Lower cost alternatives for molecular diagnosis of COVID-19: conventional RT-PCR and SYBR Green-based RT-qPCR

Abstract

In March 2020, WHO declared a pandemic state due to SARS-CoV-2 having spread. TaqMan-based real-time RT-qPCR is currently the gold standard for COVID-19 diagnosis. However, it is a high-cost assay, inaccessible for the majority of laboratories around the world, making it difficult to diagnose on a large scale. The objective of this study was to standardize lower cost molecular methods for SARS-CoV-2 identification. E gene primers previously determined for TaqMan assays by Colman et al. (2020) were adapted in SYBR Green assay and RT-PCR conventional. The cross-reactivity test was performed with 17 positive samples for other respiratory viruses, and the sensibility test was performed with 8 dilutions (10 based) of SARS-CoV-2 isolated and 63 SARS-CoV-2-positive samples. The SYBR Green assays and conventional RT-PCR have not shown amplification of the 17 respiratory samples positives for other viruses. The SYBR Green-based assay was able to detect all 8 dilutions of the isolate. The conventional PCR detected until 10⁷ dilution, both assays detected the majority of the 63 samples, 98.42% of positivity in SYBR Green, and 93% in conventional PCR. The average Ct variation between SYBR Green and TaqMan was 1.92 and the highest Ct detected by conventional PCR was 35.98. Both of the proposed assays are less sensitive than the current gold standard; however, our data shows a low sensibility variation, suggesting that these methods could be used by laboratories as a lower cost molecular method for SARS-CoV-2 diagnosis.

Keywords: COVID-19 pandemic; Conventional PCR; Molecular diagnoses; SARS-CoV-2; SYBR Green assay.

Fig. 1

Analytical sensitivity of the tested molecular diagnostic methods using the Brazilian SARS-CoV-2 isolate. a Standard curve for absolute quantification by TaqMan real-time PCR. Amplification plot and linear regression of standard curve, slope − 3.494, R2 0.99, Eff% 93.284. b Melting curve and linear regression of the standard curve, slope − 3.497, R2 0.99, Eff% 9.168. c and d Agarose gels of one-step RT-PCR and two-steps RT-PCR, respectively. The 600-bp marker in the ladder (LD) is displayed in the gel. The arrow marks the amplified fragment of 113 base pairs (bp) amplified with the SARS-CoV-2-specific E gene primers. Each dilution is displayed in the top of gels as well as the cycle threshold (Ct) values, at the bottom of the pictures. The observed amplifications are indicated by the red boxes

Fig. 2

Comparison of Ct values obtained by TaqMan, SYBR Green, and one-step/two-step RT-PCR assays. a Ct values of 63 clinical samples detected with TaqMan and SYBR Green RT-qPCR. b, c Agarose gels of amplified fragments by one-step RT-PCR and two-step PCR assays, respectively, of 14 representatives’ clinical samples. The 600-bp marker in the ladder (LD) is indicated in the picture. The arrow points to the fragment of 113 base pairs (bp) amplified with SARS-CoV-2-specific E gene primers