How to choose the right real-time RT-PCR primer sets for the SARS-CoV-2 genome detection?

Abstract

Objectives: The SARS-CoV-2 pandemic has created an unprecedented need for rapid large-scale diagnostic testing to prompt clinical and public health interventions. Currently, several quantitative reverse-transcription polymerase chain reaction (RT-qPCR) assays recommended by the World Health Organization are being used by clinical and public health laboratories and typically target regions of the RNA-dependent RNA polymerase (RdRp), envelope (E) and nucleocapsid (N) coding region. However, it is currently unclear if results from different tests are comparable. This study aimed to clarify the clinical performances of the primer/probe sets designed by US CDC and Charité/Berlin to help clinical laboratories in assay selection for SARS-CoV-2 routine detection.

Methods: We compared the clinical performances of the recommended primer/probe sets using one hundred nasopharyngeal swab specimens from patients who were clinically diagnosed with COVID-19. An additional 30 "pre-intervention screening" samples from patients who were not suspected of COVID-19 were also included in this study. We also performed sequence alignment between 31064 European SARS-CoV-2 and variants of concern genomes and the recommended primer/probe sets.

Results: The present study demonstrates substantial differences in SARS-CoV-2 RNA detection sensitivity among the primer/probe sets recommended by the World Health Organization especially for low-level viral loads. The alignment of thousands of SARS-CoV-2 sequences reveals that the genetic diversity remains relatively low at the primer/probe binding sites. However, multiple nucleotide mismatches might contribute to false negatives.

Conclusion: An understanding of the limitations depending on the targeted genes and primer/probe sets may influence the selection of molecular detection assays by clinical laboratories.

Keywords: COVID-19; Clinical performance; In silico analysis; Molecular detection; Real-time RT PCR; SARS-CoV-2.

Fig. 1

Alignments of N1 (A), N2 (B), E (C), RdRp (D) regions of SARS-CoV-2 with SARS-Coronavirus, MERS-Coronavirus and seasonal human coronaviruses genomes. The arrows indicate the regions targeted by the sets of primer/probe. Human SARS-CoV-2: hCoV-19/Belgium/CJM-0323175/2020|EPI_ISL_420432; Human SARS-CoV: SARS coronavirus NC_004718.3; Bat SL-CoV ZXC2: Bat SARS-like coronavirus isolate MG772934.1; Bat SARS-related CoV BM48-3: BGR/2008 GU190215.1 Human MERS-CoV: Middle East respiratory syndrome coronavirus NC_019843.3; Human CoV HKU1: Human coronavirus HKU1 NC_006577.2; Human CoV OC43 : Human coronavirus OC43 strain ATCC VR-759 NC_006213.1; Human CoV NL63: Human Coronavirus NL63 NC_005831.2; Human CoV 229E: Human coronavirus 229E NC_002645.1.

Fig. 2

Comparison of the viral load detected by the six RT-qPCR assays among the positive nasopharyngeal swabs (n = 84). The viral load is expressed in log copies/mL and each clinical sample is represented by a circle. The white circles represent clinical samples detected by all RT-qPCR assays while colored circles represent samples not detected by the six assays. Bars represent the median and 95 % Confidence Interval.