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About: 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 (t) 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.

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