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

13% - Genetic Testing

7% - Cancer

.

Laboratorio de Genmica Viral &

Humana - Facultad de

Medicina - Universidad

Prediction of risk Oncotype. Early detection - Fragile X.

Classification of disease Leukemias. Therapeutic homming of presumptive target.

Prediction of toxicity & response Herceptin.

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

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

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

PCR product

of 142 bp and

98 bp

Gel Agarose

Electrophoresis

uses an electrical field to

move the negatively

charged DNA toward a

positive electrode through

an agarose gel matrix

RT-PCR (Reverse Transcriptase- 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

Flowchart of Avian Influenza virus detection and analysis

Diagnostic sites for HA gene of

AI

Fragment H5a 424 bp

1 1700

H5aF H5aR H5bF H5bR

275 689

698

1253

1 2 3 4 - + M

Fragment H5b 565 bp

M + - 1 2 3 4

Diagnostic site for NA gene of

AI 1

550

N1-R

1165 1458

N1-F

M + - 1 2 3 4

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 o Multiple viruses can cause same clinical syndrome

Respiratory infections

o Modification of PCR in order to

rapidly detect result in one run

o Multiple primer in temperature-

mediated DNA Polymerase in a

thermal cycler

o 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 Annealing

Elongation End of Elongation

SYBR

Green I

SYBR

Green I

Denaturation Annealing

Elongation 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)