MOLECULAR TECHNIQUES IN

DIAGNOSTIC MICROBIOLOGY
IFURE TOM MONDAY, MD

ST. NICHOLAS UNIVERSITY, DOMINICA

OBJECTIVES
At the end of this lecture student(s) should be able to:

 Explain the importance of molecular microbiology.

 State the uses for nucleic acid based tests.

 Explain the importance of PCR.

OBJECTIVES
At the end of this lecture student(s) should be able to:
 List the REQUIREMENTS for PCR testing.

 Discuss the stages of PCR testing.

 Understand the structure of DNA.

INTRODUCTION
 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 twentieth century.
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.
MOLECULAR MICROBIOLOGY
 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
 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 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.
MOLECULAR METHODS ARE
REVOLUTIONIZING
 In clinical microbiology, this means that we
will be able to detect smaller amounts of DNA
or RNA of pathogens than is currently
possible.
 Meaning that the time required to identify and

determine the antimicrobial susceptibility of

slow-growing pathogens will be dramatically
reduced, and that the diagnosis of non
culturable organisms will become possible.
MOLECULAR METHODS ARE
REVOLUTIONIZING
 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.

 Rapid nucleic acid amplification and detection

technologies are quickly displacing the
traditional assays based on pathogen phenotype
rather than genotype.
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
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 (Cytomegalovirus) in transplanted organs

Organisms present in small volume specimens

 Intra-ocular fluid
 Forensic samples
Molecular Methods Are Necessary if The
Traditional Methods Provide Poor
Results
 Microscopy gives false positive results-
T.vaginalis, N.gonorrhoeae

 Seropositivity is common – Chlamydia sp.

 Subtyping is mandatory – HSV, HPV, HCV

 Microbial growth is slow– M. tuberculosis

Molecular Methods Are Necessary if The
Traditional Methods Provide Poor Results

 Everyorganism contains some unique,

species specific DNA sequences

 Moleculardiagnostics makes the species

specific DNA visible
POLYMERASE CHAIN
REACTION
(PCR Test)
Stages of PCR Tests
 DNA
 PCR
 Targets
 Denaturing
 Primers
 Annealing
 Cycles
 Requirements
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)
 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
 DNA is a nucleic acid that is composed of two
complementary nucleotide building block
chains.

 Thenucleotides are made up of a phosphate

group, a five carbon sugar, and a nitrogen
base.
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
 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
Example of bonding
pattern.
Primary strand

CCGAATGGGATGC
GGCTTACCCTACG

Complementary
strand
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-C-
 Amplification (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-G-
 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 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 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 DENATURING
 Denaturing is the first step in PCR, in which
the DNA strands are separated by heating
to 95°C
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
 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
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

tothe DNA to 55 Celsius.
PCR ANNEALING
PCR Stages
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 95⁰C 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.
PCR Stages
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.
PCR
 The annealing temperature (55-65°C) used should
ensure that annealing will occur only with DNA
sequences that are completely complementary.
 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.
 The annealing time is usually 15 seconds to 1

minute.
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 - Extension
 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°C since this is the

optimum reaction.
PCR - Extension
 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.
PCR CYCLES
PCR CYCLES
PCR CYCLES
PCR CYCLES REVIEW
 Denaturalization: 94°- 95°C

 Primer Annealing: 55°- 65°C

 Extension of DNA: 72°

 Number of Cycles: 25-40

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
LEADING USES FOR NUCLEIC ACID
BASED TESTS
Molecular epidemiology
 To identify point sources for hospital and

community- based outbreaks

 To predict virulence Culture confirmation
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.
TARGET AMPLIFICATION
 Target amplification requires that the DNA to
be tested for is 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
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.
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
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.

 New organisms are missed unless sequencing is done

in the lab for our molecular unknowns (which is not

practical in a clinical setting).
 May be a problem with mixed cultures – would have

to assay for all organisms causing the infection.

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.
 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.
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
PCR to RtPCR
Real-time PCR is a quantitative method for determining
copy number of PCR templates, such as DNA or cDNA,
and consists of two types:
 probe-based and intercalator-based.
 Probe-based real-time PCR, also known as TaqMan

PCR, requires a pair of PCR primers and an additional

fluorogenic oligonucleotide probe with both a reporter
fluorescent dye and a quencher dye attached.
SYBR Green Method
 The intercalator-based (SYBR Green) method
requires a double-stranded DNA dye in the
PCR which binds to newly synthesized
double-stranded DNA and generates
fluorescence
SYBR Green Method
 Both methods require a special thermocycler
equipped with a sensitive camera that monitors
the fluorescence in each sample at frequent
intervals during the PCR.
 The principle techniques underlying both RT-

PCR and real-time PCR are total RNA

isolation, reverse transcription (RT), and PCR.
 Reverse transcription involves the synthesis of DNA
from RNA by using an RNA-dependent DNA
polymerase. PCR can amplify a single or a few copies
of a piece of DNA across several orders of magnitude,
generating thousands to millions of copies of a
particular DNA sequence.
OVERVIEW OF RT - PCR
 Tissue
 Extract RNA
 Copy into cDNA (reverse transciptase)
 Do real-time PCR
 Analyze results
QUESTIONS
 ***
An outbreak of Salmonella has occurred in the community. Molecular-
based assays will be performed to determine if multiple isolates of
Salmonella taken at hospital X are related to each other and part of the
outbreak. Laboratory technologists have accumulated 10 different
cultures growing Salmonella and suspect that there may be more
isolated in the next few days. How should these organisms be handled
until the molecular-based assays can be performed in about 3 days?
A. Maintain organisms in culture growing on solid agar in an
incubator 35°-37°C.
B. Freeze suspensions of the organisms at -20°C.
C. Isolate DNA from all organisms and store at -20°C.
D. Isolate DNA from all organisms and store at 22°-25°C.
1.
Identify the most commonly used diagnostic technique in the
virus laboratory:

 a) Virus isolation in cell culture

 b) Deep sequencing (NGS)

 c) Real Time (RT) PCR

 d) Immuno-serology
2.
How rapid is 'rapid viral diagnosis' by molecular methods?

 a) Within a working day for most laboratories

 b) 25 minutes at the bedside

 c) 3 day laboratory turnarounds

 d) 1 hour laboratory turnarounds in special emergencies

3.
 How many primers are needed for a single target?

A. 2
B. 3
C. 1
D. 4
4.
 How many primers are needed for multiple targets?

A. 2
B. 3
C. 4
D. 1
5.
 What temperature is the denaturation step in which the
DNA is separated into individual strands?

A. 90 celscius
B. 70 celscius
C. 95 celcius
D. 89 celscius
E. 101 celscius
6.
 What temperature range is the annealing phase in
which primers anneal to denatured DNA strands.

A. 30
B. 14
C. 79
D. 55
7.
 How many cycles does it take to perform PCR?

A. 20 – 40
B. 50 -55
C. 60-65
D. 70-75
E. Multiple times as possible
8.
 What is the purpose of reverse transcriptase PCR?

A. To convert RNA into DNA

B. To convert bacteria to protozoa
C. To convert DNA to RNA
D. B and C
9.
The melting temperature of nucleic acid is the
temperature when:
A. All of the double-stranded DNA is single-stranded
B. Half of the double-stranded DNA is single-stranded
C. A quarter of the double-stranded DNA is single-
stranded
D. All of the single-stranded DNA is double-stranded
10.
A PCR reaction that uses more than one primer pair is
called:
A. a. Long-range PCR
B. b. Multiplex PCR
C. c. Signal amplification
D. d. Quantitative PCR