molecular biology based diagnostic methods in microbiology
DESCRIPTION
Polymerase chain reaction in Infectious DiseasesTRANSCRIPT
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
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• 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
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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.
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DR.T.V.RAO MD 72
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