Introduction:
Coronavirus is a part of SARS family virus which is known to cause respiratory diseases and viral infection and also has major health issues. The target organ is lungs, which might be affected through aerosols and infected droplets. It is a single stranded RNA virus and it provoked a large scale epidemic. Rapid multiplication of virus is also a complicated factor in the detection of the disease. Detection of virus by RT- PCR offers the option of diagnosis in the early stages of the disease. The clinical sensitivity of the first generation RT- PCR methods during the first few days of the disease has been low and better sensitivity is obtained after a week of illness. The spread control option is difficult as there is no direct contact followed by isolation and there is difficulty in handling household expenses.
As we know Coronavirus, causes an irresistible sickness and was declared as a pandemic. The virus is secured by fat protein which is has abundant RNA genome. The quick duplication of infections is additionally an entangled factor. Consequently the recognition of this sickness can be determined by different strategies like RT-PCR (Real time Polymerase Chain Reaction), CT (Computed Tomography) and so on. RT-PCR is one of them. The detection of infection by using RT-PCR in clinical samples offers the option of conclusion and its application involves genotyping and mutation detection. RT- PCR technique is found to be a standard method for diagnosing the positive cases. In this way it helps us in finding the true negativity and also rule out positive cases.
PCR in the diagnosis of corona:
It is said to be the gold standard method which helps in diagnosing positive cases of COVID 19. The virus initially results in the conversion of RNA to DNA template which is the RNA dependent DNA polymerase. The elimination of the post amplification handling helps in reduction of risk of contamination which will become faster around time and will have higher sensitivity. It also helps in distinguishing the asymptomatic viral shedding and also has sensitivity
and specificity) of detection. Due to its disadvantages of other methods, RT- PCR remains the most useful laboratory diagnostic test for the COVID 19. It decreases the probability of susceptible and infectious people coming in contact through the early assessment of cases and reduction of contact.
Advantages of RT-PCR:
In RT- PCR technique,(cDNA) complementary DNA is made by reverse transcribing of RNA template by the enzyme reverse transcriptase. Viable microorganisms further help in quantifying the virulence with toxin or stress response gene transcription and also the microbial growth. It makes easier for us to find the sensitivity and specificity of the detection. Previous research done with the PCR shows major benefits of using it. The target specificity of PCR assay helps in designing of taxonomic and functional gene quantification and also in the quantification of individual species of phylotypes.
Disadvantages of RT- PCR:
The RT- PCR are also has many disadvantages associated such as the sensitivity, reproducibility and specificity. The complication due to rapid multiplication of the virus may give false positive cases. Another problem is the inadequate availability of samples. The real time PCR is difficult in detecting the gene, the target specificity in designing of the primers. The fluorescence readings with no template controls, is difficult in dye molecule bonding with primers and dimers.
Limitations of PCR in the diagnosis of corona:
The reaction starts generating copies of the target sequence exponentially only during the exponential phase of the PCR reaction. Detection of circulating tumor cells in
blood can be done with PCR. It helps to detect small amounts of fungal DNA. Inhibitors of the polymerase response was found in the sample, reagent confinement, amassing of pyrophosphate particles and self-tempering of the aggregating item, the PCR response inevitably stops to intensify target grouping at an exponential rate and a level impact happens making the end point measurement of PCR items inconsistent.
Sample collection and maintenance:
The sample collections are obtained from appropriate tissues, cell lines and the fecal samples. It is stored in frozen level at 80 degree Celsius and transported in dry ice (interstate transport). Serums that are improved analyses for antibodies, nutrients lipids and lipoproteins are used for proteomic analyses. Digital PCR is used in genetic engineering and for medical diagnostics as it is very much portable, low cost and helps in automatic digital PCR systems
Recent advancement:
Some of the recent advances with RT-PCR works is that the MPCR and PCR - RFLP assays help in the routine diagnosis for quick and specific simultaneous detection of both M. agalactiae and M. bovis. RT- PCR helps in the replacement of traditional tests and in detection of the fecal viral pathogens. Other similar works before nCoV were done for other diseases such as HIV, Mad cow disease, Ebola, flavivirus and Zika virus. The Digital PCR in cell biology, genetic engineering, helps in the medical diagnostics that are portable with low cost of automatic digital PCR system. Some of the other alternate methods are: to generate antibodies with the spike proteins of the virus and the rapid screening for epidemiological surveillance.
Conclusion:
With this report, we came to know about the pros and cons of RT- PCR in COVID 19. Therefore, this method is very much better in handling and experience to give reduced false positive occurrences of cases. It also helps to detect the affected people rapidly. Thus RT- PCR paves the way for further research and production of better quality diagnostic kit and screening tests.
Department of Biochemistry and Biotechnology
St. Xavier’s College, Ahmedabad
Ankita Dhankhar
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