What is PCR/RT-PCR
Polymerase Chain Reaction (PCR) is at the forefront of Molecular Diagnostics Technology today. Since it’s inception in 1985, PCR has revolutionized the molecular genetic diagnostics. Real time PCR (quantitative PCR, qPCR) is now a well-established method for the detection, quantification, and typing of different microbial agents in the areas of clinical and veterinary diagnostics and food safety.
The starting material for PCR/RT-PCR is a gene or segment of DNA called the 'target sequence” which can be amplified a million fold within few hours. The PCR reaction involves alternate steps of heat denaturation of target sequence, annealing of the primers to the complementary strands of the heat denatured DNA and then extension of the target sequence flanked by the primers. In case where RNA is the genetic material, the RNA strands are first copied to form the complimentary DNA called the cDNA by using the enzyme reverse transciptase and then follow the cycles of heat denaturation, annealing and extension of the target sequence. Within a short time, exact replicas of the target sequence are produced. At the end of many cycles, the pool is greatly enriched in the small pieces of DNA that have the target sequences, and this amplified genetic information is then available for further analysis.
Why PCR and How Sensitive It Is
If the amplified target DNA sequence is of a pathogenic agent or certain cells with genomic anomalies then in combination with a sequence specific probe PCR helps in identifying specific disease strains. In particular when specific pathogens that is difficult to culture in-vitro or require a long cultivation period, PCR plays a conclusive role in confirming the pathogenesis in the specimen.
PCR technology now permeates all the molecular biology. The sensitivity of the technique that it can amplify from even a single molecule of the DNA allow the clinicians to diagnose infectious agents such as the tuberculosis, HIV, Hepatitis ,COVID 19 etc at an early stage of the infection and to maintain the progression of the disease and the response to therapeutic agents.
What is Required and Why
Due to its high sensitivity PCR techniques always require a controlled air and sterile clean room environment to pursue the PCR based diagnostics. One should have separate areas for collecting, processing and molecular diagnostics of the patient samples. The processing of the samples is always unidirectional i.e. different air controlled areas are allocated for different steps of sample processing.
For many PCR applications specifically in the diagnosis of the infectious or fatal cytological diseases it is essential that only the specific DNA template representing the clinical specimen should enter the reaction. Thus for maintaining the stringency and accuracy of the PCR reaction so as to avoid any false positive or false negative results a clean and contamination free facility is required.
From very first step of collecting the patient for isolation of DNA, amplifying it and then confirming the presence of the amplified DNA molecules a clean and safe environment has to be maintained and utmost care should be taken for selecting the consumables like plastic wares, PCR machine and the reagent kits. The equipments must be provided separately for each area.
Also the most significant thing is that the samples being handled in the diagnostics are the hazardous and infectious thus proper care must be taken for handling these samples and one should always use good quality gloves. Sometime it has been seen that wearing gloves for long time causes irritation or skin allergy to the user. This may make the user prone to caught by the fatal infections like with hepatitis or HIV etc. In such cases one should use the gloves which are free from natural rubber proteins and if possible should use the gloves treated with antiallergic compounds like aloe powder.
DNA and RNA are susceptible to degradation with enzymes like DNase and RNase which eventually may lead to false negative results similarly a false positive result may be obtained if there is the incorporation of amplicons from any previous positive sample. Amplicons are the amplified DNA fragments which may be present in the aerosol at the work environment or most possibly in the micropipettes which are routinely used for handling the amplified DNA samples. These problems may be avoided by using the DNase / RNase and pyrogen safe plasticwares; likewise we may get rid of the problems of amplicon contamination by using the sterile filtered tips with the autoclavable pipettes. The filtered tips provide a hydrophobic barrier which prevents the entry of the aerosol containing the amplicons into the channel of micropipettes.
Further for isolating the genomic material like DNA / RNA from the clinical samples it is favorable to use the pre-standardized kits of excellent quality which could give a maximum recovery of the genetic material. Using kits is safer and save a very precious time by eliminating the need of preparing and standardizing the buffer etc. and allow more samples to be processed in a shorter time. The genetic material should always be isolated and extracted in cool chain environment starting from the collection of the clinical sample to the final processing.
The next question that knocks the mind which PCR/RT-PCR machine will be suitable for the Diagnostic PCR. As the diagnostic is a very decisive application of PCR and even a slight variation in the reaction conditions may play with the life of a patient we must always keep in the consideration the uniformity of the PCR amplification reaction. Even the very small slips may accumulate to cause serious deviation in the final outcome of the PCR results. Whenever we select a PCR machine for molecular diagnostics the first and the major issue is always the uniformity of the temperature across the heating block of the PCR machine. And also for avoiding the delay in obtaining the results of the diagnostics is only possible with a PCR machine with high ramping rate, which reduces the time taken for the heating and cooling the block during the heating and cooling cycles of the reaction. Further, all the PCR tubes/plates should be with uniform wall thickness to avoid the deviation in the thermal heat transfer and uniform amplification of the DNA / RNA samples.
Finally the results of the PCR reaction can be observed either by the liquid hybridization method in which the appearance of colour is compared with the positive controls calorimetrically using ELISA reader. This requires a sensitive ELISA reader and suitable enzyme labeled probes which may increase the cost of the diagnosis. However the results can also be observed on resolving the amplified fragments by agarose gel electrophoresis and comparing the bands of the amplified DNA with a positive marker band. This requires an electrophoresis apparatus with a horizontal gel tank. To avoid any contamination due to the environmental factors it is worthwhile to use the electrophoresis apparatus which require a low buffer volume and provides a selectivity of the voltage and current for maximum resolution of the DNA bands and could prevent the diffusion of the DNA bands even after completion of the electrophoresis.
For RT-PCR ( Realtime PCR) you get the results on screen on real time and there is no need to run the electrophoresis gel .
It’s You To Decide
However one should consider ones unique requirements while establishing a PCR diagnostic laboratory and should carefully evaluate who are the potential customers; what is needed to start; how consistently it is available and then just enjoy the test of success.
- Written By - Dr. Anita Kapur (Country Head - Technical, Genaxy Scientific Pvt. Ltd.)