PCR By Dr. Ricky Mittal

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Polymerase Chain Reaction in Diagnosis of Ocular Infections Presenter - Dr. Ricky Mittal Institute of Ophthalmology JNMC, AMU Aligarh

Polymerase chain reaction : 

Polymerase chain reaction PCR is a technique for in vitro amplification of specific DNA sequences via the temperature mediated DNA polymerase enzyme by simultaneous primer extension of complementary strands of DNA. PCR is a test tube system for DNA replication that allows a "target" DNA sequence to be selectively amplified, several million-fold in just a few hours.

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Polymerase chain reaction was invented by Kary Mullis and his colleagues in the 1983. Nobel prize 1993. It has become the most widely used nucleic acid amplification technology and gold standard for amplification processes in diagnosis and research.

Why “Polymerase”? : 

Why “Polymerase”? It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase.

Why “Chain”? : 

Why “Chain”? It is called “chain” because the products of the first reaction become substrates of the following one, and so on

The “Reaction” Components : 

The “Reaction” Components 1) Target DNA - contains the sequence to be amplified. 2) Pair of Primers - oligonucleotides that define the sequence to be amplified. 3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks. 4) Thermostable DNA Polymerase - enzyme that catalyzes the reaction 5) Mg++ ions - cofactor of the enzyme 6) Buffer solution – maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme

Templates for PCR : 

Templates for PCR Dried blood Semen stains Vaginal swabs Single hair Fingernail scrapings Insects in Amber Egyptian mummies Buccal Swab Toothbrushes

Templates for PCR for ocular Diagnosis : 

Templates for PCR for ocular Diagnosis Conjuctival swab Corneal scrapings Aqueous Vitreous Sub retinal fluid Blood

The Reaction : 

The Reaction THERMOCYCLER PCR tube

PCR tube : 

PCR tube

PCR machine : 

PCR machine

Agarose gel electrophoresis : 

Agarose gel electrophoresis Agarose gel electrophoresis is the easiest and commonest way of separating and analyzing DNA The purpose of the gel might be to look at the DNA, to quantify it or to isolate a particular band The DNA is visualised in the gel by addition of ethidium bromide

Agarose Gel : 

Agarose Gel

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Denature (heat to 95oC) Lower temperature to 56oC Anneal with primers Increase temperature to 72oC DNA polymerase + dNTPs

PCR Phases : 

PCR Phases Three phases: Exponential: Exact doubling of product is accumulating at every cycle (assuming 100% reaction efficiency). The reaction is very specific and precise. Linear: The reaction components are being consumed, the reaction is slowing, and products are starting to degrade. Plateau: The reaction has stopped, no more products are being made and if left long enough, the PCR products will begin to degrade.

Heat-stable polymerase is vitalto the ease of the process… : 

Heat-stable polymerase is vitalto the ease of the process…

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The Thermus aquaticus DNA polymerase Also called Taq polymerase Not permanently destroyed at 94ºC Optimal temperature is 72ºC

Thermus aquaticus: : 

Thermus aquaticus:

Variants of PCR : 

Variants of PCR Reverse transcriptase-PCR. Nested-PCR. Hot-start PCR. Quantitative PCR. Multiplex-PCR. Mutagenesis by PCR. Allele specific PCR. Inverse PCR. Asymmetric PCR. In Situ PCR. ….

Reverse Transcriptase-PCR : 

Reverse Transcriptase-PCR RT-PCR, one of the most sensitive methods for the detection and analysis of rare mRNA transcripts or other RNA present in low abundance. RNA cannot serve as a template for PCR, so it must be first transcribed into cDNA with reverse transcriptase from Moloney murine leukemia virus or Avian myeloblastosis virus, and the cDNA copy is then amplified. The technique is usually initiated by mixing short (12-18 base) polymers of thymidine (oligo dT) with messenger RNA such that they anneal to the RNA's polyadenylate tail. Reverse transcriptase is then added and uses the oligo dT as a primer to synthesize so-called first-strand cDNA. Roche Molecular Biochemicals: PCR Application Manual. RT-PCR

Nested PCR : 

Nested PCR Nested PCR is a variation of the polymerase chain reaction (PCR), in that two pairs (instead of one pair) of PCR primers are used to amplify a fragment. The first pair of PCR primers amplify a fragment similar to a standard PCR. However, a second pair of primers called nested primers bind inside the first PCR product fragment to allow amplification of a second PCR product which is shorter than the first one. Nested PCR is a very specific PCR amplification. http://www.pcrstation.com/images/nested-pcr.gif

Hot Start PCR : 

Hot Start PCR Hot Start PCR significantly improve specificity, sensitivity and yield of PCR. Some components essential for polymerase activity is separated from the reaction mixture until the temperature in the tubes has exceeded the optimal primer annealing temperature usually 55-65 C ˚.

Hot Start PCR : 

Hot Start PCR The technique may be performed manually by heating the reaction components to the melting temperature (e.g., 95˚C) before adding the polymerase. Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step.

Quantitative PCR : 

Quantitative PCR The determination or quantitation of the number of copies of a given gene achieves accurate estimation of DNA and RNA targets. Real Time PCR Real-time PCR uses a fluorescent reporter signal to measure the amount of amplicon as it is generated. This kinetic PCR allows for data collection after each cycle of PCR instead of only at the end of the 20 to 40 cycles. www. AppliedBiosystem.com Real Time PCR

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The real time system reduces the time required for PCR amplification and analysis from hours to minutes. Monitor amplification in real-time. Quickly and accurately quantify results. Analyze melting characteristics of PCR product. Combining online detection and continuous fluorescence monitoring allowed more rapid quantification of PCR product. The recent development of real time PCR clearly demonstrates many advantages over other existing method with: high accuracy, wide dynamic range , specificity , sensitivity , reduced carry over contamination and rapid accurate and simultaneous quantification of multiple samples.

Applications of PCR : 

Applications of PCR Molecular Identification Sequencing Genetic Engineering Molecular Archaeology Molecular Epidemiology Molecular Ecology DNA fingerprinting Classification of organisms Genotyping Pre-natal diagnosis Mutation screening Drug discovery Genetic matching Detection of pathogens Bioinformatics Genomic cloning Human Genome Project Site-directed mutagenesis Gene expression studies

Applications of PCR in Ophthalmology : 

Applications of PCR in Ophthalmology Diagnosing ocular infections -PCR can diagnose almost any infection Bacterial – Gonococcus, staphylococcus, Pseudomonas, Moraxella etc Fungal - Aspergillus , Cryptococcus Sporotrichosis, Blastomycosis etc Viral - Herpes simplex Adeno virus etc

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Tubercular Chalamydia Rickettsia Protozoa ( acanthamoeba)

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Diagnosis of Bacterial & Fungal infection is easy by conventional lab. Techniques. BUT Diagnosis of Viral, tubercular, chalamydial, Rickettesial are time consuming & cumbersome

PCR in Diagnosis of Ocular T.B : 

PCR in Diagnosis of Ocular T.B CAN BE USED IN -Conjuctivitis -Scleritis -Keretitis -Uvetitis -Retinal vaculitis SPECIMENS -Conjuctival swabs -Aqueous humor -Vitreous -Subretinal Fluid -Blood

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Polymerase chain reaction (PCR) of subretinal fluid has been successful in identifying the presence of TB in cases where the culture result is negative in retinal vasculitis PCR for M tuberculosis was positive for some TB-positive corneal donors as well as TB-negative donors. However, all cultures were negative in some studies.

PCR in early identification of organism in endophthalmitis : 

PCR in early identification of organism in endophthalmitis PCR is helpful in early Identification of organism and species in cases of endophthalmitis after cataract & subsequent management Most useful in diagnosis of Viral endophthalmitis

Detection of Chlamydia trachomatis ocular infection by PCR : 

Detection of Chlamydia trachomatis ocular infection by PCR PURPOSE -correctly measure the true status of ocular Chlamydia trachomatis infection in individuals, particularly after treatment METHODS -swab specimens obtained from the upper tarsal conjunctiva

C. trachomatis: RESULT ? : 

C. trachomatis: RESULT ? -The increased sensitivity of the rRNA-based PCR test may be due to its ability to detect low levels of C. trachomatis infection in individuals, which can occur especially after antibiotic treatment

PCR for Detection of Ocular Toxoplasma gondii : 

PCR for Detection of Ocular Toxoplasma gondii Two cases are reported of intraocular inflammation in which severe Vitritis hampered the fundal view, making an accurate clinical diagnosis impossible and vitreous analysis using conventional techniques was unhelpful. PCR for Toxoplasma gondii was positive in both cases and provided the only way of Confirming the diagnosis.

PCR in the Detection of viral ocular infections : 

PCR in the Detection of viral ocular infections Ex.: The laboratory diagnosis of ocular adenovirus infection Diagnosis of herpes virus infection Two different sets of primer can be used to increase specificity of test in diagnosis of viral infections Photograph of patient with Adenovirus infection.

Detection of Protozoal infection (Acanthamoeba) : 

Detection of Protozoal infection (Acanthamoeba) Acanthamoeba keratitis is a protozoan infection caused by Acanthamoeba is often misdiagnosed as herpes simplex keratitis, bacterial keratitis, fungal keratitis Use of 18S rRNA Gene-Based PCR Assay for Diagnosis of Acanthamoeba Keratitis using corneal scraping has a higher specificity than culture or microscopy

Diagnosis of MRSA : 

Diagnosis of MRSA MRSA (super bug) infection increased drastically in last decade Early diagnosis is required as drug therapy differs

Diagnosis of rare ocular infection : 

Diagnosis of rare ocular infection Gemella heamolysans Kingella kingae Eikenella B. subtillis Onchocerciasis Pneumocystis carni

Other uses : 

Other uses Diagnosing, identification and characterization of immunodominant recombinant clones of C. trachomatis Rapid identification of resistant strain of Pseudomonas aeruginosa from ocular isolates by PCR

Advantages of PCR: : 

Advantages of PCR: Useful non- invasive procedure. Simplicity of the procedure. Sensitivity of the PCR.

Disadvantages of PCR: : 

Disadvantages of PCR: False positive results (cross contamination). False negative results (e.g. too little infectious material) Availability Cost

References : 

References www.Roche Molecular Biochemicals: PCR Applications Manual www.Roche Molecular Biochemicals: PCR Techniques www. AppliedBiosystem.COM Real Time PCR Trends in molecular medicine Vol. 8 No.6 june 2002. Quantification using real time PCR technology, application and limitation. Robert F. Weaver. Molecular Biology. Fourth Edition. Page 600. McGraw-Hill International Edition. ISBN 978-0-07-110216-2 Robert F. Mueller,Ian D. Young. Emery's Elements of Medical Genetics: ISBN. 044307125X Velasco J. A new view of malignancy New York Times. April 9, 2002.. Watson JD, Crick FHC. Molecular structure of nucleic acids. Nature. 1953;171:737–738 PubMed Stites DP . Medical immunology. Section II. Page 270 WWW.medscape.com www. pubmedcentral.nih http://en.wikipedia.org/wiki/RT-PCR http://www.ma.uni-heidelberg.de/inst/ikc/molekularbiologie/rt-pcr.jpg http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/mbglossary/mbgloss.htm http://www.dnalc.org/ddnalc/resources/electrophoresis.html

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Thanks