DR.T.V.RAO MD 1

Dr.T.V.Rao MD

POLYMERASE CHAIN REACTION IN INFECTIOUS DISEASES

BASICS

• 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 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

virulence.

MOLECULAR MICROBIOLOGY

DR.T.V.RAO MD 3

• 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

• 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

• 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

• 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

• 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

• 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

• 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

• 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

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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, Pärnu 11.09.2004

DR.T.V.RAO MD

• 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

DR.T.V.RAO MD 14

DNA MOLECULE

Adenine

Thymine

Guanine

Cytosine

DR.T.V.RAO MD 15

1. DNA

2. PCR

• Targets

• Denaturing

• Primers

• Annealing

• Cycles

• Requirements

OUTLINE

DR.T.V.RAO MD 16

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 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 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

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

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

Example of bonding pattern.

• Primary strand

CCGAATGGGATGC

GGCTTACCCTACG

• Complementary strand

DNA

DR.T.V.RAO MD 22

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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 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

• 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

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

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.

PCR TARGETS

DR.T.V.RAO MD 27

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

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

TTAACGGCCTTAA . . . TTTAAACCGGTT

AATTGCCGGAATT . . . . . . . . . .>

and

<. . . . . . . . . . AAATTTGGCCAA

TTAACGGCCTTAA . . . TTTAAACCGGTT

DR.T.V.RAO MD 30

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

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

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

• 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

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

• 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

• 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

DR.T.V.RAO MD 38

UNDERSTANDING THE PCR CYCLE

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)

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High MW Genomic DNA Isolation

Typical Procedure 1 Cell Lysis

– 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

EtOH Precipitation • 2-2.5 volumes EtOH, -20o

• high salt, pH 5-5.5

• centrifuge or ‘spool’ out

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High MW Genomic DNA Isolation

Typical Procedure 1 Cell Lysis

– 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

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

• 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 950 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 • 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

DR.T.V.RAO MD 44

• 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

DR.T.V.RAO MD 46

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 720 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

DR.T.V.RAO MD 48

PCR CYCLES

DR.T.V.RAO MD 49

PCR CYCLES

DR.T.V.RAO MD 50

• 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

• 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

• 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

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

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

• 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

• 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 • 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?

• 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

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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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

•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 ?

• 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

• Too

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 • 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

tissue

extract RNA

copy into cDNA

(reverse transciptase)

do real-time PCR

analyze results

DR.T.V.RAO MD 66

• 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

• 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

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

• 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

• 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

DR.T.V.RAO MD 72

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DR.T.V.RAO MD 73