Polymerase Chain Reaction in Infectious diseases

Polymerase chain reaction in infectious diseases basics:

Dr.T.V.Rao MD 1 Dr.T.V.Rao MD Polymerase chain reaction in infectious diseases basics

Dr.Kary Banks Mullis discovery of pcr:

Dr.Kary Banks Mullis received a Nobel Prize in chemistry in 1993, for his invention of the polymerase chain reaction (PCR). The process, which Kary Mullis conceptualized in 1983, is hailed as one of the monumental scientific techniques of the twentieth century. Dr.Kary Banks Mullis discovery of pcr Dr.T.V.Rao MD 2

Molecular microbiology :

Molecular microbiology is the branch of microbiology devoted to the study of the molecular principles of the physiological processes involved in the life cycle of prokaryotic and eukaryotic microorganisms such as bacteria, viruses unicellular algae fungi, and protozoa. This includes gene expression and regulation, genetic transfer, the synthesis of macromolecules, sub-cellular organization, cell to cell communication, and molecular aspects of pathogenicity and virulen ce. Molecular microbiology Dr.T.V.Rao MD 3

Molecular biology deals with:

Molecular microbiology is primarily involved in the interactions between the various cell systems of microorganisms including the interrelationship of DNA, RNA and protein biosynthesis and the manner in which these interactions are regulated . Molecular biology deals with Dr.T.V.Rao MD 4

Molecular methods are revolutionizing:

Molecular methods are revolutionizing The use of molecular biology techniques, such as nucleic acid probing and amplification, provides the potential for revolutionizing how we diagnose infecting pathogens and determining the relation between nosocomial isolates. In clinical microbiology, this means that we will be able to detect smaller amounts of DNA or RNA of pathogens than is currently possible, that the time required to identify and determine the antimicrobial susceptibility of slow-growing pathogens will be dramatically reduced, and that the diagnosis of nonculturable organisms will become possible. Dr.T.V.Rao MD 5

Molecular methods in Diagnosis :

Molecular methods in Diagnosis The introduction of molecular methods will not only depend on their performance for each individual microorganism, but also on the clinical relevance of the diagnostic question asked, the prevalence of the clinical problem and whether the new methods are added to the procedures in use or will replace them . Therefore no general rules can be proposed, strategies have to be elaborated for each infectious agent or clinical syndrome. Dr.T.V.Rao MD 6

Why use a molecular test to diagnose an infectious disease?:

Need an accurate and timely diagnosis Important for initiating the proper treatment Important for preventing the spread of a contagious disease Why use a molecular test to diagnose an infectious disease? Dr.T.V.Rao MD 7

When we really need molecular methods ?:

Molecular diagnosis is most appropriate for infectious agents that are difficult to detect, identify, or test for susceptibility in a timely fashion with conventional methods. When we really need molecular methods ? Dr.T.V.Rao MD 8

Molecular biology is emerging in diagnostic method:

Molecular biology is emerging in diagnostic method Diagnostic microbiology is in the midst of a new era . Rapid nucleic acid amplification and detection technologies are quickly displacing the traditional assays based on pathogen phenotype rather than genotype. The polymerase chain reaction (PCR) has increasingly been described as the latest gold standard for detecting some microbes, but such claims can only be taken seriously when each newly described assay is suitably compared to its characterized predecessors Dr.T.V.Rao MD 9

Leading uses for nucleic acid based tests:

Nonculturable agents Human papilloma virus Hepatitis B virus Fastidious, slow-growing agents Mycobacterium tuberculosis Legionella pneumophila Highly infectious agents that are dangerous to culture Francisella tularensis Brucella species Coccidioidis immitis Leading uses for nucleic acid based tests Dr.T.V.Rao MD 10

Leading uses for nucleic acid based tests:

In situ detection of infectious agents Helicobacter pylori Toxoplasma gondii Agents present in low numbers HIV in antibody negative patients CMV in transplanted organs Organisms present in small volume specimens Intra-ocular fluid Forensic samples Leading uses for nucleic acid based tests Dr.T.V.Rao MD 11

Molecular methods are necessary if the traditional methods provide poor results ?:

12 Molecular methods are necessary if the traditional methods provide poor results ? Microscopy gives false positive results - - T.vaginalis, N.gonorrhoeae Intracellular pathogens – viruses, M.genitalium Low sensitivity – Chlamydia sp.,Neisseria sp. Seropositivity is common – Chlamydia sp . Subtyping is mandatory – HSV, HPV, HCV Microbial growth is slow – M. tuberculosis The 7th Baltic Congress in Laboratory Medicine, Pärnu 11.09.2004 Dr.T.V.Rao MD

Molecular diagnostics how it works?:

Every organism contains some unique , species specific DNA sequences Molecular diagnostics makes the species specific DNA visible Molecular diagnostics how it works? Dr.T.V.Rao MD 13 The 7th Baltic Congress in Laboratory Medicine, Pärnu 11.09.2004

Understanding the basis of polymerase chain reaction:

Understanding the basis of polymerase chain reaction Dr.T.V.Rao MD 14

DNA Molecule:

DNA Molecule Adenine Thymine Guanine Cytosine Dr.T.V.Rao MD 15

Outline:

1. DNA 2. PCR Targets Denaturing Primers Annealing Cycles Requirements Outline Dr.T.V.Rao MD 16

DNA structure:

DNA structure In double stranded linear DNA, 1 end of each strand has a free 5’ carbon and phosphate and 1 end has a free 3’ OH group. The two strands are in the opposite orientation with respect to each other ( antiparallel ). Adenines always base pair with thymine's (2 hydrogen bonds) and guanines always base pair with cytosine's (3 hydrogen bonds) Dr.T.V.Rao MD 17

DNA:

DNA is a nucleic acid that is composed of two complementary nucleotide building block chains. The nucleotides are made up of a phosphate group, a five carbon sugar, and a nitrogen base. DNA Dr.T.V.Rao MD 18

DNA:

DNA has four nitrogen bases. Two are purines ( 2 ringed base ) Adenine ( A ), Guanine ( G ) Two are pyrimidine's ( 1 ringed base ) Cytosine ( C ), Thymine ( T ) DNA Dr.T.V.Rao MD 19

DNA:

These four bases are linked in a repeated pattern by hydrogen bonding between the nitrogen bases. The linking of the two complementary strands is called hybridization. DNA Dr.T.V.Rao MD 20

DNA:

A purine always links with a pyrimidine base to maintain the structure of DNA. Adenine ( A ) binds to Thymine ( T ), with two hydrogen bonds between them. Guanine ( G ) binds to Cytosine ( C ), with three hydrogen bonds between them. DNA Dr.T.V.Rao MD 21

DNA:

Example of bonding pattern. Primary strand CC G AA T GGG A T G C GG C TT A CCC T A C G Complementary strand DNA Dr.T.V.Rao MD 22

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23 Molecular diagnostics is a set of methods to study primary structure (sequence) of DNA Hybridization with complementary sequences Amplification (synthesis) of species specific sequences PCR – polymerase chain reaction The 7th Baltic Congress in Laboratory Medicine, Pärnu 11.09.2004 -A-A-T-T-C-G-C-G-A-T-G- - T-T-A-A-G-C-G-C-T-A-C - -A-A-T-T-C-G-C-G-A-T-G- -A-A-T-T-C-G-C-G-A-T-G- -A-A-T-T-C-G-C-G-A-T-G- -A-A-T-T-C-G-C-G-A-T-G- -A-A-T-T-C-G-C-G-A-T-G- Dr.T.V.Rao MD

PCR:

PCR is a technique that takes a specific sequence of DNA of small amounts and amplifies it to be used for further testing. PCR Dr.T.V.Rao MD 24

PCR Requirements:

Magnesium chloride: .5-2.5mM Buffer: pH 8.3-8.8 dNTPs: 20-200µM Primers: 0.1-0.5µM DNA Polymerase: 1-2.5 units Target DNA:  1 µg PCR Requirements Dr.T.V.Rao MD 25

PCR Targets :

The targets in PCR are the sequences of DNA on each end of the region of interest, which can be a complete gene or small sequence. PCR Targets Dr.T.V.Rao MD 26

PCR Targets:

The number of bases in the targets can vary. TT AA GG C T C G A . . . . AA TT GG TT AA The . . . . Represents the middle DNA sequence, and does not have to be known to replicate it. PCR Targets Dr.T.V.Rao MD 27

PCR Denaturing:

Denaturing is the first step in PCR, in which the DNA strands are separated by heating to 95°C . PCR Denaturing Dr.T.V.Rao MD 28

PCR Primers:

Primers range from 15 to 30 nucleotides, are single-stranded, and are used for the complementary building blocks of the target sequence . Primers range from 15 to 30 nucleotides, are single-stranded, and are used for the complementary building blocks of the target sequence . A primer for each target sequence on the end of your DNA is needed. This allows both strands to be copied simultaneously in both directions. PCR Primers Dr.T.V.Rao MD 29

PCR Primers:

PCR Primers TT AA C GG CC TT AA . . . TTT AAA CC GG TT AA TT G CC GG AA TT . . . . . . . . . .> and <. . . . . . . . . . AAA TTT GG CC AA TT AA C GG CC TT AA . . . TTT AAA CC GG TT Dr.T.V.Rao MD 30

PCR Primers:

The primers are added in excess so they will bind to the target DNA instead of the two strands binding back to each other. PCR Primers Dr.T.V.Rao MD 31

PCR Annealing :

Annealing is the process of allowing two sequences of DNA to form hydrogen bonds. The annealing of the target sequences an primers is done by cooling the DNA to 55°C. PCR Annealing Dr.T.V.Rao MD 32

PCR Taq DNA Polymerase:

Taq stands for Thermus aquaticus, which is a microbe found in 176°F hot springs in Yellow Stone National Forest . Taq produces an enzyme called DNA polymerase , that amplifies the DNA from the primers by the polymerase chain reaction, in the presence of Mg. PCR Taq DNA Polymerase Dr.T.V.Rao MD 33

ESTABLISHMENT OF A PCR LABORATORY:

ESTABLISHMENT OF A PCR LABORATORY To perform PCR for the repetitive detection of a specific sequence, three distinct laboratory areas are required. The specific technical operations, reagents ,and personnel considerations Dr.T.V.Rao MD 34

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35 PCR laboratory Sample handling DNA preparation Clean room Stock solutions Laboratory Mixing site Thermocycler Amplification Detection Documentation QC & QA Quality control & assurance R & D ( Research and development) Alternatives: - commercial kits - robots + kits No alternative Dr.T.V.Rao MD

PCR in Clinical Microbiology:

PCR in Clinical Microbiology Molecular detection has mostly come to the clinical microbiology laboratory in the form of PCR technology, initially involving single round or nested procedures with detection by gel electrophoresis. Dr.T.V.Rao MD 36

Helps Rapid Detection timely diagnosis can save several lives:

Helps Rapid Detection timely diagnosis can save several lives Polymerase chain reaction (PCR) techniques have led the way into this new era by allowing rapid detection of microorganisms that were previously difficult or impossible to detect by traditional microbiological methods. Dr.T.V.Rao MD 37

Understanding the pcr cycle :

Dr.T.V.Rao MD 38 Understanding the pcr cycle

Slide 39:

Isolation of Nucleic Acids Goals : removal of proteins DNA vs RNA isolation of a specific type of DNA (or RNA) Types of Methods : differential solubility ‘adsorption’ methods density gradient centrifugation Types of DNA : genomic (chromosomal) organellar (satellite) plasmid (extra-chromosomal) phage/viral (ds or ss ) complementary (mRNA) General Features : denaturing cell lysis (SDS, alkali, boiling, chaotropic )  enzyme treatments protease RNase ( DNase -free) DNase ( RNase -free) 39

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High MW Genomic DNA Isolation Typical Procedure Cell Lysis 0.5% SDS + proteinase K (55 o several hours) Phenol Extraction gentle rocking several hours Ethanol Precipitation RNAse followed by proteinase K Repeat Phenol Extrac-tion and EtOH ppt EtOH Precipitation 2-2.5 volumes EtOH , -20 o high salt, pH 5-5.5 centrifuge or ‘spool’ out 40

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High MW Genomic DNA Isolation Typical Procedure Cell Lysis 0.5% SDS + proteinase K (55 o several hours) Phenol Extraction gentle rocking several hours Ethanol Precipitation RNAse followed by proteinase K Repeat phenol extrac-tion and EtOH ppt Phenol Extraction mix sample with equal volume of sat. phenol soln retain aqueous phase optional chloroform/ isoamyl alcohol extraction(s)  aqueous phase (nucleic acids)  phenol phase (proteins) 41

PCR:

PCR The PCR reaction has three basic steps Denature – when you denature DNA, you separate it into single strands (SS). In the PCR reaction, this is accomplished by heating at 95 0 C for 15 seconds to 1 minute. The SS DNA generated will serve as templates for DNA synthesis. Anneal – to anneal is to come together through complementary base-pairing (hybridization). During this stage in the PCR reaction the primers base-pair with their complementary sequences on the SS template DNA generated in the denaturation step of the reaction. Dr.T.V.Rao MD 42

PCR:

PCR The primer concentration is in excess of the template concentration. The excess primer concentration ensures that the chances of the primers base-pairing with their complementary sequences on the template DNA are higher than that of the complementary SS DNA templates base-pairing back together. The annealing temperature used should ensure that annealing will occur only with DNA sequences that are completely complementary. WHY? The annealing temperature depends upon the lengths and sequences of the primers. The longer the primers and the more Gs and Cs in the sequence, the higher the annealing temperature. WHY? The annealing time is usually 15 seconds to 1 minute. Dr.T.V.Rao MD 43

PCR Cycles:

PCR Cycles Dr.T.V.Rao MD 44

PCR:

Most PCR reaction use 25 to 30 of these cycles to amplify the target DNA up to a million times the starting concentration. PCR Dr.T.V.Rao MD 45

PCR Cycles:

PCR Cycles Dr.T.V.Rao MD 46

PCR:

PCR Extension – during this stage of the PCR reaction, the DNA polymerase will use dNTPs to synthesize DNA complementary to the template DNA. To do this DNA polymerase extends the primers that annealed in the annealling step of the reaction. The temperature used is 72 0 C since this is the optimum reaction temperature for the thermostable polymerase that is used in PCR. Why is a thermostable polymerase used? The extension time is usually 15 seconds to 1 minute. The combination of denaturation, annealing, and extension constitute 1 cycle in a PCR reaction. Dr.T.V.Rao MD 47

PCR Cycles:

PCR Cycles Dr.T.V.Rao MD 48

PCR Cycles:

PCR Cycles Dr.T.V.Rao MD 49

PCR Cycles:

PCR Cycles Dr.T.V.Rao MD 50

PCR Cycles Review:

Denaturalization: 94°- 95°C Primer Annealing: 55°- 65°C Extension of DNA: 72° Number of Cycles: 25-40 PCR Cycles Review Dr.T.V.Rao MD 51

Leading uses for nucleic acid based tests:

Leading uses for nucleic acid based tests Differentiation of antigenically similar agents May be important for detecting specific virus genotypes associated with human cancers (Papilloma viruses) Antiviral drug susceptibility testing May be important in helping to decide anti-viral therapy to use in HIV infections Non-viable organisms Organisms tied up in immune complexes Dr.T.V.Rao MD 52

Leading uses for nucleic acid based tests:

Molecular epidemiology To identify point sources for hospital and community-based outbreaks To predict virulence Culture confirmation Leading uses for nucleic acid based tests Dr.T.V.Rao MD 53

Applications of PCR:

The swab specimens can be stored 2-30°C for 4 days or frozen at -20°C. The urine samples are refrigerated at 2-8°C or stored at -20°C. A target sequence is chosen for both, amplified with polymerase, and then evaluated with an enzyme immunoassay. Applications of PCR Dr.T.V.Rao MD 54

Target amplification:

Target amplification requires that the DNA to be tested for be amplified, i.e., the number of copies of the DNA is increased. To understand this we must first review the activity of the enzyme, DNA polymerase, that is used to amplify the DNA. Target amplification Dr.T.V.Rao MD 55

Polymerase template and primer requirements:

DNA polymerase cannot initiate synthesis on its own. It needs a primer to prime or start the reaction. The primer is a single stranded piece of DNA that is complementary to a unique region of the sequence to be amplified. Polymerase template and primer requirements Dr.T.V.Rao MD 56

PCR reactions in the lab:

PCR reactions in the lab We will be doing two different PCR reactions in the lab. For the first PCR reaction we will be using what are called consensus sequence primers. These are primers that will bind to unique regions of the 16S ribosomal genes found in all bacteria. The sequences of these primers are not unique to a specific kind of bacteria, but they are unique to a conserved region (consensus sequence) of DNA found in the 16S ribosomal genes of all bacteria. They will be used to amplify a portion of the 16S ribosomal gene of an unknown bacteria. Dr.T.V.Rao MD 57

PCR reactions in the lab:

PCR reactions in the lab The sequence of the amplified DNA will be determined. The identity of the unknown will be determined by searching the DNA sequence databases. Note that that DNA of all bacteria should be amplified and yield a product using these consensus primers. For the second PCR reaction we will be using primers that are unique to the genes that encode the shiga-like toxin produced by EHEC. Only the DNA of those bacteria that carry the shiga-like toxin gene will be amplified and yield a product when using these primers. For diagnostic purposes, only the second type of PCR, in which primers unique to a single type of organism or gene are used, is practical. Dr.T.V.Rao MD 58

What are the advantages of using a molecular test?:

What are the advantages of using a molecular test? High sensitivity Can theoretically detect the presence of a single organism High specificity Can detect specific genotypes Can determine drug resistance Can predict virulence Speed Quicker than traditional culturing for certain organisms Dr.T.V.Rao MD 59

Applications of PCR in optimal diagnosis in infections:

Neisseria gonorrhea and Chlamydia trachomatis are two of the most common sexually transmitted diseases. The infections are asymptomatic and can lead to pelvic inflammatory disease, salpingitis in women, epididymitis in men, infertility, and ectopic pregnancy. Applications of PCR in optimal diagnosis in infections Dr.T.V.Rao MD 60

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61 The 7th Baltic Congress in Laboratory Medicine, Pärnu 11.09.2004 Advantages Molecular methods High sensitivity and specificity Detects pathogen, not immune response Quick results High transport toleration In-house (home-brew) PCR methods Cost effective High sensitivity High quality Fast implementation of scientific discoveries Customer friendly R&D is absolutely necessary Dr.T.V.Rao MD

What are the advantages of using a molecular test?:

Simplicity Some assays are now automated What are the advantages of using a molecular test? Dr.T.V.Rao MD 62

What are the disadvantages of using a molecular tests ?:

What are the disadvantages of using a molecular tests ? Expensive So specific that must have good clinical data to support infection by that organism before testing is initiated. Will miss new organisms unless sequencing is done as we will be doing in the lab for our molecular unknowns (not practical in a clinical setting). May be a problem with mixed cultures – would have to assay for all organisms causing the infection. Dr.T.V.Rao MD 63

What are the disadvantages of using a molecular test?:

T oo sensitive? Are the results clinically relevant? What are the disadvantages of using a molecular test? Dr.T.V.Rao MD 64

Pcr to RT pcr :

Pcr to RT pcr Use of PCR in the field of molecular diagnostics has increased to the point where it is now accepted as the standard method for detecting nucleic acids from a number of sample and microbial types. However, conventional PCR was already an essential tool in the research laboratory. Real-time PCR has catalyzed wider acceptance of PCR because it is more rapid, sensitive and reproducible, while the risk of carryover contamination is minimized Dr.T.V.Rao MD 65

OVERVIEW of RT - PCR:

OVERVIEW of RT - PCR tissue extract RNA copy into cDNA (reverse transciptase) do real-time PCR analyze results Dr.T.V.Rao MD 66

Need for novel methods in diagnosis of Infections:

Identification of the infectious agent(s) is essential to provide an accurate diagnosis, appropriately manage patient care and in certain cases reduce the risk of transmission within the community and health care settings. To meet these challenges, innovative technologies have been developed that detect single pathogens, multiple syndrome related pathogens and genotypic drug resistance Need for novel methods in diagnosis of Infections Dr.T.V.Rao MD 67

Our vision to future diagnosis of infectious diseases :

Our vision to future diagnosis of infectious diseases With the ability to test for an unlimited number of potential pathogens simultaneously, next-generation sequencing has the potential to revolutionize infectious diseases diagnostics In the microbiology laboratory, this technology will likely replace the traditional “one test, one bug” approach to pathogen diagnostics The deep sequence information being generated  is rapidly surpassing our capacity to analyze the data and will necessitate the development of highly parallel computational frameworks, such as cloud computing In adapting this technology for clinical diagnostics, interpretation of data, appropriate quality control standards, and fulfilling regulatory requirements will be critical One powerful application of next-generation sequencing is  discovery of novel pathogens that may be associated with acute or chronic illnesses Dr.T.V.Rao MD 68

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Advantages Molecular methods High sensitivity and specificity Detects pathogen, not immune response Quick results High transport toleration In-house (home-brew) PCR methods Cost effective High sensitivity High quality Fast implementation of scientific discoveries Customer friendly R&D is absolutely necessary Dr.T.V.Rao MD 69

Molecular methods have limitations:

Molecular methods have limitations However, because of their high specificity, molecular methods will not detect newly emerging resistance mechanisms and are unlikely to be useful in detecting resistance genes in species where the gene has not been observed previously. Furthermore, the presence of a resistance gene does not mean that the gene will be expressed, and the absence of a known resistance gene does not exclude the possibility of resistance from another mechanism. Phenotypic antimicrobial susceptibility testing methods allow laboratories to test many organisms and detect newly emerging as well as established resistance patterns. Dr.T.V.Rao MD 70

Diagnostic microbiology changing from phenotypic methods to Molecular methods:

Diagnostic microbiology changing from phenotypic methods to Molecular methods In hospital epidemiology, the use of such techniques has already provided tests with exceptional discriminatory power. Molecular techniques allow more efficient typing of all pathogens, and permit discrimination between strains of organisms that were previously phenotypically identical or uncharacterizable. Currently, cost and complexity limit the applicability of these techniques ; however, they are likely to be developed for routine laboratory use in the next decade, and their impact will be considerable. Dr.T.V.Rao MD 71

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Created by Dr. T.V.Rao MD for ‘e’ learning resources Microbiologists in the Developing World Email doctortvrao@gmail.com Dr.T.V.Rao MD 73