role of pcr in diagnostics

Role of PCR in DiagnosticsBy Dr Tariq MahmoodAssistant Professor,Department of Biochemistry,RIMS&R, Saifai.

What is PCR?PCR or Polymerase Chain

Reaction is a relatively inexpensive and quick, in vitro method for amplification of a segment of DNA or RNA.

It was mainly used as a research technique and as a tool for obtaining a genetic profile specially from a scant sample.

PCR technique is based on use of a thermo stable DNA polymerase to “replicate” the denatured DNA molecule in a thermocycler.

Also is required a pair of primers that bind close to the segment of interest and causes the amplification of only the desired region.

Also required are the dNTPs that are added onto the growing DNA strand.

In recent years the role of PCR in diagnostics has increased.

The use of PCR in clinical settings can be broadly divided into three categories:-To amplify human genes to check for

mutations.To amplify microbial genes in a sample.To amplify human gene from a limited

sample for creating a complete DNA profile of an individual.

PCR for diagnosis of Genetic diseases

DNA can be obtained from a tissue for the analysis of a particular gene.

But the process is cumbersome and time consuming.

PCR allows us to target a single gene of interest for amplification making the process easier, cheaper and quicker.

PCR is also of great use for prenatal testing.

The samples obtained for prenatal testing by methods such as chorionic villus sampling or Amniocentesis are limited in amount.

PCR can very effectively amplify the entire genome or the gene of interest obtained from limited DNA provided by these samples.

PCR for diagnosis of infections

PCR is largely being used in clinical settings for detection and characterization of pathogens in infectious conditions.

Till recently this was done mainly on basis of culture and then morphological and biochemical characterization of the organism.

This technique, in spite of its success, is not without its limitations.

A large number of pathogens cannot be cultured in a diagnostic lab settings.

Most viruses can only be grown in cell lines that are difficult and expensive to maintain in most lab settings.

Certain common pathogenic bacteria and virus such as M. leprae, HPV, Hep C virus have frustrated all attempts to culture them.

Even certain common pathogenic bacteria such as M. leprae cannot be grown on artificial media or even in cell lines making a definitive diagnosis difficult.

Pneumonia is the number one killer among infectious diseases in US. Inspite of this fact no etiological diagnosis is made in >35% cases.

Its estimate that less than1% of all bacteria have been described till date using culture technology.

Time taken by cultures to grow can range from days to weeks.

During this time clinicians are dependent on empirical therapy.

Apart from these inherent shortcomings of culture technology, it is also highly susceptible to pre and post analytical variables.

Time constrain on collection and transport of sample.

Risk of infection to the laboratory staff is always present.

Serological methods of diagnosis have different limitations.

Acute infections are not diagnosed.

Even Widal test shows cross reactivity with malaria confounding diagnosis of fever in tropical countries.

Why PCR is superior?

High sensitivity: Can theoretically detect a single microbe in the given sample.

High specificity: As unique non conserved domains are identified in microbe there is almost nil change of misdiagnosing.

The PCR can be run to detect a single pathogen, a group of closely related pathogens (broad range PCR) or for all organisms or large number of unrelated organisms in a single run (Multiplex PCR).

The speed of diagnosis is unrivalled. Reduced from days/week to hours.

Diagnosis not dependent on whether the pathogen is cultivatable or not.

Changes in phenotypic expression of certain traits have no effect on diagnosis.

Diagnosis not dependent on microscopic diagnosis that requires experience.

The problem of transportation of live sample largely solved.

The problem of exposure of laboratory staff to dangerous pathogens is also solved.

Real Time PCR can also give a estimation of microbial load in a sample.

Limitations of PCR

Problems of False positives: It is the methods greatest drawback and is due to it’s inherent sensitivity.◦Caused by contamination of the sample.◦Due to Broad Range PCR detecting

microbial DNA present in reagents.◦In absence of clinical correlation even

DNA from dead organisms present in circulation may be detected as a false positive.

Problem of False Negatives: Do occur but not an endemic problem such as that of false positives. Causes are:◦ Improper processing of samples for DNA

extraction. ◦Samples may contain inhibitors of PCR.

Can be monitored by using a human gene as an amplification standard or use standards provided with kits.

Cannot right away comment on antibiotic sensitivity, as in culture sensitivity, though genes responsible for antibiotic sensitivity can be detected.

Sample requires special circumstances for storage. ◦ Should be frozen◦ Should be stored in a Magnesium free

environment to thwart action of nucleases.◦ Should not be fixed in Formalin.

Cost of testing is a major limiting factor. Costs of reagents alone may run into tens of thousands.

In spite of high sensitivity and specificity these limitations have held back the development of PCR as a go to method for microbial detection.

Take Home MessageThe take home message will be best

provided in form of this example of Herpes simplex virus PCR used in HS encephalitis (HSE).

Before advent of PCR the gold standard for diagnosis of HSE was a brain biopsy, an invasive procedure associated with considerable mortality and morbidity.

With the advent of PCR a lumbar puncture is suffice.

Studies have shown PCR to be more sensitive than brain biopsy in cases of HSE, with much fewer false negative cases.

So in this case not only is PCR easier to perform than the earlier gold standard test but also provides much better results with fewer false negatives and rare false positives.

Another important example is the diagnosis of M. tuberculosis infections.

Till date three established methods are used in conjunction to reach a diagnosis.◦Z H staining for AFB. ◦Radiography.◦Culture.

These methods have severe limitations right from sample collection to interpretations of the results.

Sereological and immunological methods of diagnosis have fallen by the side.

These problems have led to both missed cases in some group of patients and over treatment in others.

PCR has a better sensitivity than conventional methods for diagnosis of M. tuberculosis infections resulting in lesser false negatives.

A study in Nairobi done on 1396 suspected cases has revealed the following data:◦Sensitivity of 93% and specificity of 84%.◦PCR detected M. tuberculosis infection in

11.7% culture negative cases.◦It increased the yield of true positive cases in

comparison of culture.

Conclusion

PCR, in spite of its shortcomings, has proven itself much more sensitive than the gold standard tests in many cases.

It overcomes various barriers seen in serological and culture assays.

In hand of competent technicians its specificity can also approach gold standard methods such as culturing the pathogen with a much higher sensitivity.

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