Molecular Methods in Diagnosis of Infectious Diseases

Dr.T.V.Rao MD

Diagnostic methods in Microbiology

Task of the method to make the microorganism visible and measureable

Cultivation Bio-testing Immunological methods Biochemical methods Microscopy Molecular methods

Watson and Cricks 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 doublestranded 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, realtime 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
-A-A-T-T-C-G-C-G-A-T-G- T-T-A-A-G-C-G-C-T-A-C-

Amplification (synthesis) of species specific sequences

PCR polymerase chain reaction -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 SYBERGreen
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 Xaxis 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 630c.

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

QC & QA
Quality control & assurance

DNA preparation

No alternative
Laboratory Thermocycler Detection Documentation

Mixing site

Amplification

Clean room

R&D
(Research and development)

Stock solutions

Alternatives: - commercial kits - robots + kits

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 R&D is absolutely necessary High quality Fast implementation of scientific discoveries Customer friendly

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 doctortvrao@gmail.com