Molecular Diagnosis of Pathogen

Emerging Diseases
Emerging diseases are significant burden on global
economies and public health
Most of the emerging diseases are caused by viruses
Most of the viruses are originate from animals (zoonotic)
and the majority are from wild animals
Most emerging viruses have RNA genome and capable
of rapid mutation and adapt to new hosts

Laboratory Diagnosis of Viral

Diseases
Five approaches:
1. Identification of the virus in cell culture
2. Microscopic identification directly in the specimen
3. Serologic procedures to detect a rise in antibody titer
or the presence of IgM antibody
4. Detection of viral antigens in blood or body fluids
5. Detection of viral nuclic acids in blood or patiens
cells

Molecular Methods
Methods based on the detection of viral genome are also
commonly known as molecular methods. It is often said
that molecular methods is the future direction of viral
diagnosis.
However in practice, although the use of these methods
is indeed increasing, the role played by molecular
methods in a routine diagnostic virus laboratory is still
small compared to conventional methods.

It is certain though that the role of molecular methods will

increase rapidly in the near future.

Why molecular test?

The diagnosis of viral infections has traditionally been:
Costly,
Laborious,
Highly skilled,
Slow.
Serology is often unhelpful in the early stages of infection.
Specific antisera for the serology tests can be difficult to obtain.
PCR technology has therefore improved the detection of a
number of these viruses.

Why use a molecular test?

Need an accurate and timely diagnosis

Important for initiating the proper treatment
Important for preventing the spread of a contagious
disease

What are the disadvantages of

using a molecular test?
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.

Industry Test Volumes &

Applications
55% - Infectious

disease
23% - Blood
Screening
Prediction of risk Oncotype.
13% - Genetic
Early detection - Fragile X.
of disease Leukemias.
Testing Classification
Therapeutic homming of presumptive target.
Prediction of toxicity & response Herceptin.
7% - Cancer
.
Laboratorio de Genmica Viral &
Humana - Facultad de

Structure of Viruses

Classical Molecular Techniques

Dot-blot, Southern blot, in-situ hybridization are examples
of classical techniques. They depend on the use of
specific DNA/RNA probes for hybridization.
The specificity of the reaction depends on the conditions
used for hybridization. However, the sensitivity of these
techniques is not better than conventional viral
diagnostic methods.
However, since they are usually more tedious and
expensive than conventional techniques, they never
found widespread acceptance.

Direct probe testing

Hybridization to come together through
complementary base-pairing.
Can be used in identification.
In colony hybridization the colony is treated to
release the nucleic acid which is then denatured
to single strands.
Labeled single-stranded DNA (a probe) unique to the
organism you are testing for is added and hybridization
is allowed to occur.
Unbound probe is washed away and the presence of
bound probe is determined by the presence of the
label.

Direct probe testing

Facilities required
Minimum BSL- 2 (Biosafety Level 2) Facility
Unidirectional work flow

Clean Room
(no nucleic acid
allowed)

Nucleic Acid
Preparation
Room

Amplification
and visualization
Room

BSL3 Facility

Polymerase Chain Reaction (PCR)

PCR (Polymerase Chain Reaction) merupakan metode molekuler untuk
penggandaan DNA secara in vitro menggunakan enzim dan sepasang primer
yang bersifat spesifik terhadap DNA target.

Pertama kali dirumuskan oleh Kary Mullis pada tahun 1983, dan berkat jasa
temuannya ini ia memperoleh hadiah Nobel bidang kimia pada tahun 1993.

Perkembangan dan aplikasi PCR sangat ditunjang oleh penemuan enzim DNA
polimerase yang bersifat tahan panas. Enzim DNA polimerase yang umum
digunakan adalah Taq polimerase yang diisolasi dari bakteri Thermus
aquaticus.

PCR digunakan secara rutin pada berbagai laboratorium seperti di perguruan

tinggi, lembaga riset, industri farmasi, maupun laboratorium klinik.

Hibridisasi Primer pada DNA Templat

Komponen Pereaksi PCR

Pereaksi standar dalam PCR meliputi:
DNA templat (1 pg 1 mg),
Mg2+ (1,5 mM),
dNTP (200 mM),
primer (1 mM),
DNA polimerase (1 5 unit),
dan bufer (pH 8,3).

Proses PCR

Profil Suhu pada PCR

PCR Process

Exponential Amplification of PCR

Vizualizing DNA bands in agarose gel

Detection of HSV using PCR

Gel Agarose
Electrophoresis
uses an electrical field to
move the negatively
charged DNA toward a
positive electrode through
an agarose gel matrix

PCR product
of 142 bp and
98 bp

RT-PCR (Reverse TranscriptasePCR)

Reverse transcriptase PCR
merupakan metode amplifikasi
cDNA (complementary DNA),
yaitu DNA hasil proses
transkripsi balik menggunakan
RNA sebagai templat,
menggunakan teknik PCR.
Terdiri dari 2 tahap:
1. Sintesis cDNA
2. Amplifikasi dengan PCR

Flowchart of Avian Influenza virus

detection and analysis

Diagnostic sites for HA gene of

AI
275

689

1253

1700

698

H5aF

H5aR
H5bF

1 2

3 4 -

+ M

Fragment H5a 424 bp

H5bR

M +

- 1 2

3 4

Fragment H5b 565 bp

Diagnostic site for NA gene of

AI
1165

550

1458

N1-F

N1-R

Amplification product = 615 bp

Molecular Diagnosis of
Chikungunya Virus

600 bp

427 bp

Molecular diagnosis of ChikV using an RT-PCR assay which amplifies a

427-bp fragment of the E2 gene.

Real Time PCR

Development of Conventional PCR that allows
REAL TIME monitoring of DNA amplification
during amplification process.
Real time PCR or Quantitative PCR (qPCR).

DNA amplification is based on fluorescence as

amplification indicator. The fluorescence signal
is directly proportional with the number of PCR
product

Amplification Curve of PCR

Quantification by real time PCR

Correlation of CT with opy of

Log

Viral Load Monitoring

Monitoring viral DNA or RNA loads has become the standard of care for
several chronic viral infections:
HIV
HBV
HCV
CMV

HIV viral load testing is an integral component of the management of

HIV infection.
It is the major tool used to monitor the success of antiretroviral therapy
and to detect the emergence of viral resistance
HIV viral loads also predict progression of disease, and gives prognostic
information

HBV/HCV viral load monitoring

HCV RNA viral loads are used to monitor response to
combination interferon-a & ribavirin therapy.
Patients who remain negative for HCV RNA 6 months after
combination therapy usually achieve a sustained virological
response
If HCV RNA is undetectable after 12 weeks of therapy there is
a 75% chance of sustained virological response.

In HBV carriers with active liver disease VL determine the

need and effectiveness of either interferon-a or lamivudine
antiviral therapy.

CMV viral load monitoring

CMV infection is serious in bone marrow, solid organ
transplant recipients and HIV-infected patients.
Poor sensitivity of traditional culture methods (& very slow).
VL testing is currently the accepted standard for monitoring
the emergence of CMV infection during immunosuppression.
Allows pre-emptive therapy prior to the emergence of clinical
disease with high sensitivity when compared to culture.

Sequence Analysis of
Neuraminidase Gene

T substitution at nucleotide 763 changing codon CAC for 274H to TAC for
274Y
Raw sequencing traces revealed the presence of a minor
subpopulation of wildtype 274 H among predominating 274Y

H5N1 Viral RNA Load from 8

patients

Multiple Alignment of DNA and

protein sequences

Multiplex PCR
Multiplex PCR

Multiple viruses can cause

same clinical syndrome

Respiratory infections
o

Modification of PCR in order to

rapidly detect result in one run

Multiple primer in temperaturemediated DNA Polymerase in a

thermal cycler

Commercial assays to detect

up to 18 respiratory viruses in 1
test.

Bacteriological applications
Speed and resolution of molecular methods have firmly established
their utility in several applications:

Detection of fastidious (hard to culture) bacteria.

Rapid detection of severe bacterial diseases.
Assessment of antibiotic resistance.
Identification of bacterial etiology through broad-spectrum PCR

Fastidious bacteria - Mycobacteria

M. tuberculosis is one of the few examples where conventional
culture remains more sensitive than molecular testing.
Difficulties in DNA extraction from the bacterial cells.

Despite this limitation, it allows confirmation of acid-fast bacilli with up

to 98% sensitivity in pulmonary tuberculosis within a day (versus two
or more weeks by culture).

Traditional methods for detecting rifampicin & isoniazid resistance

require culture, delaying the diagnosis & increasing the risk of
transmission of resistant disease in the community.

A multiplex PCR for the sequencing of rpoB and hsp65 gene

allows same day results of most multi-drug resistant strains.

Rapid bacterial detection

Severe infectious diseases sometimes require a

prompt & unequivocal diagnosis so as to guide
therapeutical & preventitive measures (both
individually and population-wide).
Meningococcal disease has devastating
consequences and requires early diagnosis
for correct antibiotic therapy as well as early
chemoprophylaxis for close contacts.

Multiplex kits for the detection of common

causes of meningitis.

Mycology/Parasitology applications

Molecular testing not as frequently applied to

eukaryotic infections.

Pneumocystis jiroveci causes severe

pneumonia in immunosuppresed patients but
detection is limited to microscopy of
respiratory specimens.

Immunofluorescence is more sensitive but is more expensive and needs

specialised facilities.

Mycology/Parasitology
applications

Another mycological example is the use of

18S rRNA gene PCR to detect Aspergillus
spp. infection in neutropenic patients.

Disease is notoriously difficult to diagnose

due to the poor sensitivity of culture in early
disease (and the difficulty in obtaining
histopathological specimens in patients
with reduced platelet counts).

Early treatment is essential for the best

outcomes resulting in empiric use of costly
and toxic antifungal therapy.

Emerging infectious disease

surveillance

Rapid and reliable aetiological diagnosis

underpins the effective management of
contagious diseases.

Laboratorio de Genmica Viral &

Humana - Facultad de
Medicina - Universidad
Autnoma de San Luis Potos

Perancangan Primer

Spesifik
18-30 basa
Kandungan G+C: 40-60%
Pasangan primer memiliki nilai Tm yang
setara. Tm (0C) = 2 (A + T) + 4 (G + C).

Contoh Pasangan Primer

Suhu
Annealing

RT-PCR
Reverse transcriptase PCR merupakan metode
amplifikasi cDNA (complementary DNA), yaitu
DNA hasil proses transkripsi balik
menggunakan RNA sebagai templat,
menggunakan teknik PCR.
Terdiri dari 2 tahap:
1. Sintesis cDNA
2. Amplifikasi dengan PCR

Transkripsi dan
Pemrosesan
Transkrip

Sintesis cDNA

Tiga Jenis Primer untuk RT-PCR

Komponen Pereaksi RT-PCR

Pereaksi untuk sintesis cDNA antara lain
mengandung sampel RNA, dNTP, primer,
bufer, ditriotreitol (DTT), inhibitor
ribonuklease, dan enzim reverse
transcriptase.
Pereaksi untuk amplifikasi cDNA: ion
Mg2+, dNTP, sepasang primer, DNA
polimerase, dan bufer,serta cDNA sebagai
DNA templat.

Real Time PCR

Teknik real time PCR:
merupakan hasil pengembangan PCR konvensional yang
memungkinkan dilakukan pemonitoran amplifikasi DNA pada saat
proses amplifikasi tersebut berlangsung (real time).
Real time PCR (juga disebut PCR kinetik) bersifat kuantitatif.
Amplifikasi DNA dideteksi berdasarkan pancaran sinar flourescen
yang digunakan sebagai indikator amplifikasi DNA. Sinyal
flourescen yang terpancar berbanding lurus dengan jumlah
amplikon atau produk PCR.
Melalui perekaman jumlah emisi flourescen untuk tiap siklus maka
dimungkinkan untuk memonitor reaksi PCR pada fase eksponensial
yaitu fase pada saat peningkatan jumlah produk PCR berbanding
lurus dengan jumlah templat target (DNA sampel), sehingga real
time PCR bersifat kuantitatif.

Kurva Amplifikasi DNA dalam PCR

Log Amplikon vs Jumlah Siklus

Hubungan antara CT dengan Log

Jumlah Salinan

Prinsip kerja probe TaqMan

Prinsip Kerja Probe SYBR Green I

SYBR
Green I

Denaturation

Elongation

Annealing

End of Elongation

Materi Tambahan

Teknik Blot
Pustaka Gen
Hibridisasi DNA
DNA Typing

Western Blot

Southern Blot

Nothern Blot

Construction of gene library

Colony
Hybridization

DNA Fingerprinting
Variable Number of Tandem Repeat
(VNTR) loci are chromosomal regions
in which a short DNA sequence motif
(such as GC or AGCT) is repeated a
variable number of times end-to-end at
a single location (tandem repeat).

VNTR

In this example, Locus A is a tandem repeat of the motif GC: there are four
alleles, with two, three, four, or five repeats (A2, A3, A4, and A5,
respectively). Locus B is a tandem repeat of the motif AGCT: there are only
two alleles, with two or three repeats (B2 and B3, respectively).

The example shows a DNA fingerprint that includes both loci

simultaneously. Individual #1 is heterozygous at Locus A (A2 / A5)
and homozygous at Locus 2 (B2 / B2: note that this genotype gives a
single-banded phenotype in the fingerprint). Individual #2 is
heterozygous at both loci: (A4 / A3 and B3 / B2) respectively). The two
individuals are distinguishable at either locus. Typical fingerprints
include a dozen or more VNTR loci.`

Example 2

Finger Printing

DNA Footprinting

DNA Footprinting (2)