The document discusses the increasing role of PCR in medical diagnostics. It begins by explaining what PCR is and how it works to amplify DNA segments. It then describes the three main uses of PCR in clinical settings: 1) to detect genetic mutations, 2) to detect microbial genes in samples, and 3) to amplify human DNA from limited samples. The rest of the document provides examples of how PCR has improved the diagnosis of genetic diseases and infections compared to previous methods. It concludes that while PCR has limitations, it has proven more sensitive than gold standard tests in many cases by overcoming barriers of other diagnostic techniques.

1. Role of PCR in Diagnostics
By Dr Tariq Mahmood
Assistant Professor,
Department of Biochemistry,
RIMS&R, Saifai.

2. 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.

3.  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.

5.  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.

6. 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.

7.  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

8. 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.

9.  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.

10.  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.

11.  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.

12.  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.

13.  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.

14. 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.

15.  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.

16.  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.

17.  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.

18. 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.

19.  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.

20.  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.

21.  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.

22. Take Home Message
 The 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.

23.  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.

24.  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.

25.  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.

26.  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.

27. 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.

28. THANK YOU