tetano
Editor, Senior Moderator
Microbiol Spectr
. 2022 Aug 11;e0135822.
doi: 10.1128/spectrum.01358-22. Online ahead of print.
High-Throughput COVID-19 Testing of Naso-Oropharyngeal Swabs Using a Sensitive Extraction-Free Sample Preparation Method
Todd M Pryce[SUP] 1 [/SUP], Erin J Haygarth[SUP] 1 [/SUP], Jessica Bordessa[SUP] 1 [/SUP], Courtney T Jeffery[SUP] 1 [/SUP], Ian D Kay[SUP] 1 [/SUP], James P Flexman[SUP] 1 [/SUP], Peter A Boan[SUP] 1 2 [/SUP]
Affiliations
Abstract
High-throughput diagnostic assays are required for large-scale population testing for severe acute respiratory coronavirus 2 (SARS-CoV-2). The gold standard technique for SARS-CoV-2 detection in nasopharyngeal swab specimens is nucleic acid extraction followed by real-time reverse transcription-PCR. Two high-throughput commercial extraction and detection systems are used routinely in our laboratory: the Roche cobas SARS-CoV-2 assay (cobas) and the Roche MagNA Pure 96 system combined with the SpeeDx PlexPCR SARS-CoV-2 assay (Plex). As an alternative to more costly instrumentation, or tedious sample pooling to increase throughput, we developed a high-throughput extraction-free sample preparation method for naso-oropharyngeal swabs using the PlexPCR SARS-CoV-2 assay (Direct). A collection of SARS-CoV-2-positive (n = 185) and -negative (n = 354) naso-oropharyngeal swabs in transport medium were tested in parallel to compare Plex to Direct. The overall agreement comparing the qualitative outcomes was 99.3%. The mean cycle of quantification (C[SUB]q[/SUB]) increase and corresponding mean reduction in viral load for Direct ORF1ab and RdRp compared to Plex was 3.11 C[SUB]q[/SUB] (-0.91 log[SUB]10[/SUB] IU/mL) and 4.78 C[SUB]q[/SUB] (-1.35 log[SUB]10[/SUB] IU/mL), respectively. We also compared Direct to a four-sample pool by combining each positive sample (n = 185) with three SARS-CoV-2-negative samples extracted with MagNA Pure 96 and tested with the PlexPCR SARS-CoV-2 assay (Pool). Although less sensitive than Plex or Pool, the Direct method is a sufficiently sensitive and viable approach to increase our throughput by 12,032 results per day. Combining cobas, Plex, and Direct, an overall throughput of 19,364 results can be achieved in a 24-h period. IMPORTANCE Laboratories have experienced extraordinary demand globally for reagents, consumables, and instrumentation, while facing unprecedented testing demand needed for the diagnosis of SARS-CoV-2 infection. A major bottleneck in testing throughput is the purification of viral RNA. Extraction-based methods provide the greatest yield and purity of RNA for downstream PCR. However, these techniques are expensive, time-consuming, and depend on commercial availability of consumables. Extraction-free methods offer an accessible and cost-effective alternative for sample preparation. However, extraction-free methods often lack sensitivity compared to extraction-based methods. We describe a sensitive extraction-free protocol based on a simple purification step using a chelating resin, combined with proteinase K and thermal treatment. We compare the sensitivity qualitatively and quantitatively to a well-known commercial extraction-based system, using a PCR assay calibrated to the 1st WHO international standard for SARS-CoV-2 RNA. This method entails high throughput and is suitable for all laboratories, particularly in jurisdictions where access to instrumentation and reagents is problematic.
Keywords: PlexPCR; SARS-CoV-2; cobas; extraction-free; high-throughput.
. 2022 Aug 11;e0135822.
doi: 10.1128/spectrum.01358-22. Online ahead of print.
High-Throughput COVID-19 Testing of Naso-Oropharyngeal Swabs Using a Sensitive Extraction-Free Sample Preparation Method
Todd M Pryce[SUP] 1 [/SUP], Erin J Haygarth[SUP] 1 [/SUP], Jessica Bordessa[SUP] 1 [/SUP], Courtney T Jeffery[SUP] 1 [/SUP], Ian D Kay[SUP] 1 [/SUP], James P Flexman[SUP] 1 [/SUP], Peter A Boan[SUP] 1 2 [/SUP]
Affiliations
- PMID: 35950846
- DOI: 10.1128/spectrum.01358-22
Abstract
High-throughput diagnostic assays are required for large-scale population testing for severe acute respiratory coronavirus 2 (SARS-CoV-2). The gold standard technique for SARS-CoV-2 detection in nasopharyngeal swab specimens is nucleic acid extraction followed by real-time reverse transcription-PCR. Two high-throughput commercial extraction and detection systems are used routinely in our laboratory: the Roche cobas SARS-CoV-2 assay (cobas) and the Roche MagNA Pure 96 system combined with the SpeeDx PlexPCR SARS-CoV-2 assay (Plex). As an alternative to more costly instrumentation, or tedious sample pooling to increase throughput, we developed a high-throughput extraction-free sample preparation method for naso-oropharyngeal swabs using the PlexPCR SARS-CoV-2 assay (Direct). A collection of SARS-CoV-2-positive (n = 185) and -negative (n = 354) naso-oropharyngeal swabs in transport medium were tested in parallel to compare Plex to Direct. The overall agreement comparing the qualitative outcomes was 99.3%. The mean cycle of quantification (C[SUB]q[/SUB]) increase and corresponding mean reduction in viral load for Direct ORF1ab and RdRp compared to Plex was 3.11 C[SUB]q[/SUB] (-0.91 log[SUB]10[/SUB] IU/mL) and 4.78 C[SUB]q[/SUB] (-1.35 log[SUB]10[/SUB] IU/mL), respectively. We also compared Direct to a four-sample pool by combining each positive sample (n = 185) with three SARS-CoV-2-negative samples extracted with MagNA Pure 96 and tested with the PlexPCR SARS-CoV-2 assay (Pool). Although less sensitive than Plex or Pool, the Direct method is a sufficiently sensitive and viable approach to increase our throughput by 12,032 results per day. Combining cobas, Plex, and Direct, an overall throughput of 19,364 results can be achieved in a 24-h period. IMPORTANCE Laboratories have experienced extraordinary demand globally for reagents, consumables, and instrumentation, while facing unprecedented testing demand needed for the diagnosis of SARS-CoV-2 infection. A major bottleneck in testing throughput is the purification of viral RNA. Extraction-based methods provide the greatest yield and purity of RNA for downstream PCR. However, these techniques are expensive, time-consuming, and depend on commercial availability of consumables. Extraction-free methods offer an accessible and cost-effective alternative for sample preparation. However, extraction-free methods often lack sensitivity compared to extraction-based methods. We describe a sensitive extraction-free protocol based on a simple purification step using a chelating resin, combined with proteinase K and thermal treatment. We compare the sensitivity qualitatively and quantitatively to a well-known commercial extraction-based system, using a PCR assay calibrated to the 1st WHO international standard for SARS-CoV-2 RNA. This method entails high throughput and is suitable for all laboratories, particularly in jurisdictions where access to instrumentation and reagents is problematic.
Keywords: PlexPCR; SARS-CoV-2; cobas; extraction-free; high-throughput.