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.

Evaluation of SYBR Green real time PCR for detecting SARS-CoV-2 from clinical samples

“…This small difference in the detection limit indicates that the specificity of the assay is mainly determined by the ORF1b-nsp14 primer set. Other groups proposed other assays for detecting SARS-CoV-2 based on SYBR Green with similar ( Dorlass et al, 2020 ; Won et al, 2020 ) or even higher detection limit than us ( Meza-Robles et al, 2020 ). In contrast, the SYBR Green assay for N region seems to be not suitable for SARS-CoV-2 diagnosis, at least, in our experimental conditions.…”

“…For SARS-CoV-2 detection, the WHO recommended routine confirmation of cases of COVID-19 based on detection of unique sequences of virus RNA by nucleic acid amplification test, such as RT-qPCR, with confirmation by nucleic acid sequencing when necessary ( World Health Organization, 2020 ). Previous work have attempted to assess the analytical sensitivity and specificity of the different sets of primers and probes available (either commercially or in-house developed) ( Barra et al, 2020 ; Nalla et al, 2020 ; Chu et al, 2020 ; Corman et al, 2020 ; Jung et al, 2020 ; Vogels et al, 2020 ) as well as adapted to SYBR Green ( Dorlass et al, 2020 ; Meza-Robles et al, 2020 ; Won et al, 2020 ).…”

Diagnosis of SARS-CoV-2 infections in times of material shortage

“…The intercalating dye-based assays for hRNAse P and E genes were as sensitive as the hydrolysis probe-based assays, but the RdRp gene showed ten times less sensitivity. Dorlass and collaborators (12) also reported good performance of E gene primers published by Corman et al (8) using intercalating dye-based assays in nasopharyngeal swabs collected in hospitals when compared to probe-based assay. Only one SARS-CoV-2 RT-qPCR positive individual who was positive for RNAse P by SYBR RT-qPCR was not positive for the E and RdRp genes when material from Q-tips plus classical RNA extraction was tested.…”

Evaluation of “Caterina assay”: An Alternative Tool to the Commercialized Kits Used for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Identification

“…A carful study of the stem–loop architecture in the N1 gene amplicon permitted to identify a looped-out region from the primer binding site where the oligos could perfectly anneal to the template, increasing the sensibility of the detection method ( Figure 2 ) [ 21 , 27 ]. Indeed, the reaction presented a high sensitivity (detection limit to 50 viral genomes/μL, Ct = 35.75) ( Figure 3 ), which resulted to be higher compared to other SYBR ® Green-based RT-PCR methods for SARS-CoV-2 detection (10 3 copies/µL) [ 28 ]. Specificity of detection of SARS-CoV-2 was finally confirmed by the sequencing of 50 RT-PCR amplicons coming from clinal specimens ( Figure 5 ).…”