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. Am J Clin Pathol 2021 Jun 17;156(1):24-33
Date
05/04/2021Pubmed ID
33940605Pubmed Central ID
PMC8135719DOI
10.1093/ajcp/aqab060Scopus ID
2-s2.0-85108387082 (requires institutional sign-in at Scopus site)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.
Author List
Buchan BW, Gerstbrein D, Cruz A, Hoff J, Sievert E, Ledeboer NA, Faron MLAuthors
Blake W. Buchan PhD Professor in the Pathology department at Medical College of WisconsinJessica Hoff PhD Assistant Professor in the Pathology department at Medical College of Wisconsin
Nathan A. Ledeboer PhD Vice Chair, Professor in the Pathology department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
HumansLimit of Detection
RNA, Viral
Real-Time Polymerase Chain Reaction
Sensitivity and Specificity
Specimen Handling
