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Embee Diagnostics

 

PCR

Polymerase Chain Reaction 

Polymerase Chain reaction popularly known as PCR is one of the most powerful Diagnostic tools Modern Science has ever invented. Based on DNA detection of a specific Bacteria or Virus, or a genetic disorder as of today PCR is the ultimate in Human Diagnostics. It is due to the invention of PCR only that Human genome mapping became possible.

 The techniques which have been used earlier like ELISA, RIA are no doubt good for screening but have got their own Limitations also e.g. false positivity & negativity. However in PCR all these are ruled out due to specific detection of       diseases specific DNA. Deadly Diseases like Tuberculosis which are very difficult to diagnose in early stages can be detected by PCR just on the onset of the diseases. Not only this, the biggest advantage of detecting M.Tuberculosis by PCR is the fact that all the False positives are ruled out which is today a major problem  with the existing detection methods like ELISA. E.g. a TB ELISA kit has a sensitivity & specificity of just about 70% whereas the sensitivity & specificity of PCR is 100%.

 The detection by PCR helps in two ways .i.e.

 -            Early detection

 -            Confirmed detection.

Early detection helps the patients to be put on drugs at the earliest so that the patient has to be put on the therapy for the least possible period thereby saving the cost of drugs as well as the Mental agony of the patient.

Confirmed detection helps patient not being subjected to the therapy which is not required at all.

In cases of Viral diseases like HBV, HCV or HIV, not only does PCR help in confirmed detection but the Viral loads can be monitored for the patients on therapy thereby helping the physician understand whether the patient is responding to a particular drug or not which is just simply not possible by other methodologies.

PCR detection is not limited to only the Bacterial & Viral diseases. All the genetic disorders or Chromosomal aberrations can be detected by PCR besides a whole lot  of other application in HLA etc.

The technique has gained popularity in the International Scenario due to various benefits offered by the technology. There is an ever growing demand for investigations based on  PCR & in future majority of the tests wherever there is a possibility of getting a DNA will be performed by PCR only.

PCR has three basic steps:

n          EXTRACTION OF DNA

n          AMPLIFICATION OF DNA

n          DETECTION OF DNA.

DNA EXTRACTION:

The extraction process in the past involved very tedious procedures & was a limiting factor for diagnostic PCR. But with the advancement in technology the extraction procedures have become so simple that now the DNA can be extracted from any kind of sample within 20-60 minutes with some simple reagent addition and Centrifugation e.g. a kit like DNA-Prepmate-M. The entire proceedings of the test and accuracy of the tests depend upon a good extraction system used.

 

AMPLIFICATION OF DNA:

After DNA is extracted, it can be amplified to over a billion copies from even one DNA with the aid of Thermal Cycler. This is how it becomes possible to detect the DNA as the number of copies now are enough to be detected. There are several reagents etc. required for amplification like specific Primers, Taq, dNPTS, premixes led to a total simplification of the process as well as contamination control in the methodology.

 

DETECTION OF DNA:

Detection of DNA can either be done by an Electrophoresis system or a liquid Hybridization depending upon the fact that if a simple detection and confirmation is required then it could be done by Electrophoresis also but in case of quantification, a liquid hybridization is necessary.

 

INSTRUMENTS REQUIRED FOR EXTRACTION of  DNA & RNA:

For DNA extraction a Digital Non-refrigerated centrifuge of at least 1300 rpm with RCF of about 16000g. A good and reliable centrifuge is a must because DNA extraction involves steps which are sensitive to the accurate speed & RCF 

For RNA extraction, a Digital Refrigerated centrifuge of 14000 rpm with RCF of about 15000-16000 g. A good and reliable refrigerated centrifuge is a must because RNA is sensitive to temperature & the extraction involves steps which are sensitive to the speed & RCF.

A good autoclavable set of Micro-pipettes is required for DNA or RNA extraction in the range of 2-20ml, 20-200ml, 200-1000ml,.

A variable speed of say 0-3000rpm Vortex Mixer is required which should have a heavy duty construction.

A microwave oven for Lysis of the cells will also be required.

A water bath for boiling & incubations.

 

INSTRUMENTS REQUIRED FOR AMPLIFICATION OF DNA OR RNA:

Thermal Cycler is the instrument which can amplify one DNA to almost  a billion copies. In the initial stages the amplification of DNA was done by robotic use with the help of several water baths. The invention of Thermal Cycler led to a total revolution in PCR. Today the amplification of DNA is just possible within 2 hours with the help of a thermal Cycler. Amplification process involves three steps i.e.

--   Denaturation of DNA at 96C.

         --   Anneling of Primers at 56 C.

         --   Extension of primers at 72 C. 

All these steps are performed by a Thermal Cycler.

A good autoclavable set Micro-pipettes is required for DNA or RNA extraction in the range of 2-20ml, 20-200ml, 200-1000ml,.

 

INSTRUMENTS REQUIRED FOR DETECTION OF AMPLIFIED PRODUCT:

After the amplification process for detection based on the methodology used, for detection following Instruments are required:

Gel Electrophoresis System: A small Gel electrophoresis system which is capable of providing a consistent 50 or 100 volt current. Should be flexible enough to accommodate small or large gels.

UV TRANSILLUMINATOR: This instrument is required for Visualization of the bands which will be generated by electrophoresis.

GEL DOCUMENTATION SYSTEM: This system along with the software provided with it is capable of giving a detailed analysis of the bands as well as is capable of giving a detailed results for subsequent matching or use.

ELISA READER: This is required for those PCR assays which are Based on Liquid hybridization for the calorimetric estimations.

There are few other instruments during the process for sample processing as well  as for the preparatory work e.g. 

  • LAMINAR FLOWS: required for processing samples for extraction & while preparing the reagents mixes.

  • AUTOCLAVE: For sterilizing the Triple distilled water, microfuge tubes, micro tips, pipettes etc.

  • Incubator: required for incubation in case of Liquid Hybridization.

  • -20 C Deep freezer: For storage of reagents etc.

  • +4 C 1KVA:For thermal Cycler for continuous operations.

 

SPACE REQUIRED FOR PERFORMING PCR.

Three clean rooms of approximately the size of 6x 6 feet are enough. Two rooms preferably should have a small Anti-room type of an opening but is not essential.

 

PARAMETERS AVAILABLE IN PCR:

These days availability of parameters by PCR is not a problem & a whole lot of  parameters as available.

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Feature of the month - PCR (Polymerase Chain Reaction)
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