Drawbacks of conventional methods

takes longer time delay in starting appropriate treatment needs more labour needs more resources should be confirmed by molecular methods

Classified as

Nucleic-acid based methods immunological assays miscellaneous methods

detection and manipulation of nucleic acids (DNA & RNA) & allowing microbial genes to be examined directly. Divided into three categories i) hybridization ii) amplification iii) sequencing & enzymatic digestion of nucleic acids

 Based

on the ability of two nucleic acid strands that have complementary base sequences to specifically bond with each other & form a double-stranded molecule or duplex The base adenine always binds to thymine & guanine with cytosine

 Require

one nucleic acid strand that originate from an organism of known identity (the probe) & the other strand that originate from an unknown organism to be detected (the target) Positive hybridization identifies the unknown organism as the same as the probe-source organism The single-stranded nucleic acid may be a DNA or RNA

 Production

& labeling of single-stranded probe nucleic acid Preparation of single stranded target nucleic acid Mixture & hybridization Detection of hybridization

 Both

probe and target nucleic acids are mixed in solution or solid formats Positive hybridization is detected depending upon the reporter molecule Reporter molecule may be radioactive ( 32P, 125I, 35S), biotin-avidin, digoxigenin, chemiluminescent labels

POLYMERASE CHAIN REACTION

Polymerase

Chain Reaction (PCR) was first described by Kjell Kleppe & Indian nobel prize winner Har Gobind Korana

 The

improvements provided by Dr.Kary Mullis have made PCR a central technique in biochemistry and molecular biology. Kary received a Nobel Prize in chemistry in 1993 , for his invention of the polymerase chain reaction (PCR).

 Based

on nucleic acid amplification techniques Most widely used method Nucleic acid replication occur through numerous cycles A single copy is multiplied to 107 or more copies within a short period

 PCR

involves 25 to 50 repetitive cycles cycle with 3 reactions

Each

denaturation of target nucleic acid primer annealing to single-stranded target nucleic acid extension of primer-target duplex

Cut

Paste Amplify

Heat causes DNA strands to separate

5 3 5

Denature DNA strands 94oC

5 3

 Primers (short oligonucleotides about 20 to 30 nucleotide long) containing sequences complementary to the target region are the key components to enable selective and repeated amplification. Primer nucleotide sequence depends on the intended target such as genus-specific genes or species-specific genes or virulence genes

Primers bind to the target sequence

5 3

Primers anneal 54oC

5 3
5 3 3 5

3 5

 Extension

is done by DNA polymerase which add nucleotides to the 3 terminus of each primer & produce by extension a sequence complementary to the target template Taq polymerase is the enzyme commonly used for primer extension Extension occurs at 72C

Taq Polymerase extends primer

DNA is replicated
5 3
5 3

3
3
5

Extend 72oC
5 3
5 3 3 5

3 5

Repeat denaturing, annealing, and extending 30 cycles

Cycle 1 Cycle 2
3

5
3 5 3 5

5 3

Cycle 3

3 5 3 5

5 3 5 3

 Denaturation

93 to 95C

1min

Annealing

50 to 55C

45sec

Elongation

70 to 75C

1-2min

How does PCR work?

Heat (94oC) to denature DNA strands Cool (54oC) to anneal primers to template

Warm (72oC) to activateTaq Polymerase, which extends primers and replicates DNA Repeat multiple cycles

 Amplifies

an RNA target Most useful for the viral infections Reverse transcriptase is used which directs synthesis of DNA from viral RNA template within 30 mts. Routine PCR technology is followed with the DNA

 Also

called quantitative real time polymerase chain reaction (Q-PCR/qPCR) or kinetic polymerase chain reaction standard for rapid diagnosis

Gold

 Thermocycling

or target DNA amplification & detection of target DNA by fluorescently labeled probes are both combined
vessel is not opened in between so cross contamination is greatly lessened

Reaction

 Able

to quantitate the amount of product & so determine the number of copies of target in the original specimen to complete the process is significantly less

Time

Some

systems as little as 20 to 30 mts.

 Rapidity
Sensitivity Specificity Samples

from different sources & several samples can be tested simultaneously Valuable genetic information is obtained

 Should

be carefully supervised Strict measures should be followed to prevent contamination Expensive Cannot differentiate viable & nonviable organisms

 FOR

DETECTION OF ANTIBODIES OR ANTIGENS

Techniques

for antibody detection a) Enzyme-linked immunosorbent assay b) radioimmuno assay

known antigen is incubated in wells in a plastic plate, antigens are adsorbed onto the plate microtitre plate The pts serum sample is added, incubated Washed, enzyme-labeled anto-globulin is added to the wells, incubated A chromogenic substrate is added, incubated Reaction is stopped by adding stop solution

 The

development of colour will indicate the presence of the antibody intensity of the colour can be read by ELISA readers & permits a quantitative measurement

The

 Similar

to ELISA Radiolabeled ligand is added instead of enzyme-labeled ligand The amount of radioactivity provides the titer of the antibody Measured by Geiger counter or a similar instrument

 The

antigen is an allergen The radiolabeled ligand will attach to the IgE specific for the allergen Useful in persons with Type I hypersensitivity

 Immunochromatographic

assays Sandwich-type immunoassays * ELISA * ECL (Electro-chemiluminiscence) * TRF (Time-resolved fluorescence assay) Immunofluorescence assays Flow cytometry

 Immunochromatographic

assays

simplest, within 15 mts. Sample diluted in sample buffer, applied over assay strips Least effective only one antigen is identified Low sensitivity, low specificity

Sandwich-type

very simple, very sensitive can be used for large no of small volume samples widespread use in epidemiology ECL detector antibody labeled with a chemiluminescent compound ruthenium TRF ab labeled with a lanthanide chelate

Immunofluorescence

a fluorochrome is attached to the antibody Monoclonal antibody is used Highly specific Two types direct indirect

 Immunoblotting

eg. Western blot antigens are separated by gel electrophoresis on the basis of size molecules transferred to nitrocellulose memb enzyme labeled antibody against the ag is added Used to confirm the presence of HIV1 & HIV2 ab in serum

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