tetano
Editor, Senior Moderator
J Med Virol
. 2020 Jul 23;10.1002/jmv.26328.
doi: 10.1002/jmv.26328. Online ahead of print.
COVID-19 diagnostics for resource-limited settings: Evaluation of "unextracted" qRT-PCR
Nicholas M Adams[SUP] 1 [/SUP], Mindy Leelawong[SUP] 1 [/SUP], Alison Benton[SUP] 2 [/SUP], Criziel Quinn[SUP] 2 [/SUP], Frederick R Haselton[SUP] 1 [/SUP], Jonathan E Schmitz[SUP] 2 3 4 [/SUP]
Affiliations
Abstract
The coronavirus disease 2019 (COVID-19) pandemic has created a precipitous increase in the need for molecular diagnostics. Unfortunately, access to RNA extraction reagents can represent a bottleneck for quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR)-based methodologies, stemming from both extraordinary supply-chain stresses and the global reach of the virus into resource-limited settings. To provide flexible diagnostic options for such environments, we report here an "unextracted modification" for qRT-PCR using the Centers for Disease Control's (CDC's) widely utilized primers/probe sets for severe acute respiratory syndrome coronavirus 2 (N1/N2/N3 targeting viral nucleocapsid and RP-control targeting human RNase P). This approach replaces RNA extraction/purification with a heat-inactivation step of viral transport media (VTM), followed by direct inoculation-with or without VTM spin concentration-into PCR master mixes. Using derivatives of care from our clinical workflow, we compared traditional and unextracted CDC methodologies. Although some decrease in analytic sensitivity was evident (by higher C[SUB]t[/SUB] values) without extraction, in particular for the N2 primer/probe-set, we observed high categorical positive agreement between extracted and unextracted results for N1 (unconcentrated VTM-38/40; concentrated VTM-39/41), N3 (unconcentrated VTM-38/40; concentrated VTM-41/41), and RP (unconcentrated and concentrated VTM-81/81). The negative categorical agreement for N1/N2/N3 was likewise high. Overall, these results suggest that laboratories could adapt and validate unextracted qRT-PCR protocols as a contingency to overcome supply limitations, with minimal impact on categorical results.
Keywords: COVID‐19; RNA; RT‐PCR; SARS‐CoV‐2; diagnostics; unextracted.
. 2020 Jul 23;10.1002/jmv.26328.
doi: 10.1002/jmv.26328. Online ahead of print.
COVID-19 diagnostics for resource-limited settings: Evaluation of "unextracted" qRT-PCR
Nicholas M Adams[SUP] 1 [/SUP], Mindy Leelawong[SUP] 1 [/SUP], Alison Benton[SUP] 2 [/SUP], Criziel Quinn[SUP] 2 [/SUP], Frederick R Haselton[SUP] 1 [/SUP], Jonathan E Schmitz[SUP] 2 3 4 [/SUP]
Affiliations
- PMID: 32779772
- PMCID: PMC7405028
- DOI: 10.1002/jmv.26328
Abstract
The coronavirus disease 2019 (COVID-19) pandemic has created a precipitous increase in the need for molecular diagnostics. Unfortunately, access to RNA extraction reagents can represent a bottleneck for quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR)-based methodologies, stemming from both extraordinary supply-chain stresses and the global reach of the virus into resource-limited settings. To provide flexible diagnostic options for such environments, we report here an "unextracted modification" for qRT-PCR using the Centers for Disease Control's (CDC's) widely utilized primers/probe sets for severe acute respiratory syndrome coronavirus 2 (N1/N2/N3 targeting viral nucleocapsid and RP-control targeting human RNase P). This approach replaces RNA extraction/purification with a heat-inactivation step of viral transport media (VTM), followed by direct inoculation-with or without VTM spin concentration-into PCR master mixes. Using derivatives of care from our clinical workflow, we compared traditional and unextracted CDC methodologies. Although some decrease in analytic sensitivity was evident (by higher C[SUB]t[/SUB] values) without extraction, in particular for the N2 primer/probe-set, we observed high categorical positive agreement between extracted and unextracted results for N1 (unconcentrated VTM-38/40; concentrated VTM-39/41), N3 (unconcentrated VTM-38/40; concentrated VTM-41/41), and RP (unconcentrated and concentrated VTM-81/81). The negative categorical agreement for N1/N2/N3 was likewise high. Overall, these results suggest that laboratories could adapt and validate unextracted qRT-PCR protocols as a contingency to overcome supply limitations, with minimal impact on categorical results.
Keywords: COVID‐19; RNA; RT‐PCR; SARS‐CoV‐2; diagnostics; unextracted.