PfAgo-based detection of SARS-CoV-2

Abstract

In the present study, we upgraded Pyrococcus furiosus Argonaute (PfAgo) mediated nucleic acid detection method and established a highly sensitive and accurate molecular diagnosis platform for the large-scale screening of COVID-19 infection. Briefly, RT-PCR was performed with the viral RNA extracted from nasopharyngeal or oropharyngeal swabs as template to amplify conserved regions in the viral genome. Next, PfAgo, guide DNAs and molecular beacons in appropriate buffer were added to the PCR products, followed by incubating at 95 °C for 20-30 min. Subsequently, the fluorescence signal was detected. This method was named as SARS-CoV-2 PAND. The whole procedure is accomplished in approximately an hour with the using time of the Real-time fluorescence quantitative PCR instrument shortened from >1 h to only 3-5 min per batch in comparison with RT-qPCR, hence the shortage of the expensive Real-time PCR instrument is alleviated. Moreover, this platform was also applied to identify SARS-CoV-2 D614G mutant due to its single-nucleotide specificity. The diagnostic results of clinic samples with SARS-CoV-2 PAND displayed 100% consistence with RT-qPCR test.

Keywords: Molecular diagnosis; PfAgo; SARS-CoV-2.

Graphical abstract

Fig. 1

Schematic of recommended SARS-CoV-2 PAND workflow. MB: molecular beacon; gn: newly generated guide; Q: quencher; F: fluorophore.

Fig. 2

Establishing SARS-CoV-2 PAND with single input guide. A. SARS-CoV-2 PAND with N gene as the target; B. SARS-CoV-2 PAND with ORF1ab gene as the target. Sequences of 5′-phosphorylated input guides (gN 17, gO 31) are labeled in green. Newly generated single-stranded DNAs (gns) are indicated in blue, while the complementary sequences of gns in the molecular beacons are highlighted in brown; C. the signal intensity of detecting N gene with one, two or three input guides; D. the signal intensity of detecting ORF1ab gene with one, two or three input guides.

Fig. 3

Schematic of the genome of SARS-CoV-2 and the optimized amplicons of ORF1ab and N gene. The sequences of the input guide gN-17 and gO-31 are highlighted in blue. The 3′-end of both gns are indicated in bold black letters.

Fig. 4

LoD for RT-qPCR test with Thermofisher nucleic acid detection kit (A) and SARS-CoV-2 PAND assays (B).

Fig. 5

Identifying SARS-CoV-2 and its D614G mutants with PAND in clinical samples. A. testing results of clinical samples collected in February 2020 with SARS-CoV-2 PAND; B. testing results of clinical samples collected in June 2020 with SARS-CoV-2 PAND; C. detection of nt23403 SNP in clinical samples with PAND.