Comparative analysis of primer-probe sets for the laboratory confirmation of SARS-CoV-2

Authors:  Jung et al.                   

Link to paper: https://www.biorxiv.org/content/10.1101/2020.02.25.964775v1       

Journal/ Pre-Print: BioRXiv

SUMMARY 

This study compares the sensitivity of different primer probe sets recommended by the World Health Organisation (WHO) for the confirmation of SARS-CoV-2. Sensitivity is required for the early diagnosis of SARS-CoV-2. Of the WHO recommended SARS-CoV-2 primers, ORF1 and N gene primers were assessed in this study. Using serial dilutions of SARS-CoV-2 RNA isolated from infected Vero cells, the sensitivity of different primer sets was assessed using quantitative RT-PCR. This study concluded that ORF1ab (China), 2019-nCoV_N2 (USA), 2019-nCoV_N3 (USA), and NIID_2019-nCoV_N (Japan) sets may be recommended for the laboratory confirmation of SARS-CoV-2.

RESEARCH HIGHLIGHTS

1. CT values compared at a concentration of 15 copies per reaction
2. Sensitivity of primer probe sets was consistent across serial dilutions of RNA samples.
3. ‘2019-nCoV_N2, N3’ of USA most sensitive N gene set comparable to that of

NIID_2019-nCOV_N (Japan)

1. ‘ORF1ab’ of China most sensitive ORF1 gene set
2. Appropriate combination of above best for most sensitive confirmation of SARS-CoV-2

RESEARCH IMPACT:

Provides researchers with the most sensitive primer sets for the early diagnosis of SARS-CoV-2.

METHODOLOGY:

African green monkey kidney Vero cells (ATCC CCL-81) were infected with a clinical isolate SARS-CoV-2 (BetaCoV/Korea/KCDC03/2020) provided from Korea CDC.

RNA was isolated and purified using QiAMP Qiagen kit.

qRT-PCR was used to assess primer sensitivity.

STRENGTHS AND WEAKNESSES OF THE PAPER:

Strengths:

Quality of the assays

Sensitivity of primer probe sets was consistent across serial dilutions of RNA samples.

Weaknesses:

Could have done more repeats as can’t have a loading control CT to adjust to.

Only one set of primers used to reverse transcribe RNA.