key: cord-0274675-cp6nzpyk
authors: Jain, S.; Bhowmick, A.; Pandey, A. K.
title: A rapid One-Pot RNA-isolation method for simplified clinical detection of SARS-COV-2 infection in India
date: 2022-05-27
journal: nan
DOI: 10.1101/2022.05.26.22275661
sha: 5d9fc8336c350735dc01406c483c8391dddaef74
doc_id: 274675
cord_uid: cp6nzpyk

Background: With the rapid increase in COVID-19 cases and the discovery of new viral variants within India over multiple waves, the expensive reagents and time-consuming sample pretreatment required for qPCR analysis have led to slower detection of the disease. The vast Indian population demands an inexpensive and competent sample preparation strategy for rapid detection of the disease facilitating early and efficient containment of the disease. Methods: In this study, we have surveyed the spread of COVID-19 infection over Faridabad, Haryana, India for 6 months. We also devised a simple single-step method for total RNA extraction using a single tube and compared its efficacy with the commercially available kits. Findings: Our findings suggest that determining Ct values for samples subjected to the One Pot (OP) RNA extraction method was as efficient as the commercially available kits but delivers a subtle advantage in a way, by minimizing the cost, labor, and sample preparation time. Conclusion: This novel crude RNA extraction method is stable and capable of operating in developing countries like India for low resource settings, without the use of expensive reagents and instruments. Additionally, this method can be further adapted to pooling samples strategies owing to its high sensitivity.

ice-cold ethanol solution was added to the bottom layer followed by again centrifugation at 1 2 8 14000 rpm for 5 mins. Finally, the supernatant was discarded followed by air drying of the tubes 1 2 9

for 10 mins (to remove residual ethanol). The RNA pellet was then further resuspended in 20 µl 1 3 0 of DEPC-treated water, quantified and subsequently used as a template in qRT-PCR analysis for A2m-square arrays method with two different master pools was applied in this study which . This

pooling strategy is a two-dimensional array ("A2m") with master pool testing. The first test was . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 27, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 depends on the expected prevalence rate of the disease under study and the characteristics of the 1 5 5 diagnostic test. Further, it also depends on dilution effects in the master pool, the length of the 1 5 6 sensitivity depends on both the sensitivity/ lower limit of detection of the assay and viral load in . The optimal pool size required for testing was calculated with an available online calculator (http://www.bios.unc.edu/_mhudgens/optimal.pooling.b.htm).

Therefore, according to the pooling size strategy mentioned above, the master pools size was 25.

9.7%; 18-35 years-16.2%; 36-53 years-23.8%; 54-71 years-25.4% and 72-89 years-28.8% 1 8 1 (Table 1) . Thus, our data indicate that SARS-CoV-2 is highly affecting individuals belonging to . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 27, 2022. ; https://doi.org/10.1101/2022.05.26.22275661 doi: medRxiv preprint renal disorder (14.7 %) and obesity (11.2%) ( Table 1) . Amongst the COVID-19 affected Health Organization, 2022). From this 6 months study the prevalence rate of Covid-19 infection 2 1 5 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) are more susceptible to infection than women, our data also revealed that males are more 2 1 7 predisposed to COVID-19 infection than females (Bwire, 2020). However, it was also reported . Although previous studies pointed to the fact that COVID-19 prevalence increases in 2 2 1 adolescents and youth compared to older adults, our data revealed that the rate of infection diagnosis outside the centralized testing facilities to hasten the clinical decision-making within a . Also, another CRISPR-based, FnCas9, Editor Linked Uniform . In addition, a sensitive RNA- One-pot (OP) RNA-isolation method is highly cost-effective with a cost of approximately Rs.

. CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 27, 2022. with the column method is more than 1 hour, but with this new method, the time required is of Haryana, where this study was carried out. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. chloroform, and glycogen to VTM sample containing the NP/OP swab followed by brief heating 2 7 6 at 65 and subsequent centrifugation at high speed. total patients registered in that corresponding group followed by multiplying by 100. and accurate nucleobase detection using FnCas9 and its application in COVID-19 diagnosis. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted May 27, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 

Metabolic syndrome (obesity) 11.2% (1367) Pregnant women 1.6% (89)

Competing interests