Alternative RNA extraction-free techniques for the real-time RT-PCR detection of SARS-CoV-2 in nasopharyngeal swab and sputum samples

Villota, Stephany D.; Nipáz, Victoria; Carrazco-Montalvo, Andrés; Hernandez, Sarah; Waggoner, Jesse J.; Ponce, Patricio; Coloma, Joséfina; Orlando, Alberto; Cevallos, Varsovia

doi:10.1016/j.jviromet.2021.114302

Cited by 9 publications

(9 citation statements)

References 18 publications

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“…However, this approach results in reduced sensitivity and specificity of the downstream RT-PCR process. It may require an additional 3 to 7 PCR cycles to reach the detection threshold compared to that of reactions with purified RNA 2,12 , compromising the detection of low viral loads. Still, studies reported sensitivity values ranging from 51% 38 to 91.4% 48 as commonly used measures of validity, including specificity.…”

Section: Discussionmentioning

confidence: 99%

“…Accuracy [2], Sensitivity [3] and Specificity [4] were estimated for diagnostic efficiency as indexes using a confusion matrix approach [60][61][62] . The confusion matrix and confidence interval (95%) were calculated using a diagnostic test evaluation software MedCalc version 20.027 (MedCalc Software Ltd, Ostend, Belgium).…”

Section: Rna Quantificationmentioning

confidence: 99%

“…Coronaviruses (CoV) are part of Coronaviridae family with unsegmented single-stranded positive RNA genome belonging 26 to 36 kb length with wide host range, including humans [1][2][3] . In the history of humankinds have experienced previus infection, during the 1960s CoV-virus have been describe beta-coronavirus like OC43-CoV and HKU1-CoV, and alfa-coronavirus like 229E-CoV and NL63-CoV.…”

Section: Introductionmentioning

confidence: 99%

“…Several commercial SARS-CoV-2 RT-PCR protocols employ manual extraction kits to isolate viral RNA from nasopharyngeal swabs 24,35,36 , whereby an accurate extraction, recovery and quantification determine the efficacy of RT-PCR detection 9,37 . The more common methods for viral isolation are (1) silica-based membrane 14,38 , also called solid-phase RNA extraction; (2) or-ganic extraction using phenol-guanidineIsothiocyanate (GITC) and, (3) magnetic beads 13,39 . All these methods allow cell and viral lysis using registered reagents by trademarks that has made them a limiting resource for SARS-CoV-2 diagnose mainly in the peaks of contagious in middle of 2020 and 2021 13,40 .…”

Section: Introductionmentioning

confidence: 99%

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Cost and performance analysis of efficiency, efficacy, and effectiveness of viral RNA isolation with commercial kits and Heat Shock as an alternative method to detect SARS-CoV-2 by RT-PCR

Chica

Aguilar-Mora

Ramírez-Cando

et al. 2023

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In late 2019 a new virus reported in Wuhan, China, identified as SARS-CoV-2 spread rapidly challenging the healthcare system around the world. The need for rapid, timely and accurate detection was critical to the prevention of community outbreaks of the virus. However, the high global demand for reagents during the years 2020 and 2021 generated a bottleneck in kits used for detection, greatly affecting developing countries, lagging their ability to diagnose and control the virus in the population. The difficulty in importing reagents, high costs and limited public access to the SARS-CoV-2 detection test led to the search for alternative methods. In this framework, different commercial nucleic acid extraction methodologies were evaluated and compared against heat shock as an alternative method for SARS-CoV-2 detection by RT-PCR, in order to determine the diagnostic yield and its possible low-cost compared to other methodologies. Nasopharyngeal samples were used where the diagnostic efficiency of the alternative method was 70 to 73%. The evaluation of the discriminatory efficacy of the method took the sensitivity and specificity to establish its cut-off point, being 0.73 to 0.817, which allows discriminating between COVID-19 positives and negatives. As for the diagnostic effectiveness expressed as the proportion of subjects correctly classified, it is between 80 and 84%. On the other hand, in terms of the costs necessary to carry out the detection, the alternative method is more economical and accessible in terms of direct cost close to 47 and 49 USD, and indirect cost around 35 and 50 USD compared to the commercial methods available in this comparison and evaluation, being possible its implementation in developing countries with high infection rates, allowing access to the diagnostic test with a reliable and low-cost method. Keywords: COVID-19, RT-PCR, Viral RNA.

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Section: Discussionmentioning

confidence: 99%

Section: Rna Quantificationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Cost and performance analysis of efficiency, efficacy, and effectiveness of viral RNA isolation with commercial kits and Heat Shock as an alternative method to detect SARS-CoV-2 by RT-PCR

Chica

Aguilar-Mora

Ramírez-Cando

et al. 2023

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“…Limited and inconsistent access to reagents for viral RNA extraction has resulted in the development of extraction-free methods for SARS-CoV-2 molecular detection. Such protocols utilize modified thermocycling conditions and additional master mix reagents to provide direct specimen testing, which is facilitated by relatively inhibitor-free primary clinical specimens [12][13][14][15][16]. While such techniques require less dedicated consumables and processing time, resulting nucleic acids cannot be applied to downstream molecular characterization of SARS-CoV-2 variants or further workup of negative cases.…”

Section: Plos Onementioning

confidence: 99%

SARS-CoV-2 genotyping and sequencing following a simple and economical RNA extraction and storage protocol

Hernandez

Nguyen²,

Taz³

et al. 2023

PLoS ONE

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Since the beginning of the SARS-CoV-2 pandemic, supply chain shortages have caused major disruptions in sourcing the materials needed for laboratory-based molecular assays. With increasing demand for molecular testing, these disruptions have limited testing capacity and hindered efforts to mitigate spread of the virus and new variants. Here we evaluate an economical and reliable protocol for the extraction and short-term ambient temperature storage of SARS-CoV-2 RNA. Additional objectives of the study were to evaluate RNA from this protocol for 1) detection of single nucleotide polymorphisms (SNPs) in the spike gene and 2) whole genome sequencing of SARS-CoV-2. The RNAES protocol was evaluated with residual nasopharyngeal (NP) samples collected from Emory Healthcare and Emory Student Health services. All RNAES extractions were performed in duplicate and once with a commercial extraction robot for comparison. Following extraction, eluates were immediately tested by rRT-PCR. SARS-CoV-2 RNA was successfully detected in 56/60 (93.3%) RNAES replicates, and Ct values corresponded with comparator results. Upon testing in spike SNP assays, three genotypes were identified, and all variant calls were consistent with those previously obtained after commercial extraction. Additionally, the SARS-RNAES protocol yield eluate pure enough for downstream whole genome sequencing, and results were consistent with SARS-CoV-2 whole genome sequencing of eluates matched for Ct value. With reproducible results across a range of virus concentrations, the SARS-RNAES protocol could help increase SARS-CoV-2 diagnostic testing and monitoring for emerging variants in resource-constrained communities.

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The effect of sample site and collection procedure on identification of SARS-CoV-2 infection

Davenport,

Arevalo-Rodriguez,

Mateos-Haro

et al. 2024

Cochrane Database of Systematic Reviews

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Background Sample collection is a key driver of accuracy in the diagnosis of SARS‐CoV‐2 infection. Viral load may vary at different anatomical sampling sites and accuracy may be compromised by difficulties obtaining specimens and the expertise of the person taking the sample. It is important to optimise sampling accuracy within cost, safety and accessibility constraints. Objectives To compare the sensitivity of different sampling collection sites and methods for the detection of current SARS‐CoV‐2 infection with any molecular or antigen‐based test. Search methods Electronic searches of the Cochrane COVID‐19 Study Register and the COVID‐19 Living Evidence Database from the University of Bern (which includes daily updates from PubMed and Embase and preprints from medRxiv and bioRxiv) were undertaken on 22 February 2022. We included independent evaluations from national reference laboratories, FIND and the Diagnostics Global Health website. We did not apply language restrictions. Selection criteria We included studies of symptomatic or asymptomatic people with suspected SARS‐CoV‐2 infection undergoing testing. We included studies of any design that compared results from different sample types (anatomical location, operator, collection device) collected from the same participant within a 24‐hour period. Data collection and analysis Within a sample pair, we defined a reference sample and an index sample collected from the same participant within the same clinical encounter (within 24 hours). Where the sample comparison was different anatomical sites, the reference standard was defined as a nasopharyngeal or combined naso/oropharyngeal sample collected into the same sample container and the index sample as the alternative anatomical site. Where the sample comparison was concerned with differences in the sample collection method from the same site, we defined the reference sample as that closest to standard practice for that sample type. Where the sample pair comparison was concerned with differences in personnel collecting the sample, the more skilled or experienced operator was considered the reference sample. Two review authors independently assessed the risk of bias and applicability concerns using the QUADAS‐2 and QUADAS‐C checklists, tailored to this review. We present estimates of the difference in the sensitivity (reference sample (%) minus index sample sensitivity (%)) in a pair and as an average across studies for each index sampling method using forest plots and tables. We examined heterogeneity between studies according to population (age, symptom status) and index sample (time post‐symptom onset, operator expertise, use of transport medium) characteristics. Main results This review includes 106 studies reporting 154 evaluations and 60,523 sample pair comparisons, of which 11,045 had SARS‐CoV‐2 infection....

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Alternative RNA extraction-free techniques for the real-time RT-PCR detection of SARS-CoV-2 in nasopharyngeal swab and sputum samples

Cited by 9 publications

References 18 publications

Cost and performance analysis of efficiency, efficacy, and effectiveness of viral RNA isolation with commercial kits and Heat Shock as an alternative method to detect SARS-CoV-2 by RT-PCR

Cost and performance analysis of efficiency, efficacy, and effectiveness of viral RNA isolation with commercial kits and Heat Shock as an alternative method to detect SARS-CoV-2 by RT-PCR

SARS-CoV-2 genotyping and sequencing following a simple and economical RNA extraction and storage protocol

The effect of sample site and collection procedure on identification of SARS-CoV-2 infection

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