Molecular Methods in Diagnosis of Ocular

DiseasesDr.T.V.Rao MD

Dr.T.V.Rao MD 1

Eye most Complex Organ

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Ocular Microbiology an Emerging Science

Ocular microbiology remains an applied science The advancements in molecular biology and the newer technologies pave way for better understanding of ocular diseases The developments have made major contributions in the control and probably even eradication of many types of eye infections

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From Biology to Molecular Biology

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Molecular Biology in Ocular Disorders

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Watson and Crick The structure of DNA was described by

British Scientists Watson and Crick as long double helix shaped with its sugar phosphate backbone on the outside and its bases on inside; the two strand of helix run in opposite direction and are anti-parallel to each other. The DNA double helix is stabilized by hydrogen bonds between the bases

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Watson and Crick discovers DNA Feb 28th

1953

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DNAA purine always links with a

pyrimidine base to maintain the structure of DNA.

Adenine ( A ) binds to Thymine ( T ), with two hydrogen bonds between them.

Guanine ( G ) binds to Cytosine ( C ), with three hydrogen bonds between them.

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Important Methods

1 Nucleic acid probes

2 Hybrid capture

3 Branched chain DNA

4 Situ hybridization

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Nucleic acid probe

Nucleic acid fragment, labelled by a radioisotope, biotin, etc., that is complementary to a sequence in another nucleic acid (fragment) and that will, by hydrogen binding to the latter, locate or identify it and be detected; a diagnostic technique based on the fact that every species of microbe possesses some unique nucleic acid sequences which differentiate it from all others, and can be used as identifying markers or "fingerprints."

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Hybridization probe Hybridization probe is a fragment of DNA or

RNA of variable length (usually 100-1000 bases long), which is used to detect in DNA or RNA samples the presence of nucleotide sequences (the DNA target) that are complementary to the sequence in the probe. The probe thereby hybridizes to single-stranded nucleic acid (DNA or RNA) whose base sequence allows probe-target base pairing due to complementarily between the probe and target.

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Nucleic Acid Probes Accu probes from Gene

probe, In which it contain Chemiluminescent label and target the rRNA of the Microorganisms of interest.

It reads events in vivo or during the multiplication of organisms.

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DNA is Endless structure The rungs of the ladder

can occur in any order (as long as the base-pair rule is followed)

Those 4 bases have endless combinations just like the letters of the alphabet can combine to make different words.

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DNA Replication DNA replication is

semi-conservative. That means that when it makes a copy, one half of the old strand is always kept in the new strand. This helps reduce the number of copy errors.

So we remained what we were ?

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So we remained what we were

Because of Genes

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DNA to RNA DNA remains in the

nucleus, but in order for it to get its instructions translated into proteins, it must send its message to the ribosome's, where proteins are made. The chemical used to carry this message is Messenger RNA

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Blotting technique

Southern Blot

It is used to detect DNA.

Northern Blot

It is used to detect RNA.

Western blot

It is used to detect protein.

TYPES OF BLOTTING TECHNIQUES

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Nucleic Acid Hybridizations

The hybridization of a radioactive probe to filter bound DNA or RNA is one of the most informative experiments that is performed in molecular genetics. Two basic types of hybridizations are possible.

Southern hybridization - hybridization of a probe to filter bound DNA; the DNA is typically transferred to the filter from a gel

Northern hybridization - hybridization of a probe to filter bound RNA; the RNA is typically transferred to the filter from a gel

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Western blotting Western blotting is an Immunoblot ing

technique which rely on the specificity of binding between a molecule of interest and a probe to allow detection of the molecule of interest in a mixture of many other similar molecules.

In Western blotting, the molecule of interest is a protein and the probe is typically an antibody raised against that particular protein.

The SDS PAGE technique is a prerequisite for Western blotting .

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Western Blotting

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Restriction fragment length polymorphism

Sir Alec Jeffreys developed restriction fragment length polymorphism (RFLP), which quickly became the standard technique for DNA testing throughout the 1980s. RFLP provided the world with the first form of genetic testing based on DNA, the body's genetic material

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Restriction Fragment Length Polymorphism

(RFLP) Restriction Fragment Length Polymorphism (RFLP) is a technique in which organisms may be differentiated by analysis of patterns derived from cleavage of their DNA.

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RFLP The resulting DNA

fragments are then separated by length through a process known as Agarose gel electrophoresis, and transferred to a membrane via the Southern blot procedure.

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Spoligotyping Spoligotyping, a new method for

simultaneous detection and typing of M. tuberculosis complex bacteria, has been recently developed. This method is based on polymerase chain reaction (PCR) amplification of a highly polymorphic direct repeat locus in the M. tuberculosis genome.

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Spoligotyping in Tuberculosis

The well-conserved 36-bp direct repeats are interspersed with unique spacer sequences varying from 35 to 41 bp in size. Clinical isolates of MTC bacteria can be differentiated by the presence or absence of one or more spacers.

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Results analyzed by Computer

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DNA finger printing Every one of our DNA is equal except for only about

0.10 %. DNA finger printing lies in uniqueness of those

regions of DNA that do differ from person to person. Only 5 % of our DNA code rest do not code called in

past as Junk DNA and contain repeated sequences of base pairs

Called as Variable number of tandem repeats contain 20 to 100 base pairs and the same sequence is repeated one to 3 times in a row

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Documentation of Finger Printing for Records

Finger print means translating all the variable number of tandem repeats to visible records

All VNTR is tested for restriction length polymorphism which differ from species to species.

All the obtained material is blotted to Nylon or Nitrocellulose membrane ( Southern Blotting )

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RLFP to PCR

Isolation of sufficient DNA for RFLP analysis is time-consuming and labour intensive. However, PCR can be used to amplify very small amounts of DNA, usually in 2-3 hours, to the levels required for RFLP analysis. Therefore, more samples can be analysed in a shorter time.

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Opportunistic Infections are Difficult to Diagnose

Opportunistic pathogenic agents are increasingly encountered in ocular infections due to widespread use of topical and systemic immunosuppressive agents, increasing numbers of patients with (HIV) infection and with organ transplants who are on immunosuppressive therapy and cause ocular infections due to increased use of contact lens.

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Cataract Surgery Legal Implications

The dreaded infections endophthalmitis following cataract extraction and lens implantation often are caused by opportunistic pathogens

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Diagnostic methods in microbiology

Task of the method – to make the microorganism

“visible” and “measureable”

Difficult on several Occasions• Microscopy• Cultivation• Bio-testing• Immunological

methods• Biochemical methods• Molecular methods

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Contact Lenses increases Opportunistic Infections

These opportunistic pathogens also cause ocular infections due to increased use of contact lens. The dreaded infections endophthalmitis following cataract extraction and lens

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Collection of Optimal specimen is most Important

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Culturing Continues to be Less sensitive

Culture of intraocular specimens is considered as the gold standard in the diagnosis of endophthalmitis. under the most appropriate care, traditional microbiological methods yield positive results in only 60-70% of the clinically typical cases of endophthalmitis.

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Prior Antibiotic therapy reduces the isolate rate

Prior antibiotic therapy, small number of organisms in the samples, possible localized nature of infections in the lens capsule and fastidious growth requirement of the offending organisms, the organism not being recovered in roughly around 30-40% of the cases.

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Molecular diagnostics – how it works

Every organism contains some unique,

species specific DNA sequences

Molecular diagnostics makes the species specific DNA visible

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PCR methods are rapid and sensitive

PCR, as a specific, sensitive and rapid technique in the identification of the pathogen in the clinical specimen has been developed extensively over the past decade. Its value as a clinical tool is being increasingly recognized

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Polymerase Chain Reaction Methodology A Mile stone in

Medical History He had the idea to use a pair of primers to bracket the desired DNA sequence and to copy it using DNA polymerase, a technique which would allow a small strand of DNA to be copied almost an infinite number of times.

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Dr. Kary Mullis, wins Nobel Prize in 1993

Kary received a Nobel Prize in chemistry in 1993, for his invention of the polymerase chain reaction (PCR). The process, which Kary Mullis conceptualized in 1983, is hailed as one of the monumental scientific techniques of the twentieth century.

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PCR Liberates a Innocent Prisoner

KirkBloods worth case A Waterman Imprisoned for 9 years

on wrong evidences of Rape

Unmatched DNA by PCR makes a freeman

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Locates Genes for Color Blindness

Color Blind British John Dalton died in 1844

Request his eyes to be preserved

And to be investigated why he confused scarlet with green, and pink with blue

Recent PCR studies prove Dalton lacked a gene for making one of the three photo pigments essential for normal color vision.

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Color Blindness is x linked

The genes for our red and green colour receptors are located on the X-chromosome, giving women a redundant set of receptor genes.

This is why men are far more prone to colour-blindness than women.

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DNA – RNA - DNA

In Molecular biology, the 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.

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Common Tools of Molecular Biology

Nucleic acid fractionation Polymerase chain reaction Probes, Hybridization Vector, Molecular cloning Nucleic acid enzymes Microarray DNA sequencing Electrophoretic separation of nucleic acid Detection of genes: *DNA: Southern blotting; inSitu hybridization; FISH

Technique *RNA: Northern blotting *Protein: Western blotting, immunohistochemistry

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Current Uses of molecular Biology

The most recent applied technologies, genetic engineering, DNA finger-printing in the social and forensic science, pre and postnatal diagnosis of inherited disease, gene therapy and drug Design.

Molecular biology allows the laboratory to be predictive in nature, it gives information that the patients may be at risk for disease (future).

Major tool in Diagnosis of Infectious

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Restriction Endonuclease

If two organisms differ in the distance between sites of cleavage of a particular restriction endonuclease, the length of the fragments produced will differ when the DNA is digested with a restriction enzyme. The similarity of the patterns generated can be used to differentiate species (and even strains) from one another.

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Restriction Endonuclease Restriction endonucleases are enzymes

that cleave DNA molecules at specific nucleotide sequences depending on the particular enzyme used. Enzyme recognition sites are usually 4 to 6 base pairs in length.

The recognition sequences are randomly distributed through the DNA and recognizes different nucleotide sequences, and snips through DNA molecule.

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Taq polymerase Taq polymerase is a thermostable DNA

polymerase named after the thermophilic bacterium Thermus aquaticus from which it was originally isolated by Thomas D. Brock in 1965. It is often abbreviated to "Taq Pol" (or simply "Taq"), and is frequently used in polymerase chain reaction (PCR), methods for greatly amplifying short segments of DNA

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Disadvantages of Taq Pol

Taq mis-incorporates 1 base in 104.

A 400 bp target will contain an error in 33% of molecules after 20 cycles.

Error distribution will be random.

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Thermocycler is Back bone of PCR methodology

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

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PCR - Three basic Steps

Cut

Paste

Amplify

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PCR Primers

TTAACGGCCTTAA . . . TTTAAACCGGTT

AATTGCCGGAATT . . . . . . . . . .>

and

<. . . . . . . . . . AAATTTGGCCAA

TTAACGGCCTTAA . . . TTTAAACCGGTT

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Cutting, pasting and amplifying is the basis of

Reaction

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Denaturing Template

Heat causes DNA strands to separate

3’

5’

5’

3’

Denature DNA strands 94oC

5’

3’

3’

5’

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Annealing Primers• Primers bind to the template sequence

• Taq Polymerase recognizes double-stranded substrate

3’

5’

5’

3’

Primers anneal 64oC3’

5’

5’

3’3’ 5’3’5’

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Taq Polymerase Extends

3’

5’3’ 5’3’5’

Extend 72oC

3’

5’3’ 5’3’5’

5’

3’

5’

3’

• Taq Polymerase extends primer

• DNA is replicated

Repeat denaturing, annealing, and extending 30 cyclesDr.T.V.Rao MD 60

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

The 7th Baltic Congress in Laboratory Medicine, Pärnu 11.09.2004

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PCR occurs in cycles and Multiplies the DNA

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Applications of PCR

The standard specimen procedure can quantitate HIV-1 RNA in a range of 400-75,000 copies/mL.

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Advantages of PCR

Speed Ease of use Sensitivity Robustness

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Candida infections can be specifically identified

The fragments of 125-bp (EO3) and 317 bp (HSP) specific for C. albicans were used for amplification.

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Molecular methods proving highly Sensitive

It has been postulated that DNA sequencing of the universal nested PCR product may allow identification of the causative organisms in a number of culture are few

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PCR helps in several critical Conditions

PCR has also been evaluated in the diagnosis of fungal endophthalmitis using broad range primers as well as primers specific for C. albicans. Detection of fungal DNA by PCR in intraocular specimens will prove as a useful means of diagnosing endophthalmitis. It will greatly facilitate management decisions when conventional culture is negative.

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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.

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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

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Nested polymerase chain reaction

Nested polymerase chain reaction is a modification of polymerase chain reaction intended to reduce the contamination in products due to the amplification of unexpected primer binding sites.

Nested polymerase chain reaction involves two sets of primers, used in two successive runs of polymerase chain reaction, the second set intended to amplify a secondary target within the first run products

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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.

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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

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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.

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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.

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AdvantagesMolecular 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 friendlyDoctortvrao’s ‘e’ learning series

R&D is absolutely necessary

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Polymerase Chain Reaction Available for several

infections …. Chlamydia trachomatis Slow growing Mycobacterium

tuberculosis viruses like Herpes simplex virus , Varicella Zoster virus . Adeno virus in our laboratory for

corneal specimens

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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

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Disadvantages of PCR Methods

Expensive to the Developing world Need well trained, Manpower Coordination for quality control Adoption to changing needs Timely technical support False positive results due to Amplifications

Above all dedicated Staff

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Molecular Epidemiology in Eye Care

In several world health funded projects molecular methods are used for

1 Plasmid analysis 2 Genomic fingerprinting 3 Emerging drug resistance 4 Polymerase chain reaction to identify

emerging and remerging microbes, 5 Determination of antibiotic resistance

patterns.

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Be Familiar with Molecular Biology

or ???

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Programme Created by Dr.T.V.Rao MD for Awareness of Emerging Need for

Molecular Methods in Laboratory Diagnosis Email

doctortvrao@gmail.com

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