Evaluation of a High-Definition PCR Assay for the Detection of SARS-CoV-2 in Extracted and Nonextracted Respiratory Specimens Collected in Various Transport Media

Abstract

Abstract Objectives We conducted an analytic and clinical comparison of a novel high-definition polymerase chain reaction PCR (HDPCR) assay to traditional real-time PCR (RT-PCR) for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in upper respiratory specimens. Methods Analytic performance of RT-PCR, HDPCR, and extraction-free HDPCR was established through replicate testing of a serially diluted clinical specimen containing SARS-CoV-2. A clinical comparison of all 3 assays was conducted using 351 prospectively collected upper respiratory swab specimens obtained from symptomatic and asymptomatic individuals collected in various transport media. Results RT-PCR and HDPCR assays using extracted nucleic acid demonstrated similar analytic limits of detection (LoD) and clinical performance, with 100% positive and negative agreement. Extraction-free HDPCR demonstrated a 1.5 to 2.0 log10 increase in LoD based on cycle threshold values. However, clinical performance of extraction-free HDPCR remained high, demonstrating 97.8% positive and 99.6% negative agreement with RT-PCR. An overall increase in “invalid” and “presumptive” results was observed when using the extraction-free method, but this was highly variable based on transport medium used. Conclusions HDPCR performs similar to RT-PCR for the detection of SARS-CoV-2. The use of an extraction-free HDPCR protocol maintained high clinical performance despite reduced analytic LoD, with the benefit of reduced hands-on time and cost of reagents associated with nucleic acid extraction.