MolecularMethod of Disease Diagnosis by sanju sah (1).pptx

Molecular methods of disease diagnosis involve analyzing genetic material to detect pathogens, mutations, or biomarkers. Techniques like PCR, DNA sequencing, and microarrays enable precise identification of diseases, even at early stages. These methods offer high sensitivity and specificity, facilitating personalized treatment and improving patient outcomes.

1. Molecular Methods for disease diagnosis By- Sanju Sah St. Xavier’s, College, Maitighar , Kathmandu

2. History of Diseases Diagnostics The impact of microorganisms on human health today is essential, in spite of the significant progress in disease control and understanding. The threat from different microorganisms has changed during the human history as we humans have changed lifestyle, demonstrating the intimate relation between microorganisms and their hosts.

3. History of Diseases Diagnostics The ability of a health care practitioner to efficiently deliver effective care depends upon their ability to accurately identify the cause of the patient’s problem, i.e. make the diagnosis. Over time, our understanding of the mechanistic basis of disease has increased and furthered our ability to make more accurate and specific diagnoses. Early healers needed only to feel the patient’s skin to diagnose “fever”, for example, but with further understanding of different illnesses and their symptoms, doctors gained the ability to distinguish between yellow fever, scarlet fever, and other types of infections.

4. It was the advent of modern microscopy and histopathology, or cellular pathology, in the nineteenth century, however, which enable a tremendous leap forward in our understanding of biology of disease at a cellular level. Today, cell cultures, additional antibiotic sensitivity testing and genetic information from molecular diagnostics provide critical additional information.

5. Traditional typing methods Rely upon isolation and magnification of the microorganism by culture. Organism is then identified by examination of the whole organism or by protein- or lipid- based analysis Phenotypic characteristics such as morphology (including staining), requirements for growth and metabolism of substrates (i.e. biochemical assays). Differentiating capability needs to be determine Phenotypic characteristics are not stable but can be affected by for example the technical handling, changes in gene regulation and gain or loss of plasmids.

6. Limitation of phenotypic methods Inability to grow certain fastidious pathogens Inability to maintain viability of certain pathogens in specimens during transport to laboratory Extensive delay in cultivation and identification of slowly growing pathogens Lack of reliable methods to identify certain organism grown in vitro Use of considerable time and resources in establishing the presence and identity of pathogens in specimens.

7. Molecular diagnosis Molecular diagnosis of human disorders is referred to as the detection of the various pathogenic mutations in DNA and /or RNA samples in order to facilitate detection, diagnosis, sub-classification, prognosis, and monitoring response to therapy. Molecular diagnostics combines laboratory medicine with the knowledge and technology of molecular genetics and has been enormously revolutionized over the last decades, benefiting from the discoveries in the field of molecular biology.

8. Molecular Methods for diagnosis Molecular diagnostics is the use of the molecular biology techniques to: Expand scientific knowledge of natural history of diseases, Identify individuals who are at risk of acquiring specific diseases, Diagnose infectious and other human diseases at the nucleic acid level e.g. at molecular level.

9. Molecular Diagnostics: Emergence The information revolution in molecular biology is permeating every aspect of medical practice The rate of disease gene discovery is increasing exponentially, which facilitates the understanding diseases at molecular level Molecular understanding of disease is translated into diagnostic testing, therapeutics, and eventually preventive therapies 9

10. Molecular Diagnostics: Goal To introduce essential concepts in molecular diagnostics that impact on the identification of novel markers of human diseases To develop and apply useful molecular assays to monitor disease, determine appropriate treatment strategies, and predict disease outcomes. 10

11. History of Molecular Diagnostics 1865 Gregor Mendel, Law of Heredity 1866 Johann Miescher , Purification of DNA 1953 1970 Recombinant DNA Technology 1977 DNA sequencing 1985 In Vitro Amplification of DNA ( PCR ) 2001 The Human Genome Project Watson and Crick, Structure of DNA The Molecular Biology Timeline Sickle Cell Anemia Mutation 1949

12. Molecular Diagnostics Impact Monitor diseases more accurately: Allows for early treatment and better patient care. Determine most appropriate treatment: Reduces or eliminates unnecessary treatment. Reduces or eliminates inadequate treatment. Greater cost effectiveness. Reduce patient morbidity and mortality.

13. Molecular method: Overview Non-nucleic acid-based analytic methods that detect phenotypic traits not detectable by conventional strategies have been developed to enhance bacterial detection, identification and characterization. For laboratory diagnosis of infectious diseases to remain timely and effective, strategies that integrate conventional, nucleic acid based and analytic techniques must continue to evolve.

14. Several methods that analyze microbial DNA or RNA can detect, identify and characterise infectious etiologies. Although technical aspects may differ, all molecular procedures involve the direct manipulation and analysis of genes, Understanding of nucleic acid composition and structure is required for molecular diagnostic tests. Molecular methods are classified into one of three categories: Hybridization Amplification Sequencing and enzymatic digestion of nucleic acids.

15. NUCLEIC ACID HYBRIDIZATION Hybridization methods are based on ability of two nucleic acid strands that have complementary base sequences (i.e. are homologous) to specifically bond with each other and form a double stranded molecule or duplex or hybrid. This duplex formation is driven by consistent manner in which the base adenine always bonds to thymine, while the bases guanine and cytosine always form a bonding pair.

16. Because hybridization requires nucleic acid sequence homology, a positive hybridization reaction between two nucleic acid strands, each from different source, indicates genetic relatedness between the two organisms that donated each of the nucleic acid strands for hybridization reaction

17. Hybridization assays require that one nucleic acid strand (probe) originate from an organism of known identity and other strand (target) originate from an unknown organism to be detected or identified. Positive hybridization identifies the unknown organism as being the same as the probe source organism. With a negative hybridization test, the organism remains undetected or unidentified. The single stranded nucleic acid components used in hybridization may be either RNA or DNA so that DNA-DNA, DNA-RNA and even RNA-RNA duplexes may form, depending on the specific design of the hybridization.

18. Hybridization Steps and Components Production and labeling of single-stranded probe nucleic acid Preparation of single-stranded target nucleic acid Mixture and hybridization of target and probe nucleic acid Detection of hybridization

19. Production and labeling of single-stranded probe nucleic acid Probe design (i.e., probe length and the sequence of nucleic acid bases) depends on the sequence of the intended target nucleic acid. Selection and design of a probe depends on the intended use. In the past, probes produced through a labor intensive recombinant DNA and cloning techniques with the nucleic acid sequence of interest. More recently, probes have been chemically synthesized using instrumentation

20. The design and production of nucleic acid probes is now relatively easy. Although probes may be hundreds to thousands of bases long, oligonucleotide probes (i.e., those 20 to 50 bases long) usually are sufficient for detection of most clinically relevant targets All hybridization tests must have a means to detect or measure the hybridization reaction. Accomplished with the use of a “reporter” molecule attached to the single-stranded nucleic acid probe. Probes may be labeled with a variety of molecules, but most commonly, radioactive (e.g., 32 P, 3 H, 125 I, or 35 S), biotin- avidin , digoxigenin , fluorescent, or chemiluminescent labels

21. Radioactively labeled probes is detected by the emission of radioactivity from the probe-target mixture. Nonradioactive biotinylated labelled probes involves the chemical incorporation of biotin into probe DNA which is then detected by using avidin . Avidin is biotin binding protein that has been conjugated with an enzyme such as horseradish peroxidase . When chromogenic substrate is added, the peroxidase produces a coloured product that can be detected visually or spetrophotometrically .

22. Preparation of Target Nucleic Acid Because hybridization is driven by complementary binding of a homologous nucleic acid sequence between probe and target, the target nucleic acid must have a single strand and the base sequence integrity must be maintained. Failure to meet these requirements results in negative hybridization reactions as a result of factors such as target degradation, insufficient target yield, and the presence of interfering substances such as organic chemicals (i.e., false-negative results).

23. Obtaining target nucleic acid and maintaining its appropriate conformation and sequence can be difficult. The steps in target preparation vary, depending on the organism source of the nucleic acid and the nature of the environment from which the target organism is being prepared (i.e., laboratory culture media; fresh clinical material, such as fluid, tissue, or stool; and fixed or preserved clinical material).

24. Target preparation steps involve enzymatic and/or chemical destruction of the microbial envelope to release target nucleic acid, the removal of contaminating molecules such as cellular components (protein), stabilization of target nucleic acid to preserve structural integrity and, if the target is DNA, denaturation to a single strand, which is necessary for binding to complementary probe nucleic acid. Nucleic acid extraction procedures are optimized to ensure a high degree of purity, integrity, and yield of the desired nucleic acid.

25. Mixture and Hybridization of Target and Probe The ability of the probe to bind the correct target depends on the extent of base sequence homology between the two nucleic acid strands and the environment in which probe and target are brought together. Environmental conditions set the stringency for a hybridization reaction, and the degree of stringency can determine the outcome of the reaction. As stringency increases, the specificity of hybridization increases and as stringency decreases, specificity decreases.

26. Hybridization stringency is most affected by: Salt concentration in the hybridization buffer (stringency increases as salt concentration decreases) Temperature (stringency increases as temperature increases) Concentration of destabilizing agents (stringency increases with increasing concentrations of formamide or urea)

27. Detection of Hybridization The method depends on the reporter molecule used for labeling the probe nucleic acid and on the hybridization format. Hybridization using radioactively labeled probes is visualized after the reaction mixture is exposed to radiographic film (i.e., autoradiography). Hybridization with nonradioactively labeled probes is detected using colorimetry , fluorescence, or chemiluminescence , and detection can be somewhat automated using spectrophotometers, fluorometers , or luminometers , respectively.

28. Hybridization Formats Hybridization reactions can be done using either a: Solution format or Solid support format .

29. Solution Format Probe and target nucleotide strands are placed in a liquid reaction mixture that facilitates duplex formation; hybridization occurs substantially faster than with a solid support format. However, before duplex formation can be detected, the hybridized, labeled probes must be separated from the nonhybridized , labeled probes (i.e., “background noise”). Separation methods includes enzymatic (e.g., S1 nuclease) digestion of single-stranded probes and precipitation of hybridized duplexes, use of hydroxyapatite or charge magnetic microparticles that preferentially bind duplexes, or chemical destruction of the reporter molecule (e.g., acridinium dye) attached to unhybridized probe nucleic acid. After the duplexes have been “purified” from the reaction mixture and the background noise minimized, hybridization detection can proceed by the method appropriate for the type of reporter molecule used to label the probe

30. Solid support format Either probe or target nucleic acids may be attached to a solid support matrix and still be capable of forming duplexes with complementary strands. Various solid support materials and common solid formats exist, including filter hybridizations, southern or northern hybridizations, sandwich hybridizations, and in situ hybridizations.

31. Filter hybridization Target sample is affixed to a membrane (e.g., nitrocellulose or nylon fiber filters) The membrane is then processed to release target DNA from the microorganism and denature it to a single strand. A solution containing labeled probe nucleic acid is used to ‘flood’ the membrane and allow for hybridization to occur. After a series of incubations and washings to remove unbound probe, the membrane is processed for detection of duplexes. An advantage of this method is that a single membrane can hold several samples for exposure to the same probe.

32. Southern hybridization In this method purified nucleic acid which has been digested with specific enzymes to produce several fragments of various sizes is affixed into membrane. Because fragments of different sizes migrate through the porous agarose at different rates, they can be separated over the time they are exposed to the electric field. When electrophoresis is complete, the nucleic acid fragments are stained with florescent dye ethidium bromide so that fragment may be visualized on exposure to UV light. For Southern hybridization the target nucleic acid bands are transferred to membrane that is then exposed to probe nucleic acid.

33. This method allows the determination of which specific target nucleic acid fragment is carrying the base sequence of interest. However, the labor intensity of the procedure precludes its common use in most diagnostic settings.

34. Sandwich hybridization With sandwich hybridization two probes are used One probe is attached to the solid support, is not labeled and via hybridization “captures” the target nucleic acid from the sample to be tested. The presence of this duplex is then detected using a labeled second probe that is then detected using a labeled second probe that is specific for another portion of the target sequence. Sandwiching the target between two probes decreases nonspecific reactions but requires greater number of processing and washing steps. For such formats, plastics microtiter wells coated with probes have replaced filters as the solid support material, thereby facilitating these multiple-step procedure for testing a relatively large number of specimens.

35. In Situ Hybridization Allows a pathogen to be identified in the context of the pathologic lesion being produced. This method uses patient cells or tissues as the solid support phase. Tissue specimens thought to be infected with a particular pathogen are processed maintaining structural integrity of the tissue and cells, yet allows the nucleic acid of the pathogen to be released and denatured to a single strand with the base sequence intact. Technically demanding, yet extremely useful It combines the power of molecular diagnostics with the additional information provided through histopathologic examination

36. AMPLIFICATION METHODS Although hybridization methods are highly specific for organism detection and identification, they are limited by their sensitivity; that is, without sufficient target nucleic acid in the reaction, false-negative results occur. Many hybridization methods require “amplifying” of target nucleic acid by growing target organisms to greater numbers in culture. The requirement for cultivation detracts from the potential for faster detection and identification of the organism using molecular methods.

37. Therefore, the development of molecular amplification techniques that do not rely on organism multiplication has contributed greatly to faster diagnosis and identification while enhancing sensitivity and maintaining specificity. For purposes of discussion, amplification methods are divided into two major categories: methods that use polymerase chain reaction (PCR) technology and assays that are not PCR based

38. Overview of PCR and Derivations PCR- most widely used target nucleic acid amplification method This method combines the principles of complementary nucleic acid hybridization with those of nucleic acid replication applied repeatedly through numerous cycles. Amplifies a single copy of a nucleic acid target, often undetectable by standard hybridization methods, and multiply to 10 7 or more copies in a relatively short period.

39. This provides ample target that can be readily detected by numerous methods. Conventional PCR involves 25 to 50 repetitive cycles, with each cycle comprising three sequential reactions: denaturation of target nucleic acid, primer annealing to single-strand target nucleic acid, and extension of primer -target duplex.

40. Derivations of the PCR Method Multiplex PCR Nested PCR Quantitative PCR RT-PCR Arbitrary primed PCR PCR for nucleotide sequencing.

41. Multiplex PCR More than one primer pair is included in the PCR mixture. This approach offers a couple of notable advantages. First, strategies including internal controls for PCR have been developed. For example, one primer pair can be directed at sequences present in all clinically relevant bacteria (i.e., the control or universal primers) Second, primer pair can be directed at a sequence specific for the particular gene of interest (i.e., the test primers). The control amplicon should always be detectable after PCR; absence of the internal control indicates that PCR conditions were not met, and the test must be repeated. When the control amplicon is detected, absence of the test amplicon can be more confidently interpreted to indicate the absence of target nucleic acid in the specimen rather than a failure of the PCR assay

42. Another advantage of multiplex PCR is the ability to search for different targets using one reaction. Primer pairs directed at sequences specific for different organisms or genes can be put together, avoiding the use of multiple reaction vessels and minimizing the volume of specimen required. For example, multiplexed PCR assays containing primers to detect viral agents that cause meningitis or encephalitis (e.g., herpes simplex virus, enterovirus , West Nile virus) have been used in a single reaction tube. A limitation of multiplex PCR is that mixing different primers can cause some interference in the amplification process. Optimizing multiplex PCR conditions can be difficult, especially as the number of different primer pairs included in the assay increases.

43. Nested PCR Involves the sequential use of two primer sets. The first set is used to amplify a target sequence. Amplicon obtained is then used as the target sequence for a second amplification using primers internal to those of the first amplicon . Advantage - extreme sensitivity and confirmed specificity without the need for using probes. Because production of the second amplicon requires the presence of the first amplicon , production of the second amplicon automatically verifies the accuracy of the first amplicon . Problem – procedure requires open manipulations of amplified DNA that is readily aerosolized and capable of contaminating other reaction vials

44. Arbitrary primed PCR Uses short (random) primers Not specifically complementary to a particular sequence of a target DNA. Although these primers are not specifically directed, their short sequence (approximately 10 nucleotides) ensures that they randomly anneal to multiple sites in a chromosomal sequence. On cycling, the multiple annealing sites result in the amplification of multiple fragments of different sizes. Theoretically, strains with similar nucleotide sequences have similar annealing sites and thus produce amplified fragments (i.e., amplicons ) of similar sizes. Therefore, by comparing fragment migration patterns after agarose gel electrophoresis, the examiner can judge strains or isolates to be the same, similar, or unrelated.

45. Quantitative PCR An approach that combines the power of PCR for the detection and identification of infectious agents with the ability to quantitate the actual number of targets originally in the clinical specimen. The ability to quantitate “infectious burden” has tremendous implications for studying and understanding the disease state (e.g., acquired immunodeficiency syndrome [AIDS]), the prognosis of certain infections, and the effectiveness of antimicrobial therapy.

46. Reverse transcription PCR (RT-PCR) Amplifies an RNA target. Because many clinically important viruses have genomes composed of RNA rather than DNA (e.g., the human immunodeficiency virus [HIV], hepatitis B virus), the ability to amplify RNA greatly facilitates laboratory-based diagnostic testing for these infectious agents. Reverse transcription includes a unique initial step that requires the use of the enzyme reverse transcriptase to direct the synthesis of DNA from the viral RNA template, usually within 30 minutes. Once the DNA has been produced, relatively routine PCR technology is applied to obtain amplification.

47. Real-Time PCR Most conventional PCR-based tests used in clinical laboratories were developed in-house and required dedicated laboratory space to control or reduce cross-contamination that produced false-positive results. Conventional PCR assays also require multiple manipulations, including initial amplification of target nucleic acid, detection of

48. Digital PCR ( dPCR ) an emerging real-time method that is a modification of the traditional polymerase chain reaction. In traditional PCR, multiple target sequences are amplified in a single-reaction cuvette or well. Digital PCR separates individual nucleic acid samples within a single specimen into separate regions or droplets. Each region or droplet within the sample will either contain no molecule, a single molecule, or a negative or positive reaction.

49. Therefore, the quantitation of the amplification is based on counting the regions that contain a positive amplified product. The quantitation is not based on exponential amplification in comparison to the starting quantity of the target therefore eliminates errors associated with rate of amplification changes that are affected by interfering substances and the use of a standard curve. Digital PCR provides a possible resolution for the detection of infectious agents or pathogens that are present in very low numbers in biological samples.

50. SEQUENCING AND ENZYMATIC DIGESTION OF NUCLEIC ACIDS The nucleotide sequence of a microorganism’s genome is the blueprint for the organism. Therefore, molecular methods that elucidate some part of a pathogen’s genomic sequence provide a powerful tool for diagnostic microbiology. Other methods, either used independently or in conjunction with hybridization or amplification procedures, can provide nucleotide sequence information to detect, identify, and characterize clinically relevant microorganisms. These methods include nucleic acid sequencing and enzymatic digestion and electrophoresis of nucleic acids.

51. Nucleic Acid Sequencing Nucleic acid sequencing involves methods that determine the exact nucleotide sequence of a gene or gene fragment obtained from an organism.

52. Nucleotide sequences obtained from a microorganism can be compared with an ever-growing gene sequence database for: Detecting and classifying previously unknown human pathogens Identifying various known microbial pathogens and their subtypes Determining which specific nucleotide changes resulting from mutations are responsible for antibiotic resistance Identifying sequences or cassettes of genes that have moved from one organism to another Establishing the relatedness between isolates of the same species

53. Before the development of rapid and automated methods, DNA sequencing was a laborious task only undertaken in the research setting. However, determining the sequence of nucleotides in a segment of nucleic acid from an infectious agent can be done rapidly using amplified target from the organism and an automated DNA sequencer. Because sequence information can now be rapidly produced, DNA sequencing has entered the arena of diagnostic microbiology.

54. Identification of microorganisms using PCR in conjunction with automated sequencing is slowly making its way into clinical microbiology laboratories presently, such molecular analyses are limited for the most part to research-oriented laboratories. It is becoming quite clear that combinations of phenotypic and genotypic characterization are most successful in identifying a variety of microorganisms for which identification is difficult such as the speciation of Nocardia , mycobacteria , and organisms that commercial automated instruments fail to identify or correctly identify.

55. Enzymatic Digestion and Electrophoresis of Nucleic acids This is less exacting methods of identifying and characterizing microorganism than is nucleic acid sequencing. However this methods still provide valuable information for diagnosis and control of infectious diseases. Enzymatic digestion of DNA is accomplished using restrictio endonuclease enzyme. Each restriction endonuclease enzyme recognizes a specific nucleotide sequence known as restriction site. Once restriction site is recognized, it catalyses the digestion of nucleic acid strand at that site.

56. The number and size of fragments produced by enzyme digestion depends on length of nucleic acid being digested, nucleotide sequence of the strand being digested and particular enzyme used for digestion. Following digestion, fragments are subjected to agarose gel electrophoresis, which allow them to be separated according to their size differences.

57. During electrophoresis all nucleic acid fragments of the same size comigrate as a single band. For many digestion, electrophoresis results in the separation of several different fragment sizes. The nucleic acid bands in agarose gel are stained with the fluorescent dye ethidium bromide, which allow them to be visualized on exposure to UV light.

58. The pattern obtained after gel electrophoresis are referred to as restriction pattern, and difference between microorganism restriction pattern are known as restriction fragment length polymorphisms ( RFLPs ). Because RFLPs reflect differences or similarities in nucleotide sequences, Restriction Enzyme Analysis methods can be used for organism identification, and for establishing strain relatedness within same species.