Molecular Methods In Diagnosis Of Infectious Diseases

Molecular Methods in Diagnosis of Infectious Diseases Dr.T.V.Rao MD

Diagnostic methods in M icrobiology
Task of the method – to make the microorganism “visible” and “measureable ”
Cultivation
Bio-testing
Immunological methods
Biochemical methods
Microscopy
Molecular methods

Watson and Crick’s Discovery of DNA -Path to Molecular Medicine

Traditional Microbial Diagnostic Methods
Morphology
Staining properties
Grow on different laboratory condtions and media.
Phenotyping protocols use purely biological phenomena and usually refer to study of Proteins

Disadvantages of Phenotyping Methods
Lack of Reproducibility
Poor Discriminatory power
Difficulties in Typing

Genotyping Methods
Genetic methods generally seek to detect polymorphisim at the level of nucleic acid
Genotypes are more specific, more easily quantified and standardized among the different organisms

Basic requirement
The genome is unique in each individual
Ultimate discriminatory step would be to sequence the entire genome of every organism, but not practical or economical

What is Practicable
Several methods in detecting nuclei acid polymorphisim in a chosen genetic marker are commonly used to target the genome or organism.

Complete genomic configuration

Restriction Endonuleases
It was discovered that a type of bacterial enzyme was found to have the ability to cut DNA in a test tube. These restriction endonulease, so named because they cut double stranded DNA at restricted sites, were discovered as a natural part of the bacterial machinery.

Restriction endonulease EcoRI cuts double-stranded DNA and generates sticky ends .

What is the Choice of Marker
Genetic markers are nucleotide sequences within the genome that are polymorphic (with differences) between strains of the same organisms

Choice of Markers
Markers depend on Type of study,
Recent outbreak
Rapid evolving markers.
Markers related to evolution of organism over years require more stable markers

Kary Mullis - Nobel prize in 1993
Kary Mullis shared the 1993 Nobel Prize in Chemistry with Michael Smith. Mullis received the prize for his development of the Polymerase Chain Reaction (PCR)

PCR a Revolution in Science
A process first described by Kjell Kleppe and 1968 Nobel laureate H. Gobind Khorana that allows the amplification of specific DNA sequences. The improvements provided by Mullis have made PCR a central technique in biochemistry and molecular biology

Polymerase Chain Reaction
Polymerase chain reaction ( PCR ) is a technique to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of a particular DNA sequence. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA.

Taq polymerase - backbone of PCR technology
Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase , an enzyme originally isolated from the bacterium Thermus aquaticus .

Oligonucleotide – DNA Primers
This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, by using single-stranded DNA as a template and DNA Oligonucleotide (also called DNA primers), which are required for initiation of DNA synthesis.

Amplified sequences are Blotted

PCR in Clinical Microbiology
Molecular detection has mostly come to the clinical microbiology laboratory in the form of PCR technology, initially involving single round or nested procedures with detection by gel electrophoresis.

Helps Rapid Detection
Polymerase chain reaction (PCR) techniques have led the way into this new era by allowing rapid detection of microorganisms that were previously difficult or impossible to detect by traditional microbiological methods.

Automation and Multiplex PCR
With the advent of multiplex PCR, real-time PCR and improvements in efficiency through automation, the costs of molecular methods are decreasing such that the role of molecular methods will further increase.

Progress in Molecular Methods
Molecular methods have now progressed beyond identification to detect antimicrobial resistance genes and provide public health information such as strain characterisation by genotyping.

Molecular based Tests need
Nucleic acid-based tests used in diagnosing infectious diseases use standard methods for isolating nucleic acids from organisms and clinical material and restriction endonulease enzymes, gel electrophoresis, and nucleic acid hybridization techniques to analyze DNA or RNA

Molecular diagnostics is a set of methods to study primary structure (sequence) of DNA
Hybridization with complementary sequences
Amplification (synthesis) of species specific sequences
PCR – polymerase chain reaction
-A-A-T-T-C-G-C-G-A-T-G- - T-T-A-A-G-C-G-C-T-A-C - -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-

Advances on PCR Methods
Fairly recently, a new method of PCR quantification has been invented. This is called “ real-time PCR” because it allows the scientist to actually view the increase in the amount of DNA as it is amplified.

REAL TIME ASSAYS

New Technologies – Real Time Assays
The Real Time assays are proving to better technologies
1 Rapid
2 Quantitative measurement
3 Lower contamination rate
4 Higher sensitivity
5 Higher specificity
6 Easy standardization
Now a new gold standard for rapid diagnosis of virus infection in the acute phase samples.

RT - PCR
Proving to be
Accurate
Precise
Easy to perform
RT PCR technologies are easy to transfer research Laboratory protocols to Diagnostic Laboratories

OVERVIEW of RT - PCR tissue extract RNA copy into cDNA (reverse transciptase) do real-time PCR analyze results

Real Time Reporters
All real time PCR systems rely upon the detection and quantization of fluorescent reporter, the signal of which increases in direct proportion of the amount of PCR product in a reaction.

REAL TIME PCR Cyber Green
USING SYBER® GREEN
The simplest and economical format the reporter is the double strand DNA specific dye SYBR ® Green
Called as Molecular Probe.

How SYBR Green works
SYBR green binds to double stranded DNA and upon excitation emits light
Thus as PCR product accumulates the fluoresce increases

Limitations of SYBER®Green
Advantages
Inexpensive
Easy to Use
Sensitive
Disadvantages
SYBR green will bind to any double stranded DNA in a reaction, may result in an overestimation of the target concentration

Other Emerging Alternatives
Two most popular alternatives to SYBR green are TaqMan® and Molecular Beacons .
Both technologies depend on hybridization probes relying on fluorescence resonance energy transfer.( FRET) and quantization

TaqMAN ®

TaqMAN ® Sequencing

TaqMAN ® probes

Documentation of Amplification
The light emitted from the dye in the excited state is received by a computer and shown on a graph display, such as this, showing PCR cycles on the X-axis and a logarithmic indication of intensity on the Y-axis.

Molecular Beacons
Molecular Beacons
Uses FRET Fluorescence Resonance Energy Transfer
Uses two sequence specific
Oligonucleotide labelled with fluorescent dyes

Molecular Beacons – RT PCR
Molecular beacons are designed to adopt a hairpin structure while free in solution, brining the fluorescent dye and quencher in close proximity. When a molecular beacon hybridizes to a target the fluorescent dye emits light upon irradiation, and rebind to target in every cycle for signal measurement.

Loop Mediated Isothermal Amplification (LAMP)
Loop mediated isothermal amplification is a simple, rapid, specific and cost effective nucleic acid amplification method characterized by use of 8 distinct regions on the target gene.
The amplification proceeds at a constant temperature using strand displacement reaction.

LAMP
Amplification and detection of gene can be completed in a single step, by incubating the mixture of samples, primers DNA polymerase with strand displacement activity and substrates at a constant temperature of 63 0 c.

LAMP
Compared with PCR, and real time PCR, the LAMP has advantages of reaction simplicity and detection sensitivity.
The higher sensitivity and specificity of LAMP reaction is attributed to continuous amplification under isothermal condition employing six primers recognizing eight distinct regions of the target.

Advantages of LAMP
LAMP functions on isothermal amplification.
LAMP does not require any thermal cycler and thus cane be performed even with water bath/heating block
LAMP method do not require sophisticated temperature control devices
Cost effective

Lesser False Positives in LAMP
In LAMP both amplification and detection occur simultaneously during the exponential phase without going through the plateau phase where the non spurious amplification leads to lower sensitivity and false positivity.

Loop Mediated Isothermal Amplification in Clinical Diagnosis
LAMP technology proving to be ideal in detection of DNA or RNA of the pathogenic organisms
Proving to be highly efficient in diagnosis of Viral and Bacterial infections,
LAMP is capable of detecting the presence of pathogenic agents earlier than PCR

LAMP proving an efficient Technology
A one step single tube real time accelerated reverse transcription loop mediated isothermal amplification (RT-LAMP ) assays for rapid detection of some recently emerged viral pathogen eg West Nile, SARS, Dengue, Japanese encephalitis Chikungunya Norwalk, H5N1 highly pathogenic avian influenza., and CMV,HPV,VZV

Multiplex PCR
TaqMan probes and Molecular beacons allow multiple DNA species to be measured in the same sample ( Multiplex PCR) since fluorescent dyes with different emission spectra may be attached to different probes

Uses of Automated RT - PCR
Several viral infections can be detected in acute phase serum samples.
Increasing used in for early and accurate detection of all most human viruses including
Measles, Mumps, Herpes simplex viruses, Rota viruses Noro virus,Influenzae virus type A and B, Respiratory Syncitical virus, SARS, Dengue Japanese Encephalitis, Hepatitis B and C, West Nile, Chikungunya,HIV, Avian flu virus,

Multiplex PCR in Real Time
Multiplex real time quantitative RT-PCR assays have been developed for simultaneous detection identification and quantification of HBV, HCV and HIV-! In plasma and Serum samples.

Real-Time PCR Method Molecular Beacons
Molecular beacons are short segments of single-stranded DNA (Figure 1). The sequence of each molecular beacon must be customized to detect the PCR product of interest.

Molecular methods are necessary if the traditional methods provide poor results
Microscopy gives false positive results -
- T.vaginalis, N.gonorrhoeae
Intracellular pathogens – viruses, M.genitalium
Low sensitivity – Chlamydia sp.,Neisseria sp.
Seropositivity is common – Chlamydia sp .
Subtyping is mandatory – HSV, HPV, HCV
Microbial growth is slow – M. tuberculosis

How are you going to measure the PCR product
Directly
Sybr green
Quality of primers critical
Indirectly
In addition to primers, add a fluorescently labeled hybridization probe
Many different approaches to this, see Bustin J.Mol.Endocrinol. (2000) 25:169

Importance of controls
Negative control (no DNA)
checks reagents for contamination
No reverse transcriptase control
detects if signal from contaminating DNA
Positive control
checks that reagents and primers work
especially importance if trying to show absence of expression of a gene

Standards
Same copy number in all cells
Expressed in all cells
Medium copy number advantageous
correction more accurate
Reasonably large introns
No pseudo gene
No alternate splicing in region you want to PCR

Real-time PCR applications
Quantitation of gene expression
Pathogen detection
Viral quantitation
Array verification
Drug therapy efficacy
DNA damage measurement
Quality control and assay validation
Genotyping

Establishing PCR laboratory Sample handling DNA preparation Clean room Stock solutions Laboratory Mixing site Thermocycler Amplification Detection Documentation QC & QA Quality control & assurance R & D ( Research and development) Alternatives: - commercial kits - robots + kits No alternative

Emerging Technologies in Molecular Diagnosis

QIAGEN One Step RT-PCR Kit
The QIAGEN One Step RT-PCR Kit is designed for easy and sensitive one-step RT-PCR using any RNA template. A unique enzyme combination and specially developed reaction buffer ensure efficient reverse transcription and PCR in one tube.

RT-PCR in one step The Robus™ T I Kit is base
RobusT RT-PCR Kits perform cDNA synthesis and PCR amplification of cDNA successively in a single tube during a continuous thermal cycling

Uses and Advantages in Testing by PCR Methods
Clinical diagnostics: detection and quantification of infectious microorganisms, cancer cells and genetic disorders
Capable of amplifying long targets, up to 6.0 kb
One-tube system allows rapid, sensitive and reproducible analysis of RNA with minimal risk of sample contamination
Amplifies products from a wide variety of total RNA or mRNA sources

Advantages Molecular methods
High sensitivity and specificity
Detects pathogen, not immune response
Quick results
High transport toleration
In-house (home-brew) PCR methods
Cost effective
High sensitivity
High quality
Fast implementation of scientific discoveries
Customer friendly
R&D is absolutely necessary

ESTABLISHMENT OF A PCR LABORATORY
To perform PCR for the repetitive detection of a specific sequence, three distinct laboratory areas are required. The specific technical operations, reagents ,and personnel considerations

Prevention of Contamination in PCR Laboratory
PCR contamination be considered as a form of infection . If standard sterile techniques that would be applied to tissue culture or microbiological manipulations are applied to PCR, then the risk of contamination will be greatly reduced. Above all else, common sense should prevail.

Avoiding contamination
The single most important source of PCR product contamination is the generation of aerosols of PCR amplicons that is associated with the post-PCR analysis. Methods for eliminating this aerosol range from physical design of laboratories and use of specific pipettes to chemical and enzymatic approaches. The choice of method is often dependent on the frequency of amplification of a target amplicon and the relative amounts and concentrations of the amplicons created by the PCR.

Created for Awareness on Emerging Technologies in Developing World Dr.T.V.Rao MD Email [email_address]

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Molecular Methods In Diagnosis Of Infectious Diseases

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