Comparative evaluation of a dual-target real-time RT-PCR assay for COVID-19 diagnosis and assessment of performance in pooled saliva and nasopharyngeal swab samples

Abstract

Objectives: Sensitive molecular diagnostic assays are essential for COVID-19 diagnosis. We evaluated the Hecin Scientific SARS-CoV-2 nucleic acid test kit, a dual-target real-time RT-PCR assay targeting the SARS-CoV-2 N and ORF1ab genes.

Methods: The Hecin test kit's diagnostic performance in detecting SARS-CoV-2 RNA was compared to the LightMix Modular SARS and Wuhan CoV E-gene kit (TIB Molbiol) and an in-house single-tube nested real-time RT-PCR using 296 clinical specimens, 11 proficiency testing samples, and 30 low-positive deep throat saliva and nasopharyngeal swab (NPS) samples pooled into negative samples in ratios of 1:5, 1:10, and 1:30.

Results: The limit-of-detection of the Hecin test kit was around 500 dC/mL for the N and ORF1ab targets. Sensitivity and specificity of the Hecin test kit were 98.1% (95% CI: 93.4-99.8%) and 100% (98.1-100%), respectively, when measured against the reference method. The Hecin test kit showed fair sensitivity (80%) in low-positive NPS samples pooled in ratios of 1:5 and 1:10. Its performance in pooled samples could be dramatically improved by adjusting the assay Ct cutoff.

Conclusion: The Hecin test kit enables sensitive and specific detection of SARS-CoV-2 in clinical samples and pooled samples.

Keywords: COVID-19; SARS-CoV-2; diagnostic test evaluation; pooling; real-time RT-PCR.

Figure 1.

Correlation of the Ct values of the samples found positive for SARS-CoV-2 by the Hecin test kit and LightMix E-gene kit: (A) N target of Hecin kit vs E-gene kit, (B) ORF1ab target of Hecin kit vs E-gene kit and (C) N target vs ORF1ab target of Hecin kit

Figure 2.

Comparison of the Ct values of the Hecin test kit and LightMix E-gene kit for samples positive according to the reference standard. A Ct value of 41 was assigned to samples that tested negative by either kit. **** indicates P < 0.0001