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

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.

Keywords: COVID-19; RT-qPCR; SARS-CoV-2; SYBR Green; TaqMan probe.

Fig. 1

Comparisons of standard curves of the nine primer sets with TaqMan-based and SYBR Green-based RT-qPCR using standard plasmids (blue circle: TaqMan-based RT-qPCR results included in linear regression; orange circle: TaqMan-based RT-qPCR results excluded in linear regression; green square: SYBR Green-based RT-qPCR included in linear regression; yellow square: SYBR Green-based RT-qPCR results excluded in linear regression).  a ORF1ab, b N, c nCoV_IP4, d 2019-nCoV_N1, e 2019-nCoV_N3, f NIID_2019-nCoV_2, g ORF1b-nsp14, h HKU-N, i WH-NIC N

Fig. 2

Comparisons of analytical sensitivity of the nine primer sets with TaqMan-based and SYBR Green-based RT-qPCR using SARS-CoV-2 positive, negative nasopharyngeal swabs, HCoV-229E samples, and pure water (all samples were measured in triplicates. ND: not detected. The color of the points represents the T_m of SYBR Green-based RT-qPCR products)

Fig. 3

Comparisons of analytical sensitivity of the nine primer sets with TaqMan-based and SYBR Green-based RT-qPCR using SARS-CoV-2 positive nasopharyngeal swabs from 3 patients including one infected by B.1.351 lineage (all samples were separately diluted for 3 times into 4 series of tenfold dilutions. All were measured in replicates. ND: not detected. The color of the points represents the T_m of SYBR Green-based RT-qPCR products)

Fig. 4

Comparisons of analytical sensitivity of the nine primer sets with TaqMan-based and SYBR Green-based RT-qPCR using aerosol samples collected in Wuhan (a Ct values. In this panel, the color of the points represent the T_m of SYBR Green-based RT-qPCR products. ND: not detected. b Absolute abundances in air. In both panels, W1: Wuhan aerosol sample obtained on the 11th of January, 2020; W2: Wuhan aerosol sample obtained on the 17th of January, 2020)