Comparison of analytical sensitivity and efficiency for SARS-CoV-2 primer sets by TaqMan-based and SYBR Green-based RT-qPCR

Yile Tao,Michael Huber,Michel Bielecki,Guangyu Qiu,Zhibo Gai,Zheng Ji,Yang Yue,Qiyuan Wang,Alexandra Trkola,Martin Spillman,Qingfa Chen,Jing Wang,Junji Cao

doi:10.1007/s00253-022-11822-4

Abstract

The pandemic of coronavirus disease 2019 (COVID-19) continues to threaten public health. For developing countries where vaccines are still in shortage, cheaper alternative molecular methods for SARS-CoV-2 identification can be crucial to prevent the next wave. Therefore, 14 primer sets recommended by the World Health Organization (WHO) was evaluated on testing both clinical patient and environmental samples with the gold standard diagnosis method, TaqMan-based RT-qPCR, and a cheaper alternative method, SYBR Green-based RT-qPCR. Using suitable primer sets, such as ORF1ab, 2019_nCoV_N1 and 2019_nCoV_N3, the performance of the SYBR Green approach was comparable or better than the TaqMan approach, even when considering the newly dominating or emerging variants, including Delta, Eta, Kappa, Lambda, Mu, and Omicron. ORF1ab and 2019_nCoV_N3 were the best combination for sensitive and reliable SARS-CoV-2 molecular diagnostics due to their high sensitivity, specificity, and broad accessibility.Key points• With suitable primer sets, the SYBR Green method performs better than the TaqMan one.• With suitable primer sets, both methods should still detect the new variants well.• ORF1ab and 2019_nCoV_N3 were the best combination for SARS-CoV-2 detection.

Highlights

After first being reported in Wuhan, China, in December, 2019, the pandemic of coronavirus disease 2019 (COVID19) caused by an enveloped, single-stranded, positive-sense RNA betacoronavirus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has already lasted for more than 1.5 years
This study is aimed at comparing the analytical sensitivity and efficiencies of selected primer sets in TaqMan-based and SYBR Green-based reverse transcription-quantitative polymerase chain reaction (RT-qPCR) methods applied to 23 patient samples which included two samples of the B.1.351 lineage (Beta variant), as well as a lab cultured sample of Human Coronavirus-229E (HCoV-229E) and two environmental aerosol samples collected in Wuhan before the lockdown
ORF1ab, N, WH-NIC N, and nCoV_IP4’s amplification efficiencies of the TaqMan approach were higher than the SYBR Green ones, while the other primer sets differed, yet all matched the criteria for efficient RT-qPCR (Vogels et al 2020)

Summary

Introduction

After first being reported in Wuhan, China, in December, 2019, the pandemic of coronavirus disease 2019 (COVID19) caused by an enveloped, single-stranded, positive-sense RNA betacoronavirus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has already lasted for more than 1.5 years. Mitigation approaches (e.g., promotion of hygiene, social distancing, isolation of infected people, and restricting traveling) are crucial, but not enough for several reasons: first, unlike its close relative, severe acute respiratory syndrome coronavirus (SARS-CoV) (Lu. Applied Microbiology and Biotechnology et al 2020), its transmission can occur during its possibly quite long prodromal period when those infected are mildly ill and carry on usual activities (Heymann et al 2020; Zou et al 2020). Testing for SARS-CoV-2 infection in the whole population is central to track the spread of disease as well as to inform public policies.

Objectives

Methods

Results