOR DETECTION OF PLANT PATHOGENSHARISH J
This document discusses serological methods for detecting plant pathogens. It explains that serodiagnosis involves inducing an immune response in an animal to produce antibodies against a pathogen's antigens. These antibodies can then be used to detect the presence of the pathogen. The document describes several serological testing methods including ring interface tests, microprecipitin tests, double diffusion tests, ELISA, immunosorbent electron microscopy, and immunofluorescent staining. It concludes that serodiagnosis is a sensitive tool for identifying pathogens, detecting infections, and quantifying crop diseases.
Avs molecular diagnostic techniques for detection of plant pathogensAMOL SHITOLE
PCR is a technique used to detect plant pathogens through amplification of DNA. It involves denaturing DNA, annealing primers, and polymerizing new strands of DNA. This process is repeated to exponentially increase the amount of target DNA. Nested PCR improves sensitivity by adding a second round of amplification. Other techniques like RT-PCR, IC-PCR, bio-PCR, and co-operational PCR have also been used to detect pathogens through nucleic acid amplification and analysis. PCR provides an efficient way to diagnose and study plant pathogens.
This document discusses techniques for serologically detecting plant viruses. It begins by defining serology and its use in agriculture for detecting pathogens with variable or latent symptoms. It then describes the basics of antigen-antibody reactions and the types of antigens, antibodies, and reactions. The rest of the document focuses on specific serological tests used in plant virology, including liquid phase tests like precipitation, agglutination, and immunodiffusion assays as well as solid phase tests like ELISA, SDS-PAGE, ISEM, western blotting, and dot/tissue immunobinding assays. These tests allow detection of plant viruses through the reaction of viral coat proteins or antigens with specific antibodies.
1. Advanced diagnostic techniques such as nucleic acid-based methods like PCR and hybridization methods have overcome limitations of conventional diagnostic methods by being more sensitive, specific, and rapid.
2. PCR exponentially amplifies DNA or RNA sequences, allowing single molecule detection, but cannot differentiate live from dead organisms. Hybridization methods pair complementary nucleic acid strands to detect specific sequences.
3. Other techniques discussed include flow cytometry for analyzing cell properties, Southern blotting for detecting DNA fragments, and ELISA for detecting antibodies or antigens. These techniques have various applications in microbiology, human genetics, forensics, and more.
Culture independent methods for detection & enumeration of gut microfloraAmna Jalil
This document discusses various culture-independent methods for detecting and enumerating gut microflora, including: 1) Design of PCR primers and hybridization probes to target specific species or groups; 2) PCR-ELISA to combine PCR amplification with ELISA detection; 3) Sequence analysis of randomly amplified 16S rRNA genes; 4) Temperature gradient gel electrophoresis (TGGE) and denaturing high performance liquid chromatography (DHPLC) to separate amplified DNA fragments by size; 5) Terminal restriction fragment length polymorphism (T-RFLP) to generate terminal restriction fragments for profiling; 6) Oligonucleotide arrays and quantitative real-time PCR for rapid, accurate detection of thousands of bacteria; 7) Fluorescence in
This document discusses using a bioinformatic approach for DNA signature-based diagnostics. It describes three main molecular diagnostic methods - biochemical, antibody, and DNA-based. DNA-based methods such as PCR and DNA probes are highlighted as being highly sensitive and specific. The use of DNA barcoding to identify species via unique DNA sequences is discussed. Developing DNA signatures for pathogens like Ug99 stem rust fungus is presented as an important application of bioinformatics.
Moleecular mechanism of disease diagnosisjeeva raj
This document discusses molecular techniques for disease diagnosis, including antibody-based and nucleic acid-based methods. Antibody-based methods include using polyclonal and monoclonal antibodies in techniques like ELISA and lateral flow. PCR and RAPD are described as nucleic acid-based techniques that use primers and DNA amplification to detect pathogens. DNA microarrays are also mentioned as a diagnostic tool that screens for multiple pathogens by probing arrays of known DNA sequences.
The document discusses general principles for diagnosing infectious diseases, including:
1. Physical examination, clinical diagnosis, and epidemiological assessment help identify possible pathogens.
2. Laboratory tests are needed to confirm the causative agent, including microscopic examination, culture-based methods, and immunological or molecular detection techniques.
3. Proper specimen collection, transport, and timing are important for accurate diagnostic results.
The document discusses molecular diagnostics and genetic testing techniques. It provides an overview of molecular diagnostics, their significance in medicine, and how they are transforming fields like prenatal testing, disease detection, and drug selection. It then covers various immunological diagnostic methods like ELISA, radioimmunoassay, western blotting, and their characteristics. The document also discusses molecular genetic tests, genetic alterations detected, and techniques for DNA-based diagnosis of diseases. It focuses on the principles and procedures of molecular diagnostic methods like hybridization assays and PCR and their applications in detecting pathogens and genetic disorders.
This document discusses laboratory diagnosis of viral infections. It describes River's postulates which are modified Koch's postulates used to identify viruses as the cause of disease. It also discusses indications for laboratory diagnosis such as managing diseases with available antiviral treatment. General approaches for diagnosis include direct demonstration of viruses and components, virus isolation, and detection of specific antibodies. Common methods described are microscopy, cell and tissue culture, serology including ELISA and PCR, and detection of cytopathic effects.
This document provides an overview of the history and methods of microbial identification. It discusses how identification methods have evolved from using tubed and plated media in the 1960s to now using miniaturized biochemical reactions and system-dependent approaches comparing reaction patterns to databases. Modern rapid identification approaches include varying conventional testing, unique substrates that detect activity without growth, antigen-antibody reactions, and molecular detection methods. Specific techniques like colorimetry, fluorescence, and turbidity are used to detect metabolic activity. Rapid tests for identifying common bacteria like Staphylococcus aureus and Streptococcus pyogenes using agglutination, chromogenic media, DNA probes, PCR, and immunochromatographic assays are also overviewed.
This document provides an overview of molecular pathology and various molecular techniques. It discusses the use of nucleic acid testing for clinical purposes such as diagnosis, prognosis, and pharmacogenetics/pharmacogenomics. It describes several molecular techniques including DNA sequencing, PCR, real-time PCR, FISH, microarrays, and their applications. It also discusses DNA/RNA structure, gene expression, mutations, molecular pathology specimen handling, and instrumentation.
This document discusses molecular diagnosis of tuberculosis through nucleic acid amplification (NAA) methods like polymerase chain reaction (PCR) and transcription mediated amplification (TMA). NAA allows direct detection of mycobacterial DNA or RNA from specimens before culture results. PCR amplifies a specific DNA sequence using primers and DNA polymerase, resulting in exponential multiplication of the target sequence. TMA is an isothermal method that uses RNA transcription and DNA synthesis to amplify nucleic acids. Commercial NAA tests for tuberculosis detection include Amplicor and Enhanced Mycobacterium Tuberculosis Direct test, which have reported sensitivities of 79.4-95.2% and specificities of 98.8-100% compared to culture.
Techniques for identification of bacterial and viral pathogensAmbica Bora
This document discusses various techniques for identifying bacterial and viral pathogens. It covers microscopic, histological, microbiological, immunological, and molecular techniques.
Microscopic techniques include basic staining methods to identify organisms under a microscope. Histological techniques examine stained tissue sections to detect pathogens. Microbiological techniques involve culturing samples and conducting biochemical tests on isolates.
Immunological techniques like ELISA, agglutination, and immunofluorescence use antigen-antibody reactions for detection. Molecular techniques like PCR, multiplex PCR, and DNA microarrays allow sensitive detection and differentiation of pathogens through nucleic acid analysis.
After detection, antibiotics are usually administered but alternative remedies like photoinactivation of pathogens using blue light and bacteri
Viruses. Methods of Indication & Identification. Diagnosis of Viral diseasesEneutron
This document discusses methods for indicating, identifying, and diagnosing viral diseases in the laboratory. It describes several methods for indicating viruses including observing cytopathic effects in cell cultures, hemadsorption, hemagglutination, metabolic inhibition assays, and plaque or pock formation. Identification methods covered include serological techniques like neutralization testing, complement fixation testing, hemagglutination inhibition, and enzyme-linked immunosorbent assays (ELISA). Molecular identification using PCR to detect viral nucleic acids is also mentioned. The document outlines the main stages of viral diagnosis as detection of viruses in samples, viral cultivation and identification, and serological diagnostics including various serological tests.
India is one of 12 megabiodiverse countries containing around 7-8% of the world's species. It has high biodiversity at genetic, species, and ecosystem levels due to its varied climate, geography and 10 biogeographic zones. India hosts numerous threatened species and is home to 4 of the world's 36 biodiversity hotspots. It has over 100 national parks and 500 wildlife sanctuaries covering 4.7% of its land area and has designated numerous biosphere reserves, Ramsar sites, and UNESCO World Heritage sites to protect its natural heritage.
This document outlines standard operating procedures for collecting, handling, and storing data from plant quarantine samples tested for mycotoxins. It describes filling out forms with sample information, processing samples in the lab including recording results on control logs, and using data worksheets and software to analyze results and calculate mycotoxin concentrations. All electronic records are stored securely with backups in a separate location from the original data.
This document describes a study assessing the genetic stability of taro (Colocasia esculenta) germplasm conserved in vitro. The study aims to develop an efficient micropropagation system for taro varieties, evaluate the field performance of tissue-cultured and conventionally propagated taro, and establish an in vitro germplasm collection of taro. Shoot tips from taro corms were sterilized and cultured on Z10 medium, then subcultured and transferred to the field. Genetic analysis using DNA quantification and gel electrophoresis showed the tissue-cultured plants were genetically stable. The project will produce disease-free planting materials for distribution to farmers and for genetic resource conservation.
Viruses are nucleoproteins that can only reproduce inside living cells. They have either DNA or RNA, come in various shapes, and form protective capsids around their nucleic acids. Viruses utilize host cell substances to multiply, taking up space and disrupting cellular processes. They enter plants through wounds, vectors, or infected plant parts. Viruses move between cells through plasmodesmata and are transported via phloem to growing regions. Detection methods include observing inclusion bodies under microscopes, viewing virus particles with electron microscopes, transmission to healthy plants, serology tests like ELISA, and using DNA/RNA probes.
This document is a chapter-by-chapter summary of a guide to western blotting. It introduces the principles and techniques of western blotting, including sample preparation, gel electrophoresis, protein transfer, immunodetection, data analysis, and troubleshooting. The guide is authored by Claire Moore and contains 6 chapters covering the process from start to finish.
The document discusses the two spotted spider mite, Tetranychus urticae. It describes the mite's morphology, life cycle, distribution, host plants, and economic importance. The two spotted spider mite can damage a wide range of plants by feeding on their leaves and flowers. Management strategies discussed include biological control using predator mites and insects, as well as chemical control using miticides.
Somatic ybridization and its applicationPawan Nagar
This document discusses somatic hybridization, which involves fusing plant protoplasts from two different species or varieties to create a hybrid plant. It describes the process of somatic hybridization, including isolating protoplasts, fusing them using spontaneous or induced methods, selecting hybrid cells, and regenerating plants from hybrid callus tissue. The advantages are producing novel hybrids and transferring genes between incompatible species. The limitations include low regeneration rates and viability of fused cells. Somatic hybridization has applications in crop improvement by introducing traits like disease resistance from wild relatives.
1) Anther and pollen culture techniques involve culturing microspores excised from anthers or pollen grains to produce haploid plants. This allows for the efficient production of fully homozygous lines.
2) Factors like genotype, culture medium, and pretreatments influence anther culture success. Haploids must be doubled to be fertile and useful.
3) Somatic hybridization fuses protoplasts from different plant species using techniques like PEG or electrofusion. This can combine traits not otherwise possible. Selection and regeneration are required to produce hybrid plants.
Ento 402 (storage pest)non insect pest mitesVaibhav Wadhwa
This document provides information about mites, including their classification and importance. It discusses how mites can infest and damage agricultural crops, stored produce, and furniture. Mites can cause allergies in humans. The document describes the characteristics of mites such as their body structure and life cycle. It provides details on different families of mites including phytophagous and predatory mites. Specific mite species that damage important crops are listed. The symptoms of mite damage and methods of mite management including chemical and biological controls are summarized.
The document discusses the benefits of exercise for mental health. Regular physical activity can help reduce anxiety and depression and improve mood and cognitive functioning. Exercise causes chemical changes in the brain that may help boost feelings of calmness, happiness and focus.
PHYSICAL MAPPING STRATEGIES IN GENOMICSUsman Arshad
Genetic and physical mapping are two types of genome mapping. Genetic mapping uses pedigree analysis and breeding experiments to determine sequence features, while physical mapping uses molecular techniques. Restriction mapping, radiation hybrid mapping, and STS mapping are techniques used to construct physical maps in the absence of complete DNA sequencing. Restriction mapping identifies restriction sites, radiation hybrid mapping analyzes fragments from irradiated cells hybridized with hamster cells, and STS mapping tags genomic sites using PCR primers. These physical mapping strategies provide distance and order estimates between DNA sequences to construct frameworks for sequencing.
The document provides an overview of quantitative PCR (qPCR) and its applications. It discusses:
1. The basic steps of qPCR including RNA isolation, cDNA synthesis via reverse transcription, and quantitative PCR followed by statistical analysis of the results.
2. Different methods for RNA isolation from various samples like cells, tissues, and FFPE samples.
3. Primer and probe design considerations for qPCR and how to use probe design tools.
4. Details of the qPCR process like reaction composition, controls, and statistical tests used to analyze quantitative PCR results.
Organogenesis is the process by which plant organs like roots, shoots, flowers develop from explants. It is influenced by physical factors like light and temperature, and chemical factors like cytokinins and auxins. Organogenesis has advantages like being cheap, fast, and allowing for easy scaling up. Somatic hybridization involves fusing protoplasts from two plant species using techniques like PEG-mediated fusion. It allows for transferring genes between sexually incompatible plants but has challenges like low regeneration rates and hybrid viability. It differs from organogenesis in producing bipolar embryos without connections to parent tissue.
In this slidedeck, the following topics, which are critical steps for efficient and precise gene expression studies using real-time PCR technology, are covered:
• Effect of RNA integrity on real-time PCR results – tips on how to achieve a true RNA profile suitable for real-time PCR studies
• Improved methods for cDNA synthesis, optimized for real-time PCR
• Real-time PCR analysis
• Real-time PCR essentials and background information on different quantification strategies
• SYBR Green real-time PCR – factors influencing specificity
• Introduction to probe technology
• New, fast and efficient real-time PCR solutions
This document describes research on producing double haploids in the ornamental plant Primula via anther culture. The researchers determined the optimal microspore developmental stage for culture by examining bud size and morphology. Anthers at specific stages were cultured on media with different plant growth regulator combinations. Callus formed from some anthers, and some calli regenerated shoots. The ploidy levels of regenerated plants were determined using flow cytometry and cytology. Plants with haploid, diploid and mixoploid levels were identified. The study developed a protocol for generating double haploids in Primula through microspore culture, aiming to provide a more efficient breeding method for this ornamental crop.
Hybridization is the process of breeding plants and animals to produce hybrid offspring that have desired qualities. It involves cross-breeding two parent varieties to develop a new hybrid variety that possesses traits from both parents, such as high yield, disease resistance, or climate tolerance. The process involves selecting parent plants, removing stamens from female flowers, collecting pollen from male flowers, pollinating the female flowers, collecting mature seeds, and selecting offspring with better qualities over several generations. Hybrid varieties developed through this process contribute greatly to agricultural progress as they are widely used for traits like high yield and disease resistance.
Protoplast fusion involves removing the cell walls of plant cells through enzymatic or mechanical means to create naked protoplasts. These protoplasts can then be fused using chemicals, electricity, or other methods. This allows the cytoplasms and sometimes nuclei of different plant cells to merge, creating hybrid cells. Successful fusion can generate hybrid plants through regeneration of cell walls and tissues. Protoplast fusion overcomes sexual incompatibility and is used to introduce traits like disease resistance between species. It remains a technically challenging process with limitations like genetic instability and uncertain expression of transferred traits.
This document discusses the identification and characteristics of various ticks and mites of veterinary importance. It identifies key genera of ticks, including Amblyoma, Hyalomma, Rhipicephalus, and Argas, as well as mites such as Psoroptes, Sarcoptes, Demodex, and Tunga penetrans. It describes their life cycles, medical significance as vectors of disease, and potential control methods. The conclusion is that the student successfully identified and explained the ticks and mites and their importance to animal and public health.
The document discusses haploid plant culture techniques. It describes anther/pollen culture and ovule culture as important methods to produce haploid plants. Anther/pollen culture involves culturing anthers or isolated pollen grains to develop into haploid embryos or callus. Ovule culture involves culturing isolated ovules. The document outlines factors that affect androgenesis (anther/pollen culture) and discusses using techniques like colchicine treatment or long-term callus culture to induce chromosome doubling and generate homozygous diploid plants from haploids.
This document provides an overview of molecular techniques used in microbiology. It discusses three main categories: signal amplification methods like nucleic acid probes; nucleic acid amplification techniques like PCR and its variants; and strain typing methods. PCR and its applications are described in detail, including real-time PCR. Other amplification methods and post-amplification analysis techniques like gel electrophoresis and sequencing are also summarized. Commonly used commercial systems like BioFire FilmArray that employ multiplex PCR are highlighted.
PCR (polymerase chain reaction) is a technique used to amplify specific DNA sequences. It involves repeated cycles of heating and cooling of the DNA sample to separate and copy the DNA. There are several types of PCR including nested PCR, which increases sensitivity by using two rounds of amplification, and multiplex PCR, which allows simultaneous amplification of multiple DNA sequences. PCR has many applications in medicine, forensics, and agriculture by enabling identification of pathogens and organisms, disease diagnosis, and selective isolation of DNA fragments.
PCR (polymerase chain reaction) is a technique used to amplify specific DNA sequences. It involves repeated cycles of heating and cooling of the DNA sample to separate and copy the DNA strands. There are several types of PCR including nested PCR, which increases sensitivity by using two rounds of amplification, and multiplex PCR, which allows simultaneous amplification of multiple DNA sequences. PCR and its variations have widespread applications in medicine, forensics, agriculture, and research by enabling identification of organisms, understanding disease pathology, and selective isolation of DNA fragments.
This document provides an overview of immuno-PCR (I-PCR), which combines the antibody-based specificity of ELISA with the amplification power of PCR for highly sensitive antigen detection. Key points covered include: advantages and limitations of ELISA and real-time PCR; the main steps of I-PCR including antibody-oligonucleotide conjugation; and Innova Biosciences' ThunderLink PLUS technology for easy conjugation without specialist knowledge in 30 minutes. The presentation aims to introduce audiences to I-PCR and its applications in sensitive protein detection.
Molecular testing in Fetal and Neonatal Alloimmune Thrombocytopenia Arjuna Samaranayaka
This document discusses molecular testing for neonatal alloimmune thrombocytopenia (NAIT).
NAIT occurs when a fetus inherits a platelet antigen from the father that is absent in the mother, causing the mother to produce antibodies against the antigen. These antibodies cross the placenta and destroy the fetus's platelets.
Molecular testing can identify incompatibilities by genotyping the mother, father, and fetus for platelet antigens. It has advantages over serological methods as it doesn't require fresh platelets and can detect rare antigens. Various techniques are described like PCR-SSP, PCR-RFLP, TaqMan PCR, and high-throughput methods. Molecular testing is useful for diagnosing NAIT after
1. Recombinant DNA technology involves manipulating genetic material from different sources to create chimeric DNA molecules. This is done using various tools like restriction enzymes, DNA ligase, cloning, and gene transfer methods.
2. Medical biotechnology applies recombinant DNA technology to research and produce pharmaceuticals and diagnostic products to treat and prevent human diseases. This includes producing insulin, growth hormones, antibodies, and vaccines.
3. Techniques like PCR, blotting, hybridization, and DNA sequencing are used in recombinant DNA technology applications such as gene mapping, understanding disease mechanisms, DNA-based diagnostics, forensics, and gene therapy.
This document discusses advanced diagnostic aids in periodontology. It describes conventional diagnosis methods and their limitations. Newer diagnostic methods discussed include gingival bleeding assessment, gingival temperature measurement, improved periodontal probing, digital radiography, subtraction radiography, microbiological analysis using bacterial culture, microscopy, immunodiagnostic assays, enzymatic methods, DNA probes, and methods to characterize the host inflammatory response using biomarkers. Overall, the document outlines many potential diagnostic markers and tests but notes that more research is still needed to identify simple, predictive, and cost-effective tests to complement clinical diagnosis in periodontology.
Molecular techniques in food microbiologyNajiyaNaju1
This document discusses molecular techniques that are used in food microbiology. It describes several applications of molecular methods including detecting genetically modified foods, food authenticity testing, and microbial contamination detection. Common molecular methods described are PCR, RFLP, PFGE, and DNA microarrays. Specific techniques covered in more depth include real-time PCR, multiplex PCR, plasmid profiling, ribotyping, AFLP, LAMP, FISH, and biosensors. The document provides details on the basic principles and procedures for several of these important molecular analysis methods.
This document provides an overview of polymerase chain reaction (PCR). It describes how PCR works to exponentially amplify a specific DNA sequence. The key components needed for PCR are a DNA template, primers, DNA polymerase, and dNTPs. The basic steps of PCR involve denaturing the DNA, annealing primers, and extending the primers through repeated thermal cycling. PCR has many applications including detecting infectious agents, identifying genetic mutations, and forensic analysis. Sources of error can occur from mispriming, secondary DNA structures, and primer dimer formation.
Immunohistochemistry is a technique used to identify antigens in tissue samples using antigen-antibody interactions. It has made a large impact in disease diagnosis since its development in the 1940s-1970s. The technique involves using labeled antibodies that specifically bind to antigens in tissue sections. This binding is then visualized using markers like enzymes or fluorescent dyes. Several methods have been developed to increase the signal and reduce background noise, including indirect labeling techniques and polymer-based methods. Proper tissue processing and antibody selection are important for obtaining high quality results with immunohistochemistry.
Automated Nucleic Acid Purification from Diverse Sample types using dedicated...QIAGEN
This webinar will focus on the automation of QIAGEN’s new line of DNA and RNA sample prep kits for the microbiome. We will show how automation on the QIAcube enables efficient and reliable use of these samples for sensitive downstream applications such as qPCR and NGS. In addition, you will learn how to successfully use the CLC Microbial Genomics Module for metagenome sequencing and identification of microbial composition and diversity.
Developing Ultra-Sensitive PCR Protocols for HIV Vaccine ResearchKate Barlow
This document summarizes Dr. Catherine N. Kibirige's presentation on developing ultra-sensitive PCR protocols for HIV vaccine research. It discusses (1) the challenges of HIV's genetic diversity and finding conserved sequence regions for assay design, (2) how digital droplet PCR (ddPCR) was useful for quantifying cell line standards but qPCR was better for analyzing archival patient samples due to issues with ddPCR, and (3) two successful studies using the ultra-sensitive qPCR assay - a pilot study found HIV RNA more frequently in virologically suppressed patients with high inflammation, and characterization of the IAVI Viral Inhibition Assay showed HIV RNA detection within 2 days of differences in CD8 inhibition
This document discusses biosafety testing for cell and gene therapies performed by BioReliance, a testing services division of Merck KGaA. It outlines the comprehensive testing performed at various stages of development, including testing of cell banks, viral vectors, and final drug products. Testing evaluates important product attributes like identity, purity, potency and residuals to ensure safety and quality. A wide range of assays are used to characterize products and identify potential contaminants.
RT2 Profiler PCR Arrays are a real-time PCR technology that allows researchers to study gene expression patterns across biological pathways and processes. The arrays contain pre-designed primer assays for 84 relevant genes as well as controls on a single plate in a 96-well format. The gene content of the arrays is selected based on biological relevance and published associations with relevant pathways. The primer assays on the arrays undergo extensive validation for sensitivity, specificity, reproducibility, and amplification efficiency. The PCR Array system also includes optimized components for RNA isolation, cDNA synthesis, and real-time PCR to provide a complete validated workflow for gene expression analysis from sample to results.
Methods of Laboratory diagnosis (genotypic detection) of HIVdrakmane
1. Genotypic methods of HIV detection involve detecting mutations in the viral genome that are associated with drug resistance. This is done by sequencing parts of the HIV genome, like the reverse transcriptase and protease genes.
2. There are two main approaches - phenotypic testing directly measures viral replication in the presence of drugs, while genotypic testing detects known resistance-conferring mutations. Genotypic is recommended due to being faster and less expensive.
3. Genotypic testing involves extracting HIV RNA from plasma, amplifying genes with PCR, and then sequencing the genes to identify resistance mutations compared to wild type sequences. This allows detection of resistant strains present at 25% of the total viral population.
Tools and Techniques (Molecular & Biochemical) to Study Physiological Process...Agronomist Wasim
The document discusses various molecular and biochemical tools and techniques used to study physiological processes and stress responses in plants, including DNA and RNA isolation, cDNA synthesis and library construction, RT-PCR, Northern blot, and immunoassays. It provides details on how to perform techniques like DNA isolation, RNA isolation from yeast, cDNA library construction, and the different types and applications of RT-PCR, Northern blot, and categories of immunoassays.
This document discusses the development of ELISAs to test Dasiprotimut-T, a therapeutic cancer vaccine produced by Biovest International. It describes qualitative and quantitative ELISAs developed in-house to test for antibody concentration, impurities from host cell proteins and bovine proteins. Automated systems using Tecan Freedom EVO liquid handlers were also implemented to increase throughput and reimbursement for quality control testing required by regulatory agencies. Quality control documentation, validation protocols, stability studies and inventory management are important aspects of ensuring the vaccine manufacturing process meets regulatory standards.
The document discusses the polymerase chain reaction (PCR) technique. It describes how PCR allows targeted amplification of specific DNA regions. Key steps in PCR include using DNA polymerase, primers, and thermal cycling to denature and replicate the DNA. The document outlines different types of PCR like quantitative, multiplex, and nested PCR. It also discusses how PCR has revolutionized fields like molecular archaeology by enabling the cloning of ancient DNA. PCR is now widely used in applications like forensics, disease diagnosis, and genome sequencing.
Exploiting Artificial Intelligence for Empowering Researchers and Faculty, In...Dr. Vinod Kumar Kanvaria
Exploiting Artificial Intelligence for Empowering Researchers and Faculty,
International FDP on Fundamentals of Research in Social Sciences
at Integral University, Lucknow, 06.06.2024
By Dr. Vinod Kumar Kanvaria
How to Manage Your Lost Opportunities in Odoo 17 CRMCeline George
Odoo 17 CRM allows us to track why we lose sales opportunities with "Lost Reasons." This helps analyze our sales process and identify areas for improvement. Here's how to configure lost reasons in Odoo 17 CRM
How to Build a Module in Odoo 17 Using the Scaffold MethodCeline George
Odoo provides an option for creating a module by using a single line command. By using this command the user can make a whole structure of a module. It is very easy for a beginner to make a module. There is no need to make each file manually. This slide will show how to create a module using the scaffold method.
বাংলাদেশের অর্থনৈতিক সমীক্ষা ২০২৪ [Bangladesh Economic Review 2024 Bangla.pdf] কম্পিউটার , ট্যাব ও স্মার্ট ফোন ভার্সন সহ সম্পূর্ণ বাংলা ই-বুক বা pdf বই " সুচিপত্র ...বুকমার্ক মেনু 🔖 ও হাইপার লিংক মেনু 📝👆 যুক্ত ..
আমাদের সবার জন্য খুব খুব গুরুত্বপূর্ণ একটি বই ..বিসিএস, ব্যাংক, ইউনিভার্সিটি ভর্তি ও যে কোন প্রতিযোগিতা মূলক পরীক্ষার জন্য এর খুব ইম্পরট্যান্ট একটি বিষয় ...তাছাড়া বাংলাদেশের সাম্প্রতিক যে কোন ডাটা বা তথ্য এই বইতে পাবেন ...
তাই একজন নাগরিক হিসাবে এই তথ্য গুলো আপনার জানা প্রয়োজন ...।
বিসিএস ও ব্যাংক এর লিখিত পরীক্ষা ...+এছাড়া মাধ্যমিক ও উচ্চমাধ্যমিকের স্টুডেন্টদের জন্য অনেক কাজে আসবে ...
A workshop hosted by the South African Journal of Science aimed at postgraduate students and early career researchers with little or no experience in writing and publishing journal articles.
A review of the growth of the Israel Genealogy Research Association Database Collection for the last 12 months. Our collection is now passed the 3 million mark and still growing. See which archives have contributed the most. See the different types of records we have, and which years have had records added. You can also see what we have for the future.
Strategies for Effective Upskilling is a presentation by Chinwendu Peace in a Your Skill Boost Masterclass organisation by the Excellence Foundation for South Sudan on 08th and 09th June 2024 from 1 PM to 3 PM on each day.
A Strategic Approach: GenAI in EducationPeter Windle
Artificial Intelligence (AI) technologies such as Generative AI, Image Generators and Large Language Models have had a dramatic impact on teaching, learning and assessment over the past 18 months. The most immediate threat AI posed was to Academic Integrity with Higher Education Institutes (HEIs) focusing their efforts on combating the use of GenAI in assessment. Guidelines were developed for staff and students, policies put in place too. Innovative educators have forged paths in the use of Generative AI for teaching, learning and assessments leading to pockets of transformation springing up across HEIs, often with little or no top-down guidance, support or direction.
This Gasta posits a strategic approach to integrating AI into HEIs to prepare staff, students and the curriculum for an evolving world and workplace. We will highlight the advantages of working with these technologies beyond the realm of teaching, learning and assessment by considering prompt engineering skills, industry impact, curriculum changes, and the need for staff upskilling. In contrast, not engaging strategically with Generative AI poses risks, including falling behind peers, missed opportunities and failing to ensure our graduates remain employable. The rapid evolution of AI technologies necessitates a proactive and strategic approach if we are to remain relevant.
This presentation includes basic of PCOS their pathology and treatment and also Ayurveda correlation of PCOS and Ayurvedic line of treatment mentioned in classics.
This presentation was provided by Steph Pollock of The American Psychological Association’s Journals Program, and Damita Snow, of The American Society of Civil Engineers (ASCE), for the initial session of NISO's 2024 Training Series "DEIA in the Scholarly Landscape." Session One: 'Setting Expectations: a DEIA Primer,' was held June 6, 2024.
How to Add Chatter in the odoo 17 ERP ModuleCeline George
In Odoo, the chatter is like a chat tool that helps you work together on records. You can leave notes and track things, making it easier to talk with your team and partners. Inside chatter, all communication history, activity, and changes will be displayed.
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5. Processing of Legume Germplasm in PEQ Greenhouses and Containment Facility
Glycine max
Pisum sativum
Containment Facility
6. Symptoms only serve as a guide but
indispensable
• Similar symptoms can be produced by different viruses
• Symptoms may be extremely variable; the same virus can
produce a range of symptoms
• Lack of symptoms does not necessarily mean that no viruses
are present.
• Mixed infection result in more severe symptoms
• Symptoms are only indicative, not confirmatory
• Reduction in growth
• Colour deviation
• Necrosis
• Malformation
16. Necrotic Local Lesions by
BCMV on Chenopodium
amaranticolor
Systemic Infection of SMV on
Nicotianatabacumxanthi
Infectivity Assay Contd…
17. Biochemical Technique
Staining of inclusion bodies
• observation under light microscope
• inclusion bodies are viral aggregates or proteins
induced in cytoplasm or nucleus
• staining by Azure A and O-G combinationst
CPMVCMVBYMVTMV
19. • Reveals shape and size of
the particle
• Gives idea of the group to
which the virus belongs
• Very expensive
equipment, often not
available
Physical- Electron Microscopy
21. Serological Techniques
•Based on antigen - antibody reaction
• Antigen: a protein or polysachharide which induces
the formation of antibodies when injected into a
warm-blooded animal
• Antibody: a specific protein formed in the blood of
warm-blooded animals in response to injection of a
protein or polysaccharide
• Antiserum: blood serum containing antibodies
28. Advantages of Monoclonal Antibodies
• Unlimited quantities of the same antibody
in a reproducible manner
• Ability to produce MAbs for indefinite time
period by cryopreservation of hybridomas
for unlimited periods
30. DAS - ELISA DAC - ELISA
Indirect DAC - ELISADirect DAS - ELISA
Variants of ELISA
31. Antibody
coated
well
Antigen
binds to
antibody
A second
antibody,
linked to
enzyme,
binds to
immobilized
antigen
Substrate is
added and
converted by
enzyme into
coloured
product; the
rate of colour
formation is
proportional to
the amount of
antigen
E E E
S
SE
Wash WashWash
Double Antibody Sandwich - ELISA
32. Requirements for ELISA
• Antibodies
• Positive control
• Negative control
• Buffer control
• Each sample in duplicate wells
35. Mechanization
• Sample preparation, tissue grinders of various sorts for handling a
few samples to 1,00,000 samples
• Plate readers to quantify the results and make statistical analysis
possible.
• Robotics to wash and load the microtitre plates.
• These developments made ELISA a cost-effective method of
detection
Tissue Grinder Plate Washer
Courtesy: Dr Robert Martin, Corvallis, USA
36. Advantages of ELISA
• Reasonably sensitive
• Less susceptible to ‘false positives’
• Low per sample cost
• Handles large number of samples
• Can be subjected to automation
• Detection kits available commercially
− A boon for technicians
37. Dot Immunobinding Assay (DIBA)
• A variant of ELISA
• Nitrocellulose membrane as solid support
• Crude antisera can be used
• Stains used for revealing the reaction
• Very useful for field work
38. Tissue Blotting Immunoassay
(=Tissue Print Immunoassay, Tissue Print
Immunoblotting)
• Similar to DIBA
• Reactants can be reused
• Easily applicable for field sampling
• Samples can be prepared with virtually no equipment almost anywhere
• Qualitative test
Role Tear Blot
Courtesy: Dr Robert Martin, Corvallis, USA
41. We use…
ELISA Diagnostic Kits: virus-specific antisera
(from Agdia/ Bio-Rad/ Bioreba/ Loewe/ Neogen)
– Seed directly used in ELISA for detecting
viruses
– Grow-out tests in a greenhouse, followed
by testing the seedlings by ELISA
48. Genome Organization of Potyviruses
• Single-stranded, positive sense RNA, about 10 kb
• Genome is expressed as a single polyprotein
• 3' end of the RNA has a poly (A) tail: about 200 ‘A’ s
• CP ORF is at the 3' end of the RNA; is used for delineating
potyviruses into species
• The conserved poly (A) tail and CP region are widely
used as targets for RT-PCR
49. RT- PCR Using Specific Primers
• Utilize 3'poly A tail of the genome: oligo dT
primer is used for 1st
strand synthesis
• Upstream and downstream primers are specific to
BCMV, BCMNV, PSbMV and SMV
• Design primers based on conserved sequences of
known isolates
• Clone and sequence
50. Details of the Primers Used
Name Sequence Product
size
Specificity
B-V9260
(Upstream)
5'GTG GTA CAA TGC TGT GAA GG3' 800 bp BCMV
B-C10060
(Downstream)
5'GGA ACA ACA ARC ATT GCC GT3' 800 bp BCMV
A-V9144
(Upstream)
5'CTT GGC TCG CTA TGC ATT CG3' 467 bp BCMNV
A-C9611
(Downstream)
5'ATA TTC ATA CCC GCA CCT C3' 467 bp BCMNV
PSbMV-V9350
(Upstream)
5'GGG ATG TGG ACA ATG ATG GA3' 568 bp PSbMV
PSbMV-C9918
(Downstream)
5'TCC AGA AAG CCC TAC TGCC3' 568 bp PSbMV
SMV-V8728
(Upstream)
5'TTT GAC CAC TTG CTT GAG TA3' 544 bp SMV
SMV-C9272
(Downstream)
5'TGC CTT TCA GTA TTT TCG GAG TT3' 544 bp SMV
52. Singleplex RT-PCRSingleplex RT-PCR
((Five Viruses of Quarantine Significance for
India))
ArMV
519 bp
203 bp
BPMV and GFLV
203 bp GFLV
61 bp BPMV
CLRV
283 bp
ToRSV
330 bp
Multiplex RT-PCRMultiplex RT-PCR
(Viruses of Quarantine Significance
for India)
283 bp CLRV
203 bp ArMV
330 bp ToRSV
ArMV, CLRV, ToRSV
CLRV, GFLV, ToRSV
283 bp CLRV
203 bp GFLV
330 bp ToRSV
53. Combination of Serology and PCR Immuno Capture
RT-PCR (IC-RT-PCR)
• one of the biggest problems with PCR assays from plant
tissue is inhibitors.
• immunocapture can be used to remove inhibitors.
• antibodies trap the virus
• detergent decapsidates it
• cDNA synthesized
• scope for routine use
• no RNA extraction
54. Advantages of PCR
• Highly sensitive (can detect picogram quantities of
target nucleic acid)
• Process is automated: very rapid, it takes 2 hrs or less
for the test
• Versatile: can be used for detecting RNA or DNA
• Very useful where ELISA is not effective (viroids,
geminiviruses)
55. Real-time PCR
Real-time PCR monitors the fluorescence emitted during
the reaction as an indicator of amplicon production at
each PCR cycle (in real time) as opposed to the end point
detection
57. * based on the detection and quantitation of
a fluorescent reporter
* the first significant increase in the amount of PCR product
(CT
- threshold cycle) correlates to the initial amount of
target template
Real-time PCR Principles
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61. Three general methods for the quantitative detection:
1. DNA-binding agents (SYBR Green)
2. Hydrolysis probes (TaqMan, Beacons, Scorpions)
3. Hybridisation probes (Light Cycler)
Real-time Principles
62. • Emits a strong fluorescent signal upon binding to
double-stranded DNA.
• During the extension phase, more and more SYBR
Green will bind to the PCR product, resulting in an
increased fluorescence.
• Consequently, during each subsequent PCR cycle
more fluorescence signal will be detected.
I. SYBR Green
(double-stranded DNA binding dye)
73. II. Hydrolysis Probe Chemistry
• Hydrolysis probe is conjugated with a quencher
fluorochrome, which absorbs the fluorescence of the
reporter fluorochrome as long as the probe is intact.
• However, upon amplification of the target sequence, the
hydrolysis probe is displaced and subsequently hydrolyzed
by the Taq polymerase.
• This results in the separation of the reporter and quencher
fluorochrome and consequently the fluorescence of the
reporter fluorochrome becomes detectable.
• During each consecutive PCR cycle this fluorescence will
further increase because of the progressive and
accumulation of free reporter fluorochromes.
74. TaqMan Probes
FRET
DNA Polymerase 5' exonuclease activity
* Tm value 100
C higher than primers
* runs of identical nucleotides (no consecutive Gs)
* G+C content 30-80%
* more Cs than Gs
* no G at the 5' end
ABI Primer Express Software Tutorial (www)
75. Mocellin et al. Trends Mol Med 2003 (www)
DNA Polymerase 5' Exonuclease Activity
104. * General screening prior to moving to probe based assays
* When the PCR system is fully optimized -no primer
dimers or non-specific amplicons, e.g. from genomic DNA
When to Choose SYBR Green
105. * no post-PCR processing of products
(high throughput, low contamination risk)
*not influenced by non-specific amplification
* confirmation of specific amplification by melting point analysis
•amplification can be monitored real-time
* most specific, sensitive and reproducible
* * ultra-rapid cycling (30 minutes to 2 hours)
Real-time PCR Advantages
106. * not ideal for multiplexing
* setting up requires high technical skill and support
* high equipment cost
Real-time PCR Disadvantages
107. FTA Technology
• FTA (Flinders Technology Associates) is a trademark of
Whattman Inc. and is patented in the U.S.
• Cotton-based cellulose membrane containing lyophilized
chemicals that lyses many bacteria and viruses.
• Chemical treatment , unique to whattman that allows for the
rapid isolation and protection of nucleic acid at room
temperature.
• Used for efficient sampling and recovery of viral pathogens
from infected leaf tissue and their-subsequent molecular
analysis for geminiviruses in maize, cassava, tomato, and also
in TMV, PVY and TEV.
108. • Retaining integrity of viral pathogens within the
sampled plant tissues is often a limiting factor,
especially when sample size is large and when
working in regions remote from laboratory
facilities.
Why FTA Technology?
109. Advantages of FTA Cards
• Captures nucleic acid in one easy step.
• Nucleic acid collected on FTA-cards are stable for an year at room
temp.
• Do not require organic solvents in extraction of nucleic acids.
Involves non-organic chemicals in further process of nucleic acid
extraction.
• Do not require refrigeration and centrifugation facility during
complete process.
• Available in variety of configuration to meet application
requirement.
• Suitable for virtually any cell type like blood, culture cells, plant
material, bacteria, plasmids, virus particle, M13 plaque, solid
tissues.
• Total cost of one reaction is approximately 0.75 US$, thus, it is
cost effective technology.
110. FTA Cards in Areas Other than Virology
• Transgenics
• Genomics
• Diagnostics
• Animal identification
• Plasmids screening
• Drug discovery
• Forensic sciences
• Transfusion medicine
112. • DNA Microarrays are small, solid supports onto which the
sequences from thousands of different genes are immobilized/
attached, at fixed locations.
• The supports themselves are usually glass microscope slides
(organo-functional alkoxysilane), the size of two side-by-side
fingers
• but can also be silicon chips or nylon membranes.
• This electronic device is able to map entire genetic material and
can scrutinize tens of thousands of genes at once.
• The DNA is printed, spotted, or actually synthesized directly onto
the support.
DNA Microarrays
113. DNA Microarrays Contd…
• Each spot on an array is associated with a
particular virus/ fungus/bacterium.
• Each color in an array represents either healthy
(control) or diseased (sample) tissue.
• Depending on the type of array used, the
location and intensity of a color will tell us
whether the virus is present in either the
control/ sample DNA.
114. DOT-BLOT
ssDNA on membrane
Hybridized with labeled probe
Autoradiography
DNA-CHIPS
Oligonucleotides on the chip
Tag with fluorescent
dye/radiolabeled
Hybridized with test DNA sample
Check the hybridization for
fluorescence & scanned on
computer/Autoradiography
115. • Durable
• Low background noise
• Many probes can be labeled with different
fluoresces
• Washing -- improve reproducibility
• Flatness, rigidity and transparency -- improve
image acquisition and image processing
• Reusable
Advantages of Glass Slides
117. Gene Chip Instrument System Provides a Complete Solution for
the Analysis of Complex Genetic Information
GeneChip Hybridization
Oven 320
GeneChip Fluidics Station 400 Hewlett-Packard
GeneArray Scanner
119. Helicase Dependent Amplification (HDA)
61 bp BPMV
• No denaturation, helicase does
the job
• Strands of double stranded DNA
are separated by a DNA helicase
• Entire reaction at 65o
C for 11/2
hr
• Primer temperature and
annealing temperature are same
• Thermal cycler/ water bath/
incubator
• Kit: Biohelix
Detection of BPMV using HDA
Bean pod mottle virus, not reported from India
120. Loop Mediated Isothermal Amplification
(LAMP)
Detection of HPV using LAMP
High plains virus, not reported from India
• Four primers recognizing 6 distinct regions
on the target
• Only one enzyme, BST DNA polymerase
• BST DNA polymerase has strand
displacement activity
• Reaction under isothermal condition, 60-
65o
C for 30-60 minutes
• Terminate reaction by incubating at 80o
C for
5 min. or 95o
C for 2 min.
• Thermal Cycler (Heat block)/ Incubator with
hot bonnet
• Turbidity of Magnesium pyrophosphate (by
product) changes after amplification
• Turbidimeter/ visible
• Kit: EIKEN, Chemical Co. Ltd., Japan
121. Serology after PCR
• Thirty years later, the use of serology for detection
assays is still increasing for disease management
applications.
• Increases in the number of assays, formats and the
diversity of pathogens being detected.
• Formats are available for the scientists, farmers.
123. Germplasm Collections Infected with Seed-
transmitted Viruses
IndiaV. radiataULCV
IndiaV. mungo
USAGlycine maxSMV
India (ICRISAT)Arachis hypogaeaPeMoV
USALens culinaris
Canada, France, India, New
Zealand, USA, UK,
Pisum sativumPSbMV
IranV. unguiculataCABMV
IndiaVigna mungo
USAPhaseolus vulgarisBCMV
CountryGermplasmVirus
124. Guidelines for Safe Movement of Germplasm
by Bioversity International (formerly IPGRI)
• Aromatic Plants – Vanilla
• Cereals – Small Grain Temperate Cereals
• Industrial Crops – Sugarcane
• Legumes – Legume
• Roots, Tubers and Aroids – Cassava, Edible Aroid, Potato, Sweet
Potato, Yam
• Temperate Fruits – Grapevine, Small Fruit, Temperate Fruits
• Tree Species – Acacia spp., Eucalyptus spp., Pinus spp.
• Tropical Fruits – Cacao, Citrus, Coconut, Musa spp.
• Vegetables - Allium spp.
Source: http://www.bioversityinternational.org/scientific_information/themes/germplasm_health/
125. Analysis of Risk of Introducing Plant Viruses
along with the Germplasm
• Plant Quarantine Order (Regulation of Import into India) 2003
- Schedule IV, V, VI
• Check-list of Seed-transmitted Viruses of Legumes
• Potential Quarantine Pests of Cereals
• Potential Quarantine Pests of Legumes – being edited
• Check-list of Seed-transmitted Viruses of Non-legumes
• Crop Protection compendium by CAB International
• Plant Viruses Online (http://image.fs.uidaho.edu/vide/refs.htm)
• Descriptions of Plant Viruses (http://www.dpvweb.net/)
• ICTV dB Descriptions
(http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/index.htm)
126. Seed-transmitted Viruses of Legumes Not Known to
Occur in India
1. Artichoke yellow ring spot
virus
12. Lucerne Australian latent virus
2. Bean mild mosaic virus 13. Mulberry ringspot virus
3. Bean pod mottle virus 14. Pea early browning virus
4. Broad bean mottle virus 15. Pea enation mosaic virus
5. Braod bean stain virus 16. Peanut stunt virus
6. Broad bean true mosaic virus 17. Raspberry ring spot virus
7. Cherry leaf roll virus 18. Red clover mosaic virus
8. Clover yellow mosaic virus 19. Red clover vein mosaic virus
9. Cocoa necrosis virus 20. Satsuma dwarf virus
10. Cowpea mottle virus 21. Tomato ring spot virus
11. Cowpea severe mosaic virus 22. Vicia cryptic virus
127. Seed-transmitted Viruses of French Bean
Virus reported world over India On French bean in India
Alfalfa mosaic virus + -
Artichoke yellow ringspot virus - -
Bean common mosaic virus + +
Bean common mosaic necrosis virus + +
Bean mild mosaic viris - -
Bean pod mottle virus - -
Bean yellow mosaic virus + +
Broad bean wilt virus + -
Cacao necrosis virus - -
Cherry leaf roll virus - -
Cowpea mild mottle virus + -
Cowpea severe mosaic virus - -
Cucumber mosaic virus + +
128. Seed-transmitted Viruses of French Bean Contd..
Virus reported world over India On French bean in India
Pea early-browning virus - -
Peanut mottle virus + -
Red clover vein mosaic virus - -
Satsuma dwarf virus - -
Southern bean mosaic virus + -
Soybean mosaic virus + -
Tobacco necrosis virus + -
Tobacco rattle virus + -
Tobacco streak virus + -
Tomato aspermy virus + -
Tomato black ring virus + -
Urd bean leaf crinkle virus + +?
Total 9 (not
present)
20 (not present)
129. Growing-on Test of Legume Germplasm in
Post-entry Quarantine Greenhouse
130. Crop No. of
Samples
Crop No. of
Samples
Glycine spp. 2,268 V. mungo 9
Lathyrus spp. 15 V. radiata 383
Phaseolus spp. 1,599 V. unguiculata 645
Pisum sativum 542 Vicia spp. 117
Vigna spp. 36 V. faba 828
Total 6,442
Legumes Processed against
Seed-transmitted Viruses (2000-2012)
131. Detection of Plant Viruses in Exotic Germplasm Imported
into India (2000-2012)
Virus Crop Source of Import
Alfalfa mosaic
virus
Glycine max AVRDC (Taiwan), IITA (Nigeria), Brazil,
Myanmar, Sri Lanka, USA
Phaseolus vulgaris♣
CIAT (Colombia), Canada, Kenya, USA
Pisum sativum♣
USA
Vigna radiata♣
Japan
V. unguiculata CIAT (Colombia), IITA (Nigeria), USA
Bean common
mosaic virus
G. max♣
AVRDC (Taiwan), IITA (Nigeria), Brazil,
Thailand, USA
P. vulgaris CIAT (Colombia), CIS Hungary, Kenya,
USA
V. radiata AVRDC (Taiwan), Japan, USA
V. subterranea Ghana
Bean common
mosaic necrosis
virus
P. vulgaris CIAT (Colombia), Kenya, Russia
♣
Virus present in India but not recorded on the host on which intercepted
132. Virus Crop Source of Import
Bean yellow mosaic virus Glycine max IITA (Nigeria), Myanmar, USA
Phaseolus
vulgaris
CIAT (Colombia)
Pisum sativum USA
Vicia faba ICARDA (Syria), Bulgaria, Spain
Blackeye cowpea mosaic
virus, now a strain of
BCMV
Vigna
subterranea
Ghana
Broad bean stain virus* Vicia faba ICARDA (Syria), Bulgaria
Brod bean wilt virus V. faba ICARDA (Syria)
P. sativum USA
Cherry leaf roll virus* G. max AVRDC, Sri Lanka, Thailand, USA
P. vulgaris CIAT (Colombia), Sri Lanka
Cowpea aphid-borne
mosaic virus
Glycine max♣
AVRDC (Taiwan), IITA (Nigeria),
Myanmar, Sri Lanka, Thailand, USA
Vigna radiata♣
AVRDC (Taiwan)
V. unguiculata IITA (Nigeria), Eritrea, Guyana,
Philippines, USA
*Virus not yet reported from India; ♣
Virus present in India but not recorded on the host on which intercepted
Detection of Plant Viruses Contd….
133. Detection of Plant Viruses Contd….
Virus Crop Source of Import
Cowpea mosaic virus Vigna radiata♣
USA
V. unguiculata IITA (Nigeria)
Cowpea mottle virus V. unguiculata Philippines
V. subterranea Ghana
Cucumber mosaic virus Glycine max AVRDC (Taiwan), IITA (Nigeria), Brazil,
Myanmar, Sri Lanka, USA
Phaseolus
vulgaris
CIAT (Colombia)
V. radiata USA
V. unguiculata IITA (Nigeria), USA
Grapevine fan leaf virus G. max AVRDC (Taiwan)
Pea seed-borne mosaic
virus
Pisum sativum AVRDC (Taiwan), Australia, Bulgaria, Colombia,
Eritrea, Germany, Holland, Nepal, Russia, Syria,
USA
Vicia faba♣
ICARDA (Syria), Bulgaria, Spain
Raspberry ring spot
virus*
G. max AVRDC (Taiwan), Sri Lanka, Thailand, USA
Southern bean mosaic
virus
G. max♣
AVRDC, IITA (Nigeria), Sri Lanka, Thailand, USA
P. vulgaris♣
CIAT (Colombia)
*Virus not yet reported from India; ♣
Virus present in India but not recorded on the host on which intercepted
134. Interception of Plant Viruses Contd….
Virus Crop Source of Import
Soybean mosaic virus Glycine max AVRDC (Taiwan), IITA (Nigeria), Australia, Brazil,
Hungary, Sri Lanka, Thailand, USA
Phaseolus vulgaris♣
CIAT (Colombia)
Tobacco necrosis virus Pisum sativum♣
USA
Tobacco rattle virus P. vulgaris CIAT (Colombia)
Tobacco ring spot virus G. max IITA (Nigeria), Myanmar
Tobacco streak virus G. max♣
AVRDC (Taiwan), Australia, Brazil, Sri Lanka,
Thailand, USA
P. sativum♣
USA
Vigna unguiculata♣
CIAT (Colombia)
Tomato aspermy virus P. vulgaris CIAT (Colombia)
Tomato black ring virus G. max♣
AVRDC (Taiwan), Brazil, Sri Lanka
P. vulgaris♣
CIAT (Colombia)
Vigna unguiculata♣
IITA (Nigeria)
Tomato ring spot virus* G. max AVRDC (Taiwan), Sri Lanka, Thailand, USA
*Virus not yet reported from India; ♣
Virus present in India but not recorded on the host on which intercepted
138. Variability in Plant Viruses
Alfalfa mosaic virus, Bean yellow mosaic virus
• Numerous strains known
Bean common mosaic virus
• Ten different strains reported
Cherry leaf roll virus
• Wide range of serological variants exist
• Type (cherry) strain, Elm mosaic strain, Rhubarb strain, Golden
elderberry strain Red elder ringspot strain, Dogwood ringspot
strain, Birch strain, Walnut ringspot strain, walnut yellow vein
strain, Blackberry strain and red raspberry strains.
139. Variability in Plant Viruses Contd……
Cowpea aphid- borne mosaic virus
Strains
• European (type) strain, African (neotype) strain, African mild
strain and African vein-banding strain, South African
Passiflora strain, Zimbabwe strain, Brazilian strain and
Moroccan strain.
Serotypes
• Seven distinct CABMV serotypes reported
Pathotypes
• Considerable evidence of pathogenic variability reported
Pea seed-borne mosaic virus
• Four pathotypes viz., P1, P2, P3 and P4 known on pea
140. Southern bean mosaic virus
• Strain B
• Severe bean mosaic strain or Mexican strain
• Resistance-breaking strains
Soybean mosaic virus
• Seven pathotypes representing seven strain groups (G1–G7) in
the United States
• CN-18, a new strain of SMV was reported from Korea
Raspberry ringspot virus
• Many minor variants occur
• Three important strains: The Scottish strain, the type strain; The
English strain, differs from the Scottish strain serologically and
in vector relations; The Lloyd George yellow blotch (LG) strain
Variability in Plant Viruses Contd……
141. Variability in Plant Viruses Contd…….
Tobacco ringspot virus
• Many variants reported, based primarily on differences in
symptomatology
• Many natural antigenic variants also reported
Tobacco streak virus
• Many variants exist
• Number of strains known in India
• Recently found to infect Bt cotton also
142. Tomato black ring virus
• Tomato black ring strain (The type strain)
• Lettuce ringspot strain
• Potato bouquet strain of Köhler
• Potato pseudo -aucuba strain of Köhler
• Beet ringspot strain
• Celery yellow vein strain
Tomato ringspot virus
• Tobacco strain = tobacco ringspot virus No. 2 (The type strain)
• Peach yellow bud mosaic strain
• Grape yellow vein strain
Variability in Plant Viruses Contd……
144. Challenges in Virus Diagnosis in
Plant Quarantine
• Sample size
• Detecting an unknown/ exotic virus
• Part of the planting material to be tested
• Availablity of antisera/ primers/ sequences
• Post-entry quarantine
• Urgency of clearance of the sample
• Conformity to International Standards
145. Technique alone is not enough
We need a strategy covering
Simultaneous detection of
fungi, bacteria, viruses,
nematodes, insect pests,
weeds……
This slide starts a series of illustrations that are useful to explain the SYBR green detection chemistry. Do not belabor each slide. Go through them rapidly, with brief comments as required to clarify the activity of each molecule.
In this slide, Taq has bound to template DNA and is synthesizing a new strand. SYBR Green will bind selectively to the double-stranded DNA.
The larger, yellow sphere is Taq polymerase
The green hexagon is SYBR Green
The multi-colored sticks are nucleotides
As new DNA is made, SYBR Green begins to bind to the double-stranded DNA.
Dye molecules are bound to the length of the DNA molecule and are ready to generate a fluorescent signal.
For SYBR-Green, the opportunity for detection comes at the end of the extension step. Blue light excites the dye and raises it to a higher energy level.
When the dye molecules drop to a lower energy level, they release the energy as fluorescent light.
When the dye molecules drop to a lower energy level, they release the energy as fluorescent light.
Before we can understand the next two detection chemistries, we need to understand a concept called “FRET”, which stands for Fluorescence Resonance Energy Transfer.
Big Picture: with FRET a fluorescent signal becomes possible when two different dye molecules are in close proximity. When one dye becomes excited by a light source it can transfer this excitation energy to a second dye causing it to raise to a higher energy level. When the second dye drops to a lower energy level, it gives off fluorescent light.
A blue light source excites the green dye, raising it to a higher energy level.
When the dye molecule drops to a lower energy level, it releases energy, which is absorbed by the second dye molecule.
The red dye is now raised to a higher energy level.. When it drops to a lower energy level it will give off light.
The red dye emits a red fluorescent signal.
Hybridization probe chemistry for the LightCycler depends on FRET. A pair of hybridization probes are designed to bind to adjacent sequences on the target sequence. One member of the pair is labeled on the 3’-end with a green dye. The other member of the pair is labeled on the 5’-end with a red dye. When the pair of probes anneals to the target sequence...
…the two green and red dyes are adjacent to each other. As long as the two probe molecules remain bound next to each other, FRET can happen. If one or both of the probe molecules becomes unbound, FRET cannot happen.
When both of the hybridization probes are bound, FRET occurs exactly as described in previous series of slides. Here a blue light provides energy to excite the green dye.
As described previously
As described previously.
As described previously
It is important to note, that during the PCR process,the hybridization probes are reused from one cycle to the next. During the extension step, Taq polymerase synthesizes new DNA. When it “bumps” up against a pair of hybridization probes, the probes are displaced.
Taq has now displaced the second hybridization probe. The red and green dyes are now separated. FRET is no longer possible until the probes hybridize during the next annealing step.
As described previously
The LightCycler determines the cycle number where each reaction begins to enter the log-linear phase and matches this value up with the fluorescence data for that sample at that cycle number.
The relationship between these two values is used to calculate how much DNA was made for each reaction.
The LightCycler determines the cycle number where each reaction begins to enter the log-linear phase and matches this value up with the fluorescence data for that sample at that cycle number.
The relationship between these two values is used to calculate how much DNA was made for each reaction.
As mentioned earlier, one of the key LC features is that it is an on-line, real-time instrument. This provides the benefit of:
constant feedback of reaction data
allowing a retrospective data analysis