MOLECULAR METHODS IN

DIAGNOSIS AND
MONITORING OF
INFECTIOUS DISEASES
Fera Ibrahim
Dept. Of Microbiology, Medical Faculty,
University of Indonesia
 LABORATORY DIAGNOSIS OF
INFECTIOUS DISEASES
DIRECT
Isolation of the agents
Detection of Antigens
Detection of nucleic acids
INDIRECT
Detection of Antibodies
Histopathology
 NUCLEIC ACIDS
The Basics of DNA and RNA
 NUCLEIC ACIDS
The Basics of DNA and RNA
 NUCLEIC ACIDS
The Basics of DNA and RNA
 Transcription and Translation
in Prokaryotes
5 3

3 5

RNA
Pol.

Ribosome

mRNA
Ribosome
5
Transcription and Translation in Viruses
 Indications for molecular testing
To detect slow growing, fastidious or uncultivable
microorganisms
To determine the cause of significant outbreaks

- Rapid
- Easier to use
- Less expensive

To detect and charaterize more common

microorganisms
Development of molecular assays
for infectious diseases
SIGNAL AMPLIFICATION METHODS
Nucleic acid Probes
Hybrid Capture
Branched DNA
In Situ Hybridization
NUCLEIC ACID AMPLIFICATION
Basics of the Polymerase Chain reaction (PCR)
Other methods of Nucleic Acid Amplification
Modifications of PCR
 Development of molecular assays
for infectious diseases
POSTAMPLIFICATION ANALYSIS
Traditional Methods of Detection
Reverse Hybridization
DNA Sequencing
REAL TIME NUCLEIC ACID AMPLIFICATION
Methods of Detecting the Products of Real Amplification
STRAIN TYPING
Non amplification Based Typing
Amplification Based Typing
 SIGNAL AMPLIFICATION
METHODS
Nucleic acid Probes
DNA Probes that contain a chemiluminescent label
Target the rRNA (rRNA > rDNA genes)
Clinical Applications :
Direct detection of bacteria in clinical specimens
A rapid and specific method of identifying microorganisms in culture
(sensitivities and specificities 90s to 100%)
The advantage is time savings, the disadvantage is higher cost
N.gonorrhoeae, C.trachomatis, Group A and B Streptococcus, M.tbc, M.
Kansasii, Mgordonae, M.avium intracellulare complex, Campylobacter,
Haemophilus influenzae, streptococcus pneumoniae, Staphylococcus aureus,
Listeria monocytogenes
Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis
Hybridization
Hybridization
 SIGNAL AMPLIFICATION
METHODS
Hybrid Capture
The RNA probes used are complementary to the DNA molecule
from the microorganism of interest in a tube or microtiter plate
A monoclonal antibody recognizes and immobilizes a DNA-RNA
hybrid, another antibody labeled with reporter molecule
recognizes that complex. A reporter generate a signal in the form
of light.
The amount of signal generated in the assay is proporsional to the
amount of target present
Clinical Applications :
The detection of high risk HPV subtypes, Cytomegalovirus, HBV
N. Gonorhoeae, C. Trachomatis (more sensitive than culture)
The hybrid capture is less sensitive and specific than PCR
Hybrid Capture
 SIGNAL AMPLIFICATION

METHODS
Branched DNA (bDNA)
The organism-specific bDNA oligonucleotides probes hybridize to
the target nucleic acid molecule and this complex is captured onto a
solid substrate. Branched DNA oligonucleotide reporter molecules
which are conjugated to reporter enzymes are added and generate a
chemiluminescent signal after the addition of appropriate substrate.
Clinical Applications
The detection and quantification of pathogens such as HIV, HBV,
and HCV
It has become the standard of care to monitor the viral loads of
patients infected with HIV (75-500 copies of HIV1 RNA)
Although the sensitivity of the bDNA assays may be slightly less
than that for PCR, there is overall a good correlation between
viral loads generated by these different assays.
Branched DNA Signal Amplification
Assay
 SIGNAL AMPLIFICATION
METHODS
In Situ Hybridization
This method has been used in the molecular pathology for the
detection of chromosomal translocation, gene amplification and
for the identification of infectious agents
FISH ! oligonucleotide probe is labeled with a fluorophore,
direct fluorescence microscopy
Chromogenic in situ hybridization (CISH) ! oligonucleotide is
labeled with an enzymes, traditional light microscopy
The microbial rRNA may be used as the target for hybridization
probe ! rRNA >rDNA (more sensitive); rRNA ~ a viable
organism
PNA ISH ! a peptide nucleic acid probe (better than DNA
probe with regard to penetration ! hybridization on intact
organism
A battery probes ! bacterial meningitis, community acquired
pneumonia
 SIGNAL AMPLIFICATION
METHODS
In Situ Hybridization
Clinical Applications :
Direct detection of bacteria, mycobacterium, fungi and
parasites : bacteria in water samples and biofilm (Legionella,
E coli); Helicobacter pylori in gastric biopsies; Legionella
pneumophila in fixed respiratory specimens; Chlamydia, M
tbc, M leprae (cannot be cultivated on artificial media); S.
aureus, P. Aeruginosa, S. maltophilia, B.cepacia and H.
Influenza in specimen patient with cystic fibrosis
To differentiate viruses that produce identical cytophatic
effects (HSV and VZV) and the high and low risk HPV
subtypes, to detect viruses with neoplasia : HPV, EBV,
HHV8
Fungi and parasites : Aspergillus Vs
Fusarium/Pseudallescheria!pathogen and different
antifungal susceptibility profile ; Pneumocystis jiroveci; to
rapid identify and differentiate Candida albicans, C.
Glabrata, C. Krusei and C. Parapsilosis in blood culture
In Situ Hybridization
In situ RNA hybridization (ISH)
for FISH, CISH applications
NUCLEIC ACID AMPLIFICATION
Basics of the Polymerase Chain reaction (PCR)
First described by Kary Mullis in 1983
Exploits the basic biochemistry of DNA replication with the end
goal of amplification of a particular portion of DNA ! usually
contains diagnostically useful information
PCR reaction consist of the target DNA, oligonucleotide DNA
primers, the four nucleotide triphosphate, thermostable DNA
polymerase, MgCl2 and water
Three phase : DNA denaturation, primers annealing, primers
extention ! DNA synthesis
Clinical Applications
To detect organisms that could not be cultured,were fastidious or
slow growing
Almost every microorganism of clinically interest has been
detected and studied by PCR ! commercial assays, FDA
approved
NUCLEIC ACID AMPLIFICATION
Other methods of Nucleic Acid Amplification
The nucleic acid sequence based amplification (NASBA)
Isothermal assays ! use 3 enzymes : Reverse Transcriptase (RT), Rnase H
and T7 DNA dependent RNA polymerase
RT make a cDNA copy of target molecule (usually RNA), The primer
contains the T7RNA polymerase binding sequence on the 5 end of the
molecule, DNA-RNA hybrid is hydrolized by Rnase H, 2nd primer then
binds and creates the complementary strand of the DNA ! this complete
cDNA serves as the template for T7RNA polymerase which transcribes
numerous copies of RNA ! to be detected by a variety of methods
Transcription mediated amplification (TMA) ! sda
Strand-displacement amplification (SDA)
An isothermal reaction that relies on the ability of the DNA polymerase to
displace one strand of DNA at the site of a single strand nick and proceed
with DNA replication or amplification in this assays
Require special conditions and endonucleases that will generate a nick in
only one of the strands of the double stranded DNA molecule
NUCLEIC ACID AMPLIFICATION
Other methods of Nucleic Acid Amplification
Ligase Chain Reaction (LCR)
The ligation or connection of two probes that anneal contiguously on the
template DNA strand
N. Gonnorhoeae, Chlamydia trachomatis
Clinical Applications
FDA approved commercial assays available for the detection of N.
Gonorrhoeae and C trachomatis that use TMA and SDA; HIV viral load that
use NASBA technology; CMV pp67 assay for monitoring CMV level in a
bone marrow transplant recipient (replace antigenemia assays); for the rapid
detection of enterovirus from the cerebrospinal fluid (CSF)
NUCLEIC ACID AMPLIFICATION
Modifications of PCR
RT-PCR (Reverse Transcription PCR)
Make DNA copy from RNA
To detect RNA viruses, mRNA, rRNA (~viable organisms)
Clinical Applications
To detect dengue virus , HIV, Hantavirus, SARS
To detect rRNA bacteria, parasites and fungi
Broad range PCR
The primers of assay are designed to detect all the organisms in the group of
interest and exclude as many organisms that are not in this group
The members of a larger group may be detected in a single reaction
Clinical Applications
To detect the enteroviruses capable of causing aseptic meningitis
To charaterize mycobacteria by rpoB gene ! provide information
regarding the resistance of mycobacteria to rifampin
NUCLEIC ACID AMPLIFICATION
Modifications of PCR
Multiplex PCR
An alternative method to broad range PCR for the detection of multiple
pathogens in one reaction
Clinical Applications
To detect different microorganisms that cause the same types of
diseases ex To detect S. pneumoniae, H influenzae and N.
Meningitiditis the most common causes of bacterial menigitis
To detect the agents of atypical and typical bacterial pneumonia
Nested / Seminested PCR
Modification of PCR designed to increase the sensitivity of the assay
reaction
This modification consist of two primers sets directed against the same target
Clinical Applications
To detect microorganisms that may be in low quantity in the blood and
tissue such as Rikettsia, Bartonella
POLYMERASE CHAIN REACTION (PCR)
Molecular detection of HTLV 1

Agarose(gel(electrophoresis(and(Southern
blot(of(samples(amplied(by(SYBR(Green
real(<me(PCR(assay.(In(the(upper(part(of(the
gure:(the(two(posi<ve(HTLVHI(samples
(lanes(1(and(11),(the(samples(with(HTLVHI
indeterminate(or(posi<ve(serological(assays
(lanes(210),(MTH2(representa<ve(cells
scalar(dilu<ons((from(102(to(104;(lanes(12
14)(and(molecular(weight(markers((lane(15)
are(shown.(The(boUom(of(the(gure(shows
the(Southern(blot(assay.(The(nonHspecic
bands(exhibited(in(the(HTLVHI(nega<ve
samples((lane(79)(represent(nonHspecic
products(that(both(did(not(hybridize(with
the(HTLVHI(specic(internal(probe(and
showed(an(unrelated(mel<ng(temperature
in(SYBR(Green(real(<me(PCR.
 CMV$detec)on$by$PCR$technique
M""1"""2""""3

1353b
1078b
p 872b
p
p
603b
p
310b
p

Line"1"="positve"DNA"CMV
Line"2"="H2O,"PCR"reac=on"control
Line"3"="H2O
M"""""""""="Marker"phiX"174"DNA/haeIII
CMV$detec)on$by$PCR$technique
K+ K- S M Kbp

1.0
0.5
0.4
0.3
0.2 M = DNA Ladder
S = Sample
K- = Negative control
0.1 K+ = Positive control
Kbp = Kilo base pair
HIV 1 detection by RT-PCR
technique
k+ k- S M

M: DNA Ladder
S: Plasma sample
k+: Positive control

k-: Negative control

 POST AMPLIFICATION
Traditional Methods of Detection
ANALYSIS
Gel Electrophoresis/Southern Blot Analysis
The separation of the DNA in gel electrophoresis is largely due to size
The negatively charged DNA migrate toward the anode,
Visual comparison of the migration of the amplicon and the migration of the
DNA molecules of varying sizes in a ladder ! the user can estimate of the
size of the DNA molecule amplified
The amplified product was transferred to nitrocellulose paper and a
radiolabelled oligonucleotide probe added ! exposed to X-ray film
Enzymatic detection of Amplified products
The enzymatic reaction for the detection of the amplified product is
performed in a microtiter plate
The amount of signal generated is proportional to the amount of amplicon
present, when used with calibrated standards, quantitative information may
be obtained
Clinical Applications
Many of the commercially available systems use a colorimetric reaction for
the detection of the amplified products (Southern Blot ! labor intensive
and time consuming)
Gel
Electrophoresis
 HIV 1 detction by RT-PCR
technique

Electrophore*c+gel+prole+of+RT1PCR+products+generated+using+the+Applied+Biosystems+ViroSeq+HIV11+genotyping
system.+FiFeen+members+of+the+HIV11+subtype+panel+at+5,000+RNA+copies/ml+were+subjected+to+sequence
analysis+by+the+ViroSeq+assay+using+the+model+3100+gene*c+analyzer.+The+gel+prole+for+puried+amplied
product+is+shown+and+is+representa*ve+of+all+of+the+HIV11+subtypes+tested.+Lanes+1+and+12+contain+two+amounts
of+the+DNA+mass+ladder.+Lanes+3+and+4+contain+the+two+subtype+D+isolates+(46+and+44+ng).+Lanes+5,+6,+and+7
contain+subtypes+F,+H,+and+A+(74,+70,+and+53+ng).+Lane+8+contains+the+posi*ve+control+provided+in+the+kit+(84+ng).
Lane+10+contains+the+nega*ve+control.
Cockerill FR III. Arch Pathol Lab Med.
2003;127:1112 (www)
HPV DETECTION BY PCR and SB
HYBRIDIZATION TECHNIQUE

Southern)blot)hybridiza1on)of)PCR)products,)amplied)by)SPF1/2)(top)row),)GP51/61)(middle)row),)and)My11/09)(boIom)row).)PCR
products)from)10Kfold)dilu1ons)of)plasmids,)containing)genomic)sequences)of)HPV)genotypes)16,)35,)and)45)were)analyzed.)Lanes)1
to)5)represent)PCR)products)star1ng)with)100)fg,)10)fg,)1)fg,)100)ag,)and)10)ag)of)HPV)DNA,)respec1vely.
 GP5/6&and&GP5&+/6&+&PCR&on&1&ng&of&DNA
of&22&cloned&HPVs&diluted&in&100&ng&of
human&placental&DNA.&PCR&products&are
shown&aEer&gel&electrophoresis&(upper
panels)&or&aEer&Southern&bloJng&and
hybridizaMon&with&the&HPV&cocktail&probe
under&low&stringency&condiMons&(lower
panels).&The&level&of&the&150&bp&PCR
products&is&indicated&at&the&right.

A&combinaMon&of&the&general&primers&GP5&and&GP6,
originally&selected&from&the&HPV&L1&region&on&the&basis
of&sequence&informaMon&from&HPVT6,&T&11,&T&16,&T&18,&T31
and&T33,"was"found"to"amplify"target&DNA&of&at&least&27
mucosotropic&HPV&genotypes&under&condiMons&that&allow
mismatch&acceptance
 POST AMPLIFICATION
ANALYSIS
Reverse Hybridization
To immobilize all the probes of interest on a nitrocellulose strip
and then apply the amplicon to the strip and determine which
probe hybridized with the amplicon
Opposite to how a southern blot is performed
The assay has the advantage of using a chromogenic reaction ,
rather than radioactivity to detect hybridization
Easier to perform than DNA sequencing, to asses only the
sequence for which there are probes and does not afford the
opportunity to evaluate new mutations
Clinical applications
Line probe assays (LiPA, Innogenetics), Reverse Blot strips (Roche),
INNO-LiPA (Bayer)
To differentiate the various genetic subtypes ! HCV, HPV
To detect mutations associated with organisms resistence to antiviral agents
or antibiotic ! HIV, M. tbc
DNA Sequencing
 HPV DETECTION BY
HIBRIDIZATION TECHNIQUE

FIG.%1.%HPV%genotyping%of%PCR%product%by%reverse%line%blot%method.%SchemaAc%of%the%reverse%line%blot%genotyping
assay%from%L1%consensus%primerCgenerated%PCR
products.%The%drawing%represents%the%detecAon%of%a%hypotheAcal%mixed%infecAon%of%HPV%16,%31,%and%11.
 1=HPV 16
2=HPV 18
3=HPV 26
1=HPV 16 4=HPV 31
5=HPV 33
2=HPV 18 6=HPV 35
3=HPV 26 7=HPV 39
HPV 16 8=HPV 45
4=HPV 31 HPV 18 9=HPV 51
10=HPV 52
5=HPV 33 HPV 26 11=HPV 55
6=HPV 35 HPV 31 12=HPV 56
7=HPV 39 HPV 33 13=HPV 58
14=HPV 59
8=HPV 45 HPV 35 15=HPV 68
HPV 45 16=MM4
9=HPV 51 17=MM7
10=HPV 52 HPV 51 18=MM9
11=HPV 55 HPV 52 19=High B Globin
HPV 55 20=Low B Globin
12=HPV 56
HPV 56
13=HPV 58 HPV 58
14=HPV 59 HPV 59
15=HPV 68 HPV 68
16=MM4 MM4
17=MM7 MM7
18=MM9 MM9
19=High B Globin HPV 6
20=Low B Globin HPV 11
21=HPV 6 HPV 40
22=HPV 11 HPV 42
HPV 53
23=HPV 40
HPV 54
24=HPV 42 HPV 57
25=HPV 53 HPV 66
26=HPV 54 MM8 High B Globin
Low B Globin
27=HPV 57 K562 HPV 6
28=HPV 66 No DNA HPV 11
29=MM8 HPV 40
HPV 42
HPV 53
HPV 54
HPV 57
HPV 66
MM8
Probe&layout&of&the&HPV&genotyping&strip.&(a)&HPV&genotyping&strips&(n"5"28)"hybridized"with"the"HPV"L1"consensus"PCR"product"generated"from"the"HPV
targets&indicated&to&the&right.&Fi;y&microliters&of&PCR&product&generated&from&amplica@on&of&106&HPV&plasmid&targets&(with&the&excep@on&of&HPV&51
and&68,which&were&amplied&with&103&plasmid&targets)&in&a&background&of&human&cellular&DNA&(12.5&ng/PCR)&was&hybridized&to&the&HPV&genotyping
strips&and&detected&by&the&previously&described&reverse&line&blot&method.&(b)&Line&blot&genotyping&hybridiza@on&results&for&10&clinical&specimens&in&the
previously&described&study.&Fi;y&microliters&of&denatured&PCR&product&was&hybridized&to&each&strip.&The&genotyping&results&for&the&specimens&are&as
follows:&no.$333,$HPV$nega-ve;$no.$334,$HPV$nega-ve;$no.$352,$HPV$16,$26,$and$MM8;$no.$353,$HPV$16;&no.&354,&HPV&16,&51,&and&66;&no.&355,&HPV
nega@ve;&no.&357,&HPV&39;&no.&359,&HPV&MM7;&no.&361,&HPV&16&and&52;&and&no.&373,&HPV&18,&56,&and&58.
 POST AMPLIFICATION
ANALYSIS
DNA Sequencing
Traditional DNA sequencing
Use traditional Sanger sequencing or sequencing by termination
Radioactive reporter molecules ! fluorescently labeled reporter
molecules, automated
Clinical Applications
HCV, HIV genotyping, to determine the presence of acquired
mutations in the genome of HIV ! resistance to antiretroviral agent,
resistance associated mutation in CMV
Identify bacteria : Mycobacteria, Nocardia and fungi ! rDNA genes
as the genetic targets
Sequencing by synthesis (Pyrosequencing)
Sequencing by synthesis ! sequencing based on nucleotide incorporation
into the newly synthetized strand of DNA
The pyrophospate is converted into light by the enzymatic reaction ! the
light generated is recorded by the instrument and the sequence is determined
The limitations are its ability to generate only relatively short sequence (~
30bp)
 POST AMPLIFICATION
DNA SequencingANALYSIS
Sequencing by synthesis (Pyrosequencing)
Clinical Applications
The identification and typing of bacteria : H. Pylori, Listeria
monocytogenes
To identify mycobacteria, Nocardia and fungi ! the 16S rDNA gene
To detect resistance associated genetic mutations
Microarray analysis
A wide variety of microarrays ! gene chips ! hybridization sites !
signal generated may be fluorescent or electrical
Data generated require computers and advanced software packages for data
analysis
The advantage of microarrays is its ability to examine thousands of
hybridization reactions simultaneously.
To detect a multitude of signals simultaneously and genetic (DNA
differences or differences in expression (mRNA)
Products of broad range PCR or RT-PCR are applied to microarrays
 POST AMPLIFICATION
DNA Sequencing
ANALYSIS
Microarray analysis
Clinical Applications
Microarrays are very useful for research and discovery ! have not
yet been introduced into routine use in the clinical microbiology
laboratory for diagnosis or assessment of patient with infections
To identify bacteria, mycobacteria, fungi and viruses
To detect the genetic determinants of resistance
To study host response to infection
To discover new drugs that may be useful to cure infections
To study HIV viral gene expression and the changes into the host cell
expression profile after infection
DNA$$SEQUENCING
 DNA$$SEQUENCING
Automasi Susunan DNA

Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or

Pyro Sequencing
cDNA Microarray analysis

Adapted from Bryant et al., 2004, Lancet infectious

disease
Clinical Cancer Research 11:2625-2636,
2005
 REAL TIME NUCLEIC ACID
AMPLIFICATION
Evidence of amplification occurs during the PCR
reaction in real time
More rapid than traditional methods of amplicon
detection, significant reduction of in the chance of
amplicon contamination
Methods of Detecting the Products of Real
Amplification :
SYBR Green, a dye that binds to the minor groove of
double stranded DNA ! generate fluoresecence when
bound to DNA, nonspecific detection
 REAL TIME NUCLEIC ACID
AMPLIFICATION
Hybridization Probes
Taqman or Hydrolysis Probes, hydrolisis oligonucleotide probe is
labelled with both a fluorophore and a quencher molecule.
Fluorescence Resonance Enargy Transfer (FRET) probes
Molecules Beacon, like Taqman
Clinical Application
To detect and differentiate microorganisms ~ traditional
PCR
To rapidly detect newly recognized and emerging viral
pathogens (SARS)
The quantitative assays ! to predict disease and
monitor the viral load in response to therapy
REAL TIME NUCLEIC ACID
AMPLIFICATION
Methods of Detecting the Products of Real Amplification
SYBR Green
Hybridization Probes
 Real-time PCR Principles
* based on the detection and quantitation of a fluorescent reporter
* the first significant increase in the amount of PCR product (CT -
threshold cycle) correlates to the initial amount of target template
(www)
 STRAIN TYPING
Non amplification Based Typing
Pulsed Field Gel Electrophoresis (PFGE)
The restriction endonucleases cut the chromosomal DNA into a variety of
pieces, depending on the number of restriction sites present in that particular
strain
The fragments of chromosomal DNA are separated using specialized type of
electrophoresis ! profile
Amplification Based Typing
PCR Restriction Fragment Length Polymorphism (PCR-RFLP) !
product PCR ! enzymes restriction ! profile
Repetitive PCR (repPCR) ! repetitive DNA elements in
prokaryotic genomes form the basis for repPCR ! the
hybridization sites for the primers used in the repPCR
Clinical Applications
To investigate outbreaks in hospital, in cities and across the nation
Pulsed Field Gel Electrophpresis
PFGE Results
Restriction Fragment Length
Polymorphism (RFLP)
RFLP
RFLP Analysis
RFLP results
RFLP results
THANK YOU