POLYMERASE CHAIN REACTION

IN INFECTIOUS DISEASES BASICS Dr.T.V.Rao MD

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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.
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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 DR.T.V.RAO MD ce.

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 .

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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.

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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.

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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
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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.
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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

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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

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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

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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, Prnu 11.09.2004
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MOLECULAR DIAGNOSTICS HOW IT WORKS?

Every organism contains some unique,species specific DNA sequences Molecular diagnostics makes the species specific DNA visible

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UNDERSTANDING THE BASIS OF POLYMERASE CHAIN REACTION

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DNA MOLECULE
Adenine Thymine

Guanine
Cytosine

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OUTLINE
1. DNA 2. PCR Targets Denaturing Primers

Annealing
Cycles

Requirements

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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)

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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.
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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 )

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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.
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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.

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DNA
Example of bonding pattern.

Primary strand
CCGAATGGGATGC

GGCTTACCCTACG
Complementary strand

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Molecular diagnostics is a set of methods to study primary structure (sequence) of DNA

Hybridization with complementary sequences
-A-A-T-T-C-G-C-G-A-T-G- T-T-A-A-G-C-G-C-T-A-CAmplification (synthesis) of species specific sequences

PCR polymerase chain reaction -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-GThe 7th Baltic Congress in Laboratory Medicine, Prnu 11.09.2004
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PCR
PCR is a technique that takes a specific sequence of DNA of small amounts and amplifies it to be used for further testing.
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PCR REQUIREMENTS
Magnesium chloride: .5-2.5mM

Buffer: pH 8.3-8.8
dNTPs: 20-200M Primers: 0.1-0.5M DNA Polymerase: 1-2.5 units Target DNA: 1 g

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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.
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PCR TARGETS
The number of bases in the targets can vary. TTAAGGCTCGA . . . . AATTGGTTAA The . . . . Represents the middle DNA sequence,

and does not have to be known to replicate it.

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PCR DENATURING
Denaturing is the first step in PCR, in which the DNA strands are separated by heating to 95C.
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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.

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PCR PRIMERS
TTAACGGCCTTAA . . . TTTAAACCGGTT
AATTGCCGGAATT . . . . . . . . . .>

and
<. . . . . . . . . . AAATTTGGCCAA

TTAACGGCCTTAA . . . TTTAAACCGGTT
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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.
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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 55C.
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PCR TAQ DNA POLYMERASE

Taq stands for Thermus aquaticus, which is a microbe found in 176F 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.

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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
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PCR laboratory
Sample handling DNA preparation

QC & QA
Quality control & assurance No alternative Detection Documentation

Laboratory Mixing site

Thermocycler Amplification

Clean room Stock solutions

R&D
(Research and development)

Alternatives: - commercial kits - robots + kits

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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.

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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.
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UNDERSTANDING THE PCR CYCLE

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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 (extrachromosomal) 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)
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High MW Genomic DNA Isolation

Typical Procedure
1 Cell Lysis

EtOH Precipitation
2-2.5 volumes EtOH, -20o high salt, pH 5-5.5 centrifuge or spool out

0.5% SDS + proteinase K (55o several hours)

2 Phenol Extraction

gentle rocking several hours

3 Ethanol Precipitation 4 RNAse followed by proteinase K 5 Repeat Phenol Extrac-tion and EtOH ppt

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High MW Genomic DNA Isolation

Typical Procedure
1 Cell Lysis

Phenol Extraction
mix sample with equal volume of sat. phenol soln retain aqueous phase optional chloroform/isoamyl alcohol extraction(s)

0.5% SDS + proteinase K (55o several hours)

2 Phenol Extraction

gentle rocking several hours

3 Ethanol Precipitation 4 RNAse followed by proteinase K 5 Repeat phenol extrac-tion and EtOH ppt

aqueous phase (nucleic acids)

phenol phase (proteins)

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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.

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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?
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The annealing time is usually 15 seconds to 1 minute.

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PCR CYCLES

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PCR
Most PCR reaction use 25 to 30 of these cycles to amplify the target DNA up to a million times the starting concentration.
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PCR CYCLES

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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.
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PCR CYCLES

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PCR CYCLES

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PCR CYCLES

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PCR CYCLES REVIEW

Denaturalization: 94- 95C Primer Annealing: 5565C Extension of DNA: 72 Number of Cycles: 25-40

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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

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LEADING USES FOR NUCLEIC ACID BASED TESTS

Molecular epidemiology

To identify point sources for hospital and communitybased outbreaks

To predict virulence Culture confirmation

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APPLICATIONS OF PCR
The swab specimens can be stored 2-30C for 4 days or frozen at -20C. The urine samples are refrigerated at 2-8C or stored at -20C. A target sequence is chosen for both, amplified with polymerase, and then evaluated with an enzyme immunoassay.
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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.

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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.
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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.

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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.
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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

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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.

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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 R&D is absolutely necessary High quality Fast implementation of scientific discoveries Customer friendly
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WHAT ARE THE ADVANTAGES OF USING A MOLECULAR TEST?

Simplicity Some assays are now automated

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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.

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WHAT ARE THE DISADVANTAGES OF USING A MOLECULAR TEST?

Too

sensitive? Are the results clinically relevant?

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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

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OVERVIEW OF RT - PCR
tissue extract RNA

copy into cDNA (reverse transciptase)

do real-time PCR

analyze results
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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
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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

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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 R&D is absolutely necessary High quality Fast implementation of scientific discoveries Customer friendly
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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.

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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.

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Created by Dr. T.V.Rao MD for e learning resources Microbiologists in the Developing World Email
doctortvrao@gmail.com

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