Impact of the Nucleic Acid Extraction Method and the RT-qPCR Assay on SARS-CoV-2 Detection in Low-Viral Samples

Komiażyk, Magdalena; Walory, Jarosław; Kozińska, Aleksandra; Waśko, Izabela; Baraniak, Anna

doi:10.3390/diagnostics11122247

Cited by 12 publications

(11 citation statements)

References 24 publications

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“…In nasopharyngeal swab samples, transported in viral transport medium and/or saline solution, Pi-Lise and QuickExtract were also efficient for nucleic acid extraction from samples with high viral load (CT ≤ 30.0) and for samples with low viral load (CT ≥ 30.0). In a study by Komiazyk et al, difficulty in extracting RNA from samples with low viral load was reported, in particular, due to operational difficulties in conventional extraction techniques (by column or organic solvents) [23]. Due to several steps in the extraction using column or solvent, and also the final eluting step in which the final concentration is dependent on the water volume added, the viral RNA concentration may be reduced, impairing the detection by RT-qPCR.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2022

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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.

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Section: Discussionmentioning

confidence: 99%

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

et al. 2022

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“…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 ].…”

Section: Discussionmentioning

confidence: 99%

“…Komiazyk et al showed that the lower the viral load, the more difficult it is to interpret the results. This appears to be due to problems with available diagnostic tests failing this low viral load, which is still capable of infection [ 18 ]. With CT ≥ 35, the test also reacts with some portion of the virus, while the virus itself is no longer present; therefore, the subject is no longer contagious.…”

Section: Discussionmentioning

confidence: 99%

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

Scarabotto¹,

Balestro²,

Gagliardi³

et al. 2022

Diagnostics

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Background: Rapid diagnosis of COVID-19 is essential in order to restrict the spread of the pandemic, and different approaches for SARS-CoV-2 testing have been proposed as cost-effective and less time-consuming alternatives. For virus detection, the real-time reverse transcriptase–polymerase chain reaction (RT-PCR) technique is still the “gold standard” for accuracy and reliability, but its performance is affected by the efficiency of nucleic acid extraction methods. Objective: In order to improve the SARS-CoV-2 diagnostic workflow, we compared a “standard” commercially available kit, based on viral RNA extraction from human swab samples by magnetic beads, with its technological evolution. The two methods differ mainly in their time consumption (9 vs. 35 min). Methods: We adopted the MAGABIO PLUS VIRUS DNA/RNA PURIFICATION KIT II (BIOER), defined as “standard”, with the automatic extractor BIOER (GenePure Pro fully automatic nucleic acid purification system) to isolate RNA from nasopharyngeal swabs for the detection of SARS-CoV-2 by RT-PCR. We tested this kit with a new faster version of the first one, defined as “rapid” (MAGABIO PLUS VIRUS RNA PURIFICATION KIT II). Results and Conclusion: The two evaluated procedures provided similar analytical results, but the faster method proved to be a more suitable tool for the detection of SARS-CoV-2 from nasopharyngeal swabs, due to a more rapid availability of results, which may contribute to improving both clinical decision making and patient safety.

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“…Virus RNA acids were isolated using two procedures, column-based extraction by Viral DNA/RNA kit (A&A Biotechnology, Gdansk, Poland) and magnetic-based method via NucleoMag Pathogen kit (Machery-Nagel, Duren, Germany) with modifications to the manufacturers’ instructions, as described previously [ 45 ].…”

Section: Methodsmentioning

confidence: 99%

Differentiation of SARS-CoV-2 Variants Using RT-qPCRs by Targeting Recurrent Mutation Sites: A Diagnostic Laboratory Experience from Multi-Center Regional Study, August 2020–December 2021, Poland

Wegrzynska

Komiażyk

Walory

et al. 2022

IJMS

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Rapid identification of SARS-CoV-2 variants is essential for epidemiological surveillance. RT-qPCR-based variant differentiation tests can be used to quickly screen large sets of samples for relevant variants of concern/interest; this study was conducted on specimens collected at 11 centers located in Poland during routine SARS-CoV-2 diagnostics between August 2020 and December 2021. A total of 1096 samples (with CT < 30) were screened for Alpha, Beta, Delta, Kappa and Omicron variants using commercial assays targeting repeat mutation sites. Variants were assigned to 434 (39.6%) specimens; the remaining 662 (60.4%) samples were not classified (no tested mutations detected). Alpha (n = 289; 66.59%), Delta (n = 115; 26.5%), Kappa (n = 30; 6.91%) and Omicron (n = 2; 0.46%) variants were identified and their distribution changed over time. The first Alpha variant appeared in October 2020, and it began to gradually increase its proportion of the virus population by June 2021. In July 2021, it was replaced by the Delta variant, which already dominated by the end of the year. The first Kappa was detected in October 2021, while Omicron was found in December 2021. The screening of samples allowed the determination of epidemiological trends over a time interval reflecting the national COVID-19 waves.

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Impact of the Nucleic Acid Extraction Method and the RT-qPCR Assay on SARS-CoV-2 Detection in Low-Viral Samples

Cited by 12 publications

References 24 publications

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

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

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

Differentiation of SARS-CoV-2 Variants Using RT-qPCRs by Targeting Recurrent Mutation Sites: A Diagnostic Laboratory Experience from Multi-Center Regional Study, August 2020–December 2021, Poland

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