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Real Time PCR: Latest advancement in diagnostics
Dr T K Ghosh
The advent of Polymerase Chain Reaction (PCR) by Kary B Mullins
in the mid 1980s revolutionised molecular biology. PCR is a fairly standard
procedure now, and its use is extremely wide-ranging. At its most basic application,
PCR can amplify a small amount of template DNA (or RNA) into large quantities
in a few hours. This is performed by mixing the DNA with premiers on either
side of the DNA (forward and reverse), Taq polymerase (of the species Thermus
aquaticus, a thermophile whose polymerase is able to withstand extremely high
temperatures), free nucleotides (dNTPs for DNA, NTPs for RNA), and buffer.
The temperature is then alternated between hot and cold to denature and reanneal
the DNA, with the polymerase adding new complementary strands each time. In
addition to the basic use of PCR, specially designed primers can be made to
legate two different pieces of DNA together or add a restriction site, in addition
to many other creative uses. Clearly, PCR is a procedure that is an integral
addition to the molecular biologist’s toolbox and the method has been continually
improved upon over the years. (Purves, et al 2001) Recently, a new method of
PCR quantification, called “real time PCR” has been invented. This
allows the scientist to actually view the increase in the amount of DNA as it
is amplified. Most commonly used types are TaqMan real time PCR, molecular beacon
and SyBR Green.
Real time chemistries allow for the detection of PCR amplification during the
early phase of the reaction. Measuring the kinetics of the reaction in the early
phases of PCR provides distinct advantage over traditional PCR detection. Traditional
methods use Agarose gels for detection of PCR amplification at the final phase
or point of the PCR reaction.
The Applications
The recurrence of infectious disease is one of the main challenges
in modern medicine. Opposed to serological methods, real time PCR allows a direct
verification of the presence of viruses, bacteria and parasites in a wide range
of sample materials in short time. Hence false negative results due to the absence
of specific antibodies during window periods are considerably reduced.
Quantitative real time PCR (qr PCR) has been utilised in humans to measure viral
load for diagnosis of different disease caused by DNA viruses such as CMV, EBV
and HBV. Investigators have adopted this system to titer viral genome density
in clinical specimens as an alternative to antigenemia assays.
Tanaka and colleagues demonstrated that the CMV load in peripheral blood leukocytes
was positive in 100 per cent of immunocompromised symptomatic patients and in
24 per cent of asymptomatic patients as well as in the plasma of 76 per cent
of symptomatic patients studied, paralleling and enhancing the results of the
antigenemia assay in all cases. Moreover, the monitoring of patients with CMV
infection demonstrated that the CMV DNA copy number changed proportionally with
anti CMV therapy, confirming that this technique is quite helpful for diagnosing
the onset of CMV-related disease and monitoring virus reactivation in patients
with latent CMV infections. Other investigators quantified the serum load of
CMV and other viruses after anti-viral treatments or post transplantation using
this technique.
Although qr-PCR has been applied mostly to virus related investigations, some
authors have reported on the utilisation of this technique to study the immunological
phenomena occurring during HLA class I peptide based immunotherapy for metastatic
melanoma. Recurrence of haematological cancers has also been evaluated using
qr-PCR. To measure the risk of replace of T-lineage Acute Lymphoblastic Leukemia
(ALL) in children, these researchers investigated the detection and quantification
of residual leukemia cells that harbor the TAL-1 detection.
TAL-1 qr-PCR is a sensitive and accurate method for assessment of minimal residual
disease (MRD) in T-lineage ALL. In the wake of these results, a Japanese group
utilised qr-PCR to monitor the MRD in leukemia and lymphoma patients by assessing
PRAME (preferentially expresses antigen of melanoma) expression in peripheral
blood sample.
Moreover, PRAME positive cell lines were found to be susceptible
to lysis by specific cytotoxic T lymphocytes, suggesting that this tumor-associated
antigen can present a target for a cell mediated immune response. Since reverse
transcriptase PCR is often used for detection of micrometastasis in blood, lymph
nodes and bone marrow, some investigators analysed the expression level of cytokeratin-18
(CK18) mRNA by qr-PCR in gastrointestinal carcinoma cell lines. As high CK18
expression levels were detected in carcinoma cell lines using this technique,
compared to levels in non-epithelial cells, it was concluded that not only qualitative
but also quantitative analysis of target mRNA is important of detection of micrometastasis
in cancer.
Conclusion
Quantitative real-time PCR is a method to rapidly and precisely quantify gene
activity by detecting mRNA levels of the gene of interest. So, with the ability
to collect data in the exponential growth phase, the power of PCR have been
expanded into applications like:
- Viral quantitation
- Quantitation of gene expression
- Assay verification
- Drug therapy efficacy
- DNA damage measurement
- Quality control and assay validation
- Pathogen detection
- Genotyping
Thus applications of Real-Time PCR should allow the rapid identification of
new diagnostic targets as well as assist in the development and improvement
of treatments. For the first time in eastern India, B M Birla Heart Research
Centre, Kolkata offers Real-Time PCR to detect infectious disease profile eg.
HIV, HBV, HCV, MTb, CMV and HSV.
The author is chief pathologist, BM Birla Heart Research
Centre and Calcutta Medial Research Institute, Kolkata
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