Molecular Diagnosis
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Transcript of Molecular Diagnosis
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Molecular Diagnosis of Pathogen
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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
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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
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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.
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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.
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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
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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.
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Industry Test Volumes &
Applications 55% - Infectious disease
23% - Blood Screening
13% - Genetic Testing
7% - Cancer
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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.
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Structure of Viruses
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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.
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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.
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Direct probe testing
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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
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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.
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Hibridisasi Primer pada DNA Templat
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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).
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Proses PCR
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Profil Suhu pada PCR
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PCR Process
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Exponential Amplification of PCR
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Vizualizing DNA bands in agarose gel
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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
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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
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Flowchart of Avian Influenza virus detection and analysis
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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
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Diagnostic site for NA gene of
AI 1
550
N1-R
1165 1458
N1-F
M + - 1 2 3 4
Amplification product = 615 bp
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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.
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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
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Amplification Curve of PCR
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Quantification by real time PCR
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Correlation of CT with opy of
Log
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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
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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.
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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.
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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
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H5N1 Viral RNA Load from 8
patients
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Multiple Alignment of DNA and
protein sequences
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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.
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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
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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.
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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.
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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.
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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.
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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
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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).
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Contoh Pasangan Primer
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Suhu
Annealing
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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
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Transkripsi dan
Pemrosesan
Transkrip
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Sintesis cDNA
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Tiga Jenis Primer untuk RT-PCR
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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.
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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.
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Kurva Amplifikasi DNA dalam PCR
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Log Amplikon vs Jumlah Siklus
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Hubungan antara CT dengan Log
Jumlah Salinan
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Prinsip kerja probe TaqMan
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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
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Materi Tambahan
Teknik Blot
Pustaka Gen
Hibridisasi DNA
DNA Typing
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Western Blot
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Southern Blot
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Nothern Blot
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Construction of gene library
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Colony
Hybridization
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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).
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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).
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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.`
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Example 2
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Finger Printing
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DNA Footprinting
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DNA Footprinting (2)