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17 Jun 2021
Evaluation of seven commercial RT-PCR kits for COVID-19 testing in pooled clinical specimens
The currently spreading COVID-19 is causing havoc in the health departments. This virus initially attacks the respiratory system and gives birth to a number of respiratory syndromes. It emerged around December 2019 in the city of Wuhan in China. However, since the virus was new the scientists had to do some investigation on it using scientific tools like PCR Tubes. After considerable investigation, the WHO finally declared the pandemic on March 11 in 2020. Since there was no better and more effective way to prevent the methods, it was declared that everyone must limit their human interaction and not leave their homes unless it is extremely necessary. This was done so the ongoing chain of viral transmission could break in the middle. Currently, the test most hospitals are carrying out to diagnose the presence of pandemics is primarily composed of conventional real-time polymerase chain reaction (RT-PCR).
Testing COVID-19
There are about 350 Conventional RT-PCR COVID-19 testing kits along with the DNA Extraction Kit. Out of these 350 kits, 2o9 met the approval of the United States food and drug administration (US-FDA). RT-PCR requires a very sophisticated and well-equipped molecular laboratory with powerful manpower. Currently, a lot of the countries are going through an acute shortage of kits and the manufacturers of molecular testing are also undergoing huge stress to put up with the demand. All of this requires a bunch of novel ideas, so the reagents utilized for molecular tests could be conserved. However, it is almost frightening that the disease is still an enigma. Given the holistic view of everything, it is vital to validate the modifications required to test the protocol before universal adoption.
Where did the sampling and data collection take place? The given prospective study of observations is designed and conducted by the Department of Microbiology, Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGIMS), Lucknow, India. These studies use swabs that are collected from healthcare providers working at Rajdhani Corona Hospital.
How did RT-PCR kits get selected for testing? Before the researchers and the scientist conducted a study, a survey was done on a commercial basis. This all required a lower rate of detection, availability, and usage. The compatibility with various PCR platforms and the cost were also put into consideration.
How did RT-PCR perform on direct unpooled clinical specimens? All the samples or VTM were subjected to open within the biosafety cabinet class-II and around 300μl of total sampling were sent to further processing. In order to avoid RNA degradation, a study was also planned in such a way that the entire experiment was complete within a day. Scientists prepared 25 μl of reaction so they could the quality of SARS detection. In the process, almost all oligonucleotides were synthesized. Every sample was first screened to target the E genes, RnaseP, and RdRP as per the protocol of WHO. Furthermore, all the samples were then tested in triplicate before they could be included in any study. Once, the scientist was able to identify all the positive and negative samples by WHO protocol, a total of 40 positive and 10 negative samples were selected to further the study.
The sampling The pooling of clinical samples for RT-qPCR before the RNA extraction could take place All the positive pools were created by using the 60 μl out of the total sample of RT-PCR confirmed COVID-19-positive patient specimen. This sampling of 60 μl VTM was given to every 1 in 4 patients that were tested negative. Similar to a positive pool sampling, researchers also created the negative pools. The whole study protocol was curated that positive sampling with low and high cut-off threshold was included.
The conclusion Out of a total of 500 samplings that were tested for SARS Cov-2, sixty of them were positive while the rest were negative. The scientist then retested 10 negatives all the positive samples in a triplicate and eventually the end with around 10 negative samples and 40 positive samples for study. Turns out, there was an estimate of twenty positive samples with high viral load and a low cut-off of the threshold. Furthermore, these were seven commercial RT-PCR kits that could accurately identify the 10 negative and 40 positive samples. A positive sample with a cut of a threshold range of 35-38 was also detected by using the kits and it was identified by weak positive sampling.
Bibliography Garg, A., Patel, S. S., Singh, D. V., Arya, A. K., Vasanth, S., Pandey, A., & Srivastava, N. (2020, November 24). Evaluation of seven commercial RT-PCR kits for COVID-19 testing in pooled clinical specimens. Journal of Medical Virology. https://onlinelibrary.wiley.com/doi/full/10.1002/jmv.26691
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