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. 2021 Nov:112:264-268.
doi: 10.1016/j.ijid.2021.09.046. Epub 2021 Sep 23.

Evaluation of extraction-free RT-PCR methods for faster and cheaper detection of SARS-CoV-2 using two commercial systems

Fabio Morecchiato  1 , Marco Coppi  2 , Ilaria Baccani  1 , Niccolò Maggini  3 , Nunziata Ciccone  3 , Alberto Antonelli  2 , Gian Maria Rossolini  4
Affiliations

Evaluation of extraction-free RT-PCR methods for faster and cheaper detection of SARS-CoV-2 using two commercial systems

Fabio Morecchiato et al. Int J Infect Dis. 2021 Nov.

Abstract

Objective: When using high-throughput batched diagnostic platforms based on RT-PCR for SARS-CoV-2 detection, avoidance of the conventional nucleic acid extraction step can help to reduce the turnaround time and increase processivity. This approach can also spare reagents and plasticware, which have experienced a shortage during the initial waves of the pandemic, reducing the overall testing costs.

Methods: This study evaluated the performance of extraction-free protocols based on simple dilution of the specimen in sterile RNAse free water (with or without a heating step) in comparison to standard RNA extraction protocols, using two commercial kits for molecular detection of SARS-CoV-2 (Allplex™ SARS-CoV-2 assay and Allplex™ SARS-CoV-2/FluA/FluB/RSV assay) in nasopharyngeal swabs (NPS).

Results: Compared with conventional protocols, extraction-free protocols based on sample dilution without a heating step exhibited a lower analytical sensitivity: 74.0% and 82.1% with the Allplex™ SARS-CoV-2 assay (tested with 139 NPS samples) and the Allplex™ SARS-CoV-2/FluA/FluB/RSV assay (tested with 69 NPS samples), with a mean increase of Ct values of +2.04 and +1.32, respectively. Most false negative results were observed with sampled low viral load. Including a step of heat exposure did not improve but actually decreased the analytical sensitivity of the assay.

Conclusions: Results confirmed that extraction-free protocols could be a faster and cheaper approach to SARS-CoV-2 detection in NPS samples, which could improve processivity of diagnostic platforms.

Keywords: SARS-CoV-2; diagnostic platform; extraction-free method; molecular diagnostics; real-time polymerase chain reaction.

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Conflict of interest statement

Declaration of Competing Interest Dr. Antonelli reports personal fees from Accelerate diagnostics, personal fees from Menarini, personal fees from Seegene, outside the submitted work. Prof. Rossolini reports grants, personal fees and non-financial support from Accelerate Diagnostics, personal fees from Becton Dickinson, grants and personal fees from bioMeriéux, grants and personal fees from Cepheid, grants and personal fees from Elitech, grants and personal fees from Merck, grants and personal fees from Nordic Pharma, personal fees from Pfizer, grants from Seegene, grants and personal fees from Shionogi, personal fees and other from Venatorx, grants and personal fees from Zambon, personal fees from Roche, personal fees from Thermo Fisher, personal fees and non-financial support from Beckman Coulter, grants, personal fees and non-financial support from Menarini, grants from Arrow, grants from Symcel, personal fees from QPex, grants from DID, grants from Hain Lifescience GmbH, grants from GenePoc, grants from SetLance, grants and personal fees from Angelini, grants from Qvella, grants from Qlinea, personal fees from Qiagen, grants from Biomedical Service, grants from Liofilchem, outside the submitted work. Dr. Baccani reports other from Diesse-Diagnostica Senese, outside the submitted work. All other authors have nothing to disclose.

Figures

Figure 1
Figure 1
Comparison of each gene Ct with positive samples obtained with the STARMAG protocol (on x axis), the EX-FREE protocol and the EX-FREE-HEAT protocol using the ALLPLEX-COV kit. Negative targets were plotted as Ct >40.
Figure 2
Figure 2
Comparison of each target gene Ct with positive samples obtained with the STARMAG protocol (on x axis) and the EX-FREE protocol using the ALLPLEX-COV-FLU kit. Negative targets were plotted as Ct >40.
Figure 3
Figure 3
Endogenous IC Ct values obtained with ALLPLEX-COV-FLU and the two analytical protocols.
See this image and copyright information in PMC

References

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