Comparison of Rapid Nucleic Acid Extraction Methods for SARS-CoV-2 Detection by RT-qPCR

Abstract

Since 2020, humanity has been facing the COVID-19 pandemic, a respiratory disease caused by the SARS-CoV-2. The world’s response to pandemic went through the development of diagnostics, vaccines and medicines. Regarding diagnostics, an enormous challenge was faced due to shortage of materials to collect and process the samples, and to perform reliable mass diagnosis by RT-qPCR. In particular, time-consuming and high cost of nucleic acid extraction procedures have hampered the diagnosis; moreover, several steps in the routine for the preparation of the material makes the extracted sample susceptible to contamination. Here two rapid nucleic acid extraction reagents were compared as extraction procedures for SARS-CoV-2 detection in clinical samples by singleplex and multiplex RT-qPCR analysis, using different transport media, samples with high and low viral load, and different PCR machines. As observed, rapid nucleic acid extraction procedures can be applied for reliable diagnosis using a TaqMan-based assay, over multiple platforms. Ultimately, prompt RNA extraction may reduce costs with reagents and plastics, the chances of contamination, and the overall time to diagnosis by RT-qPCR.

Aligning monitoring of virus barriers in potable reuse with unit process treatment mechanisms and time scales: A critical review and research needs

“…The interest in dPCR and qPCR has led to improved methods and significant reductions in the cost and time associated with processing a sample (Silva et al, 2022; Stahl et al, 2021). These methods can be adapted to any pathogen whose genetic sequence is known, and future development is expected to detect other common waterborne enteric viruses.…”

Comparison of Two RNA Extraction Methods for the Molecular Detection of SARS-CoV-2 from Nasopharyngeal Swab Samples

“…More specifically, a new, faster technique of nucleic acid extraction was compared to the “reference” method, which represents the evolution. As already reported in the literature, the impact of RNA extraction is fundamental in SARS-CoV-2 detection [ 18 , 19 ] and, on the contrary, in diagnostics, the data about time consumption are also very important [ 20 ]. In fact, other papers have already demonstrated that performing a combination of rapid and quality RNA isolation is possible [ 20 ].…”

“…As already reported in the literature, the impact of RNA extraction is fundamental in SARS-CoV-2 detection [ 18 , 19 ] and, on the contrary, in diagnostics, the data about time consumption are also very important [ 20 ]. In fact, other papers have already demonstrated that performing a combination of rapid and quality RNA isolation is possible [ 20 ].…”

“…Reliable and strict, this new application can be suggested to be a precious tool for the detection of SARS-CoV-2 from nasopharyngeal swabs in a pandemic context, where it is relevant to obtain rapid and accurate results to diagnose patients and employ rapid public health actions. As already published, the setting up of rapid nucleic acid extraction procedures may be very useful for reliable diagnosis [ 20 ]. In reality, this faster method for RNA extraction (a) reduces the turnaround time, (b) improves outcomes and workflow, and (c) accelerates clinical decision making.…”

Evaluation of DNA Extraction Methods for Reliable Quantification of Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa

“…A considerable number of comparative studies have been performed, focusing on the yield of two to three (and rarely more) extraction methods to determine the optimal method for extracting nucleic acids from a given pathogen. Manual and automated approaches have been studied to determine the most efficient methods for viral nucleic acid yields for both RNA viruses (e.g., noroviruses, HIV, and SARS-CoV-2) and DNA viruses (e.g., cytomegalovirus, hepatitis B) [ 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 ], as well as for bacterial DNA extraction from complex samples (e.g., faeces, blood, urine, sputum, and amniotic fluid) [ 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 ]. These comparisons are primarily based on differences in the number of qPCR cycles used, although there have also been studies in which comparisons have been based on spectrophotometry or fluorimetric detection [ 24 , 25 ] or PCR [ 26 ].…”