Documentos de Académico
Documentos de Profesional
Documentos de Cultura
takes longer time delay in starting appropriate treatment needs more labour needs more resources should be confirmed by molecular methods
Classified as
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 (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
Each
denaturation of target nucleic acid primer annealing to single-stranded target nucleic acid extension of primer-target duplex
Cut
Paste Amplify
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
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
3
3
5
Extend 72oC
5 3
5 3 3 5
3 5
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
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
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
Techniques
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
THANK YOU