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PCR-ELISA

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PCR–ELISA
By
Hanif Alfavian
INTRODUCTION
 The PCR, with its extraordinary sensitivity, is the method
of choice for the detection of nucleic acids present in very
low concentrations in biological specimens.pcr have
problem such as use Ethidium bromide and Time
consuming.
 The need for a method to detect PCR-amplified products,
which is able to offer sensitivity, specificity and an
objective evaluation of results, has led to the coupling of
PCR with ELISA.
 PCR–ELISA was initially developed to allow the direct analysis of
labeled amplicons using ELISA but, to improve the sensitivity and
specificity of the detection, a hybridization step with specific probes
has since been added.
Difinition:
 A sensitive(200 fg and 10 cell and more than 1000 senetive to compare
with agarose) and specific PCR–ELISA is therefore based on the labeling
of amplicons during PCR, their hybridization with probes specific for the
target, capture of labeled hybrids onto microtiter plates or tubes and
detection by immunoassay.
 PCR–ELISA methods are normally developed using the conventional microtiter plate format and
colorimetric detection, as detailed in Figure.
Figure:
PCR–ELISA for detection of target is
performed by capture onto streptavidin-
coated microtiter plates of biotinylated
specific probes hybridized with digoxigenin
(Dig)-labeled amplicons (DIG detection kit).
The detection of immobilized hybridized
amplicons is performed by anti-Dig
antibodies conjugated to peroxidase or
alkaline phosphatase with final colorimetric
development.
Pcr elisa

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Pcr elisa

  • 2. INTRODUCTION  The PCR, with its extraordinary sensitivity, is the method of choice for the detection of nucleic acids present in very low concentrations in biological specimens.pcr have problem such as use Ethidium bromide and Time consuming.  The need for a method to detect PCR-amplified products, which is able to offer sensitivity, specificity and an objective evaluation of results, has led to the coupling of PCR with ELISA.
  • 3.  PCR–ELISA was initially developed to allow the direct analysis of labeled amplicons using ELISA but, to improve the sensitivity and specificity of the detection, a hybridization step with specific probes has since been added. Difinition:  A sensitive(200 fg and 10 cell and more than 1000 senetive to compare with agarose) and specific PCR–ELISA is therefore based on the labeling of amplicons during PCR, their hybridization with probes specific for the target, capture of labeled hybrids onto microtiter plates or tubes and detection by immunoassay.
  • 4.  PCR–ELISA methods are normally developed using the conventional microtiter plate format and colorimetric detection, as detailed in Figure.
  • 5. Figure: PCR–ELISA for detection of target is performed by capture onto streptavidin- coated microtiter plates of biotinylated specific probes hybridized with digoxigenin (Dig)-labeled amplicons (DIG detection kit). The detection of immobilized hybridized amplicons is performed by anti-Dig antibodies conjugated to peroxidase or alkaline phosphatase with final colorimetric development.
  • 7. Real time pcr and pcr-ELISA  Newer PCR techniques such as real-time PCR are now successfully used for the qualitative and quantitative evaluation of nucleic acids.  Real-time PCR shows similar sensitivity to the PCR–ELISA, but has the advantage of being a sealed system, decreasing the risk of carryover contamination during the reaction, which can be a problem during PCR–ELISA.
  • 8.  Real-time PCR is also more rapid, since amplification and detection of target DNA can be completed within 1 h after DNA extraction.  PCR–ELISA, however, does not require the use of expensive equipment, as real-time PCR presently does, and instead uses basic instruments present in every diagnostic laboratory.  Another advantage of PCR–ELISA is that DNA purification step from serum samples,cytological and histological samples can be omitted.
  • 9.  In addition, the typing of different genotypes of an infectious agent is more easily performed by PCR–ELISA rather than with real-time PCR, which would require multiple amplifications and/or different detector molecules.  The ideal candidate regions for PCR primer design are those identified as having the least number of nucleotide sequence variations among different genotypes.(alignment from gene bank)
  • 11.  Several popular programs are available for PCR primer and probe design, such as Clone Manager Professional Suite (Scientific & Educational Software), Oligo (MedProbe) and Primer Premier (Premier Biosoft), which are easy to use.  (i) primers should be 18–30 bp long; (ii) they should have a G/C content between 40 and 60%; (iii) they must have a Tm not lower than 50 C and not differing by more than 10 C; (iv) primers should not form possible hairpin structures; (v) they should not form homodimers and heterodimers;