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SARS-CoV2 is a member of the CoronaVirus family, it is an etiological agent of the Covid-19, which has recently taken over the whole world and has created an international public health emergency. Only in the summer of 2020, this virus has infected more than 3 million people and around 240,000 people around the world have died. It is very important to test the presence of the virus to develop containment strategies that will aim to diminish the distribution of the virus and thereby right clinical practices can be prescribed for the patients that are affected. However, it is equally important to learn properly and manage the outbreak because this still remains an obstacle for almost all the developed countries. In the early days, infection by SARS-CoV-2 mainly relied on the proper detection of the viral genome in which RT-qPCR was used. A lot of RT-qPCR based tests are now utilized in hospitals and clinics (2FDA, 2020). Other than that novel approaches and methods reported on a daily basis. All the methods and approaches need an RNA extraction step to isolate the viral genetic material prior to detection. Contact MBP Inc for viral transport medium
Sadly, the RNA extraction is a very serious bottleneck for COVID-19 diagnosis in the whole world because there are shortages in the RNA-extraction kits, which are customarily utilized in the patient samples. This is especially difficult and challenging in the developing states who lack capacities and infrastructure to make these kits domestically. Before the kits came into being, most of the RNA extraction methods were routinely used in research labs across the globe. These kits play a great role in standardizing and simplifying the molecular biology work. The RN isolation method generally takes into account three basic steps: the cell lysis, separating the RNA from other macromolecules like the DNA, lipids, proteins along RNA concentration. In order to stop the RNA degradation, cell lysis should be done under conditions that inhibit RNase activity, and this is found in abundance in a lot of regions (Nilsen, 2013). RNA is separated from other macromolecules and a lot of times it is achieved through the amalgamation of pH and organic solvents like phenol or chloroform (Chomczynski & Sacchi, 2006). The RNA concentration is usually achieved through high salt and isopropanol or through ethanol precipitation. MPB inc has been playing a significant role in assisting the end to end Covid-19 workflow. The facility prides itself in carrying out sample collection, RNA extraction, consumables and PCR kits to streamline the process of testing. Furthermore, MBP inc supplies high-quality products to ensure the accuracy of testing. Our products can be used for the ensuing purposes.
Validation of the Acid pH RNA Extraction Method In Clinical Samples
In order to validate and check the acid pH method of RNA extraction, the RT-qPCR using the TaqMan probes and primers is the best option. The nucleocapsid viral proteins called the N1 and N2 are amplified as the viral targets, and RNase P is amplified as a control. All the undetermined samples are explained as a viral load around the limit of detection (LOD) if the RT-qPCR approach. This means that the RT-qPCR method is able to detect almost 16 RNA copies per PCR reaction. The PCR test is able to detect almost 2 targets of the virus that are N1 and N2. The average Cq value of the N target is reported for dilution sets which are generally lesser than or equal to 95% positive. In the research conducted, out of the 11 undetermined samples checked, 3 of them were negative and 4 were positive. The RNA extraction procedure with acid pH described has a lot of perks over the commercial kits to test the SARS-CoV-2, especially for the current pandemic. This procedure takes very low-cost reagents and equipment and is usually found in the standard laboratories. Extraction costs are usually the biggest issue in all the clinical labs, but the cost of MBP inc’s in-house method is significantly cheaper than other extraction kits. Furthermore, the DNase treatment isn’t mandatory because the SARS-CoV-2 detection is not changed when DNA is present. The residual DNA serves as the template for RNase P gene amplification. This is because of the recent environmental issues, as this in-house method also causes low plastic contamination.
Development Of SARS-CoV-2 hid-RT-PCR
The transport media was investigated for swab collection which has an impact on RT-PCR. In order to do this, the synthetic full-genome SARS-CoV2 RNA spiked into the dilution series of three unique transport media. These transport media are used for clinical sampling when the study is carried out. The mock sample of 50,000 synthetic SARS-CoV2 RNA copied and 95% medium, corresponding to 47.5-0.05% medium in the RT-PCR reaction. All the single-reaction using RT-PCR were performed using small samples in a 20 µl reaction. Moreover, the CDC nucleocapsid 1 (N1) primer-probe set along with the recorded cycle threshold values were taken into account for the dilution series. Inhibitory effects were seen in all the media, but most importantly, there was pronounced variation between the media. The virocult and Transwab showed almost the same profiles of inhibition. The Swab had fully impeded the detection at high concentrations. However, it reached a relatively low level of inhibition as compared to Virocult and Transwab at 25% concentration. In order to test the synthetic RNA, it was seen that it did not have any lingering DNA template, which is why additional RT and qPCR reaction tests were conducted in two different steps. These methods included RT controls that show lack of DNA amplification signal, especially when the reverse transcriptase was taken out of the equation. All in all, the results tell us that there is very minimal or no inhibition of Virocult, Transwab, and Eswab at greater than 25% in the RT-PCR reaction.
References
2FDA. (2020). Coronavirus COVID-19 Diagnostic Tests and Shortages.Chomczynski, & Sacchi. (2006). The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nat. Protoc, 581-585.Nilsen. (2013). The fundamentals of RNA purification. Cold Spring Harbor Protocols, 618-624.
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