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

Expert Rev Mol Diagn. 2021 Jul;21(7):741-747. doi: 10.1080/14737159.2021.1933445. Epub 2021 Jun 4.

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.

Publication types

  • Video-Audio Media

MeSH terms

  • COVID-19 Nucleic Acid Testing*
  • COVID-19* / diagnosis
  • COVID-19* / genetics
  • Humans
  • RNA, Viral / genetics
  • Real-Time Polymerase Chain Reaction*
  • Reverse Transcriptase Polymerase Chain Reaction*
  • SARS-CoV-2 / genetics*
  • Specimen Handling*

Substances

  • RNA, Viral

Grants and funding

This study was partly supported by the donations of Richard Yu and Carol Yu, the Shaw Foundation Hong Kong, May Tam Mak Mei Yin, Michael Seak-Kan Tong, Hui Ming, Hui Hoy and Chow Sin Lan Charity Fund Limited, Chan Yin Chuen Memorial Charitable Foundation, Marina Man-Wai Lee, the Hong Kong Hainan Commercial Association South China Microbiology Research Fund, the Jessie & George Ho Charitable Foundation, Perfect Shape Medical Limited, Kai Chong Tong, and Tse Kam Ming Laurence; and funding from the Consultancy Service for Enhancing Laboratory Surveillance of Emerging Infectious Diseases and Research Capability on Antimicrobial Resistance for Department of Health of the Hong Kong Special Administrative Region Government. The funding sources had no role in the design and conduct of the study, in the collection, analysis and interpretation of data, or in the preparation, review or approval of the manuscript.