Development and implementation of a RT‐qPCR extraction‐free protocol for the detection of SARS‐CoV‐2 and impact on the turn‐around‐time

Blairon, Laurent; Piteüs, Sébastien; Beukinga, Ingrid; Tré-Hardy, Marie

doi:10.1002/jmv.26782

Cited by 7 publications

(6 citation statements)

References 17 publications

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“…Relatively easy to implement; however, this strategy requires both a heating source and an active cooling source. [36] Nasopharyngeal swabs in universal transport media RT-PCR Combination of heat treatment (65 • C for 10 min) and increase in sample input volume.…”

Section: Sample Type Naat/detection Methods Sample Preparation Strategymentioning

confidence: 99%

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Strategies That Facilitate Extraction-Free SARS-CoV-2 Nucleic Acid Amplification Tests

Delgado-Diaz¹,

Sakthivel²,

Nguyen³

et al. 2022

Viruses

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The COVID-19 pandemic has resulted in an unprecedented global demand for in vitro diagnostic reagents. Supply shortages and hoarding have impacted testing capacity which has led to inefficient COVID-19 case identification and transmission control, predominantly in developing countries. Traditionally, RNA extraction is a prerequisite for conducting SARS-CoV-2 nucleic acid amplification tests (NAAT); however, simplified methods of sample processing have been successful at bypassing typical nucleic acid extraction steps, enabling extraction-free SARS-CoV-2 NAAT workflows. These methods involve chemical and physical approaches that are inexpensive and easily accessible alternatives to overcome extraction kit supply shortages, while offering acceptable test performance. Here we provide an overview of three main sample preparation strategies that have been shown to facilitate extraction-free SARS-CoV-2 NAATs.

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Section: Sample Type Naat/detection Methods Sample Preparation Strategymentioning

confidence: 99%

“…Similar to heat treatment, thermal shock treatment of the samples prior to SARS CoV-2 NAAT has been reported by Blairon and colleagues. This workflow involves subjecting nasopharyngeal swab eluates to a thermal shock consisting of 95 • C for 5 min followed by active cooling at 4 • C for 10 min [36].…”

Section: Heat Treatment Of Respiratory Samplesmentioning

confidence: 99%

Strategies That Facilitate Extraction-Free SARS-CoV-2 Nucleic Acid Amplification Tests

Delgado-Diaz¹,

Sakthivel²,

Nguyen³

et al. 2022

Viruses

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“…The trial was approved and authorized from the Interprovincial Ethical Commission, Area 1, of A.O.U Foggia, ASL FG, ASL BAT (Authorization n°: 34/CE/2022 of February 28, 2022). The COVID-19 positive patients, subject to the release of informed consent, were recruited from March 1st, 2022 to April 1st, 2022 based on the positive rRT-PCR performed on envelope (E), nucleocapside (N), and RdRP genes ( Blairon et al, 2021 ). Specifically, 17 patients individually tested positive were retested using pooling strategy.…”

Section: Methodsmentioning

confidence: 99%

“…Two mL of Dulbecco Minimal Essential Medium (DMEM, Corning, Mediatech, Inc., 9345 Discovery Blvd, Manassa, VA 20109, United States) were added to each conical centrifuge tube containing the pool which included a total of 20 NSs (1 positive NS from each positive patient and 19 negative NSs) ( Figure 1B ), and to each conical centrifuge tube containing the single positive swab. After vortexing the tubes for 1 min, acid nucleic was extracted using STARMag 96X4 Viral DBA/RNA200C Kit, an automatic nucleic acid purification system with the convenient handling of magnetic beads, and rRT-PCR was set-up using the SARS-CoV-2 assay [Allplex™, Seegene Inc., Taewon Bldg., 91 Ogeum-ro, Songpa-gu, Seoul, Corea Republic) with CFX thermal cycler (Bio-Rad Laboratories S.r.l.Via Cellini, 18/A, 20090 Segrate (MI)—Italy] ( Blairon et al, 2021 ).…”

Section: Methodsmentioning

confidence: 99%

Severe acute respiratory syndrome coronavirus 2 detection by real time polymerase chain reaction using pooling strategy of nasal samples

Pratelli

Pellegrini²,

Ceci³

et al. 2022

Front. Microbiol.

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COVID-19 is a life-threatening multisistemic infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Infection control relies on timely identification and isolation of infected people who can alberg the virus for up to 14 days, providing important opportunities for undetected transmission. This note describes the application of rRT-PCR test for simpler, faster and less invasive monitoring of SARS-CoV-2 infection using pooling strategy of samples. Seventeen positive patients were provided with sterile dry swabs and asked to self-collected 2 nasal specimens (#NS1 and #NS2). The #NS1 was individually placed in a single tube and the #NS2 was placed in another tube together with 19 NSs collected from 19 negative patients. Both tubes were then tested with conventional molecular rRT-PCR and the strength of pooling nasal testing was compared with the molecular test performed on the single NS of each positive patient. The pooling strategy detected SARS-CoV-2 RNA to a similar extent to the single test, even when Ct value is on average high (Ct 37–38), confirming that test sensibility is not substantially affected even if the pool contains only one low viral load positive sample. Furthermore, the pooling strategy have benefits for SARS-CoV-2 routinary monitoring of groups in regions with a low SARS-CoV-2 prevalence.

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“…1 ). First-line molecular analyses are performed either by Transcription Mediated Amplification using the Aptima® SARS-CoV-2 assay kit (Hologic, San Diego, CA, USA) on a Panther platform, or by PCR using the Allplex® kit (Seegene Technologies, Seoul, South Korea) after extraction with the STARMag Viral DNA/RNA 200 C Kit (Seegene Technologies, Seoul, South Korea) on a STARLet platform (Hamilton Company, Reno, NV, USA) or without any extraction following an internal protocol( Blairon et al, 2021 ). Results that need to be answered very quickly for emergency reasons (e.g.…”

Section: Methodsmentioning

confidence: 99%

The challenge of screening SARS-CoV-2 variants of concern with RT-qPCR: One variant can hide another

Blairon

Cupaiolo

Piteüs

et al. 2021

Journal of Virological Methods

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Introduction Following the emergence of SARS-CoV-2 variants of concern (VOCs) worldwide, it is important to monitor local epidemiology to better understand the occurrence of clusters, reinfections, or infection after vaccination. Detecting mutations by specific RT-qPCR is a rapid and affordable alternative to sequencing. However, care must be taken to ensure that the techniques used are up-to-date and adapted to the variants circulating in the studied population. Material and methods All samples tested positive for SARS-CoV-2 were screened for detection of mutations of the spike protein using the Novaplex™ SARS-CoV-2 Variants I Assay from week 11 of 2021. Target sought were deletion H69/V70 and mutations N501Y and E484 K. From week 18 we used in addition the new Novaplex™ SARS-CoV-2 Variants II Assay for samples with no targets found with the Variants I assay or with the mutation E484 K alone, in order to screen the mutations L452R, K417 N/T and W152C. Results Between weeks 11 and 25, 2239 positive samples out of 54317 were tested with the Variants I Assay. Between weeks 18 and 25, 94 samples met the criteria for being tested with the Variants II Assay. Of these, 47 had the L452R mutation without the W152C mutation, typical in the B.1.617 variant. At week 25, this profile was found in 45.5% of the samples and was the most frequent. Conclusion According to our observations, variant B.1.617 has become predominant in our institution and most probably in our region. In the absence of the use of the Variants II Assay, they would have been considered wild.

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Development and implementation of a RT‐qPCR extraction‐free protocol for the detection of SARS‐CoV‐2 and impact on the turn‐around‐time

Cited by 7 publications

References 17 publications

Strategies That Facilitate Extraction-Free SARS-CoV-2 Nucleic Acid Amplification Tests

Strategies That Facilitate Extraction-Free SARS-CoV-2 Nucleic Acid Amplification Tests

Severe acute respiratory syndrome coronavirus 2 detection by real time polymerase chain reaction using pooling strategy of nasal samples

The challenge of screening SARS-CoV-2 variants of concern with RT-qPCR: One variant can hide another

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