Molecular Methods in Diagnosis of Infect

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Molecular Methods in Diagnosis of Infectious Diseases : 

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

Diagnostic methods in Microbiology : 

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 Crick’s Discovery of DNA -Path to Molecular Medicine : 

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

Traditional Microbial Diagnostic Methods : 

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 : 

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

Genotyping Methods : 

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 : 

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 : 

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 : 

Complete genomic configuration

Restriction Endonuleases : 

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. : 

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

What is the Choice of Marker : 

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 : 

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 - 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 : 

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 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 : 

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 : 

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 : 

Amplified sequences are Blotted

PCR in Clinical Microbiology : 

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 : 

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 : 

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 : 

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 : 

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

Slide 26: 

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 : 

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 : 

REAL TIME ASSAYS

New Technologies – 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 : 

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 : 

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

Real Time Reporters : 

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 PCRCyber Green : 

REAL TIME PCRCyber 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 : 

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 : 

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 : 

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®

TaqMAN® Sequencing : 

TaqMAN® Sequencing

TaqMAN® probes : 

TaqMAN® probes

Documentation of Amplification : 

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

Molecular Beacons – RT PCR : 

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 (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 : 

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 : 

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 : 

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 : 

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 : 

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 : 

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 : 

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 : 

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 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 MethodMolecular Beacons : 

Real-Time PCR MethodMolecular 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 : 

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 : 

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 : 

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 : 

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 : 

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

Slide 59: 

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 : 

Emerging Technologies in Molecular Diagnosis

QIAGEN One Step RT-PCR Kit : 

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 : 

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 : 

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

Slide 64: 

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 : 

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 : 

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 : 

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 : 

Created for Awareness on Emerging Technologies in Developing World Dr.T.V.Rao MD Email doctortvrao@gmail.com