Sequence-specific and multiplex detection of COVID-19 virus (SARS-CoV-2) using proofreading enzyme-mediated probe cleavage coupled with isothermal amplification

Abstract

The outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been challenging human health worldwide. Loop-mediated isothermal amplification (LAMP) has been promptly applied to the detection of SARS-CoV-2 owing to its high amplification efficacy and less requirement of the thermal cycler. However, the vast majority of these LAMP-based assays depend on the non-specific detection of LAMP products, which can not discern the undesirable amplificons, likely to yield unreliable results. Herein, a sequence-specific LAMP assay was reported to detect SARS-CoV-2 using proofreading enzyme-mediated probe cleavage (named Proofman), which could realize real-time and visual detection without uncapping. This assay, introducing a proofreading enzyme and the fluorogenic probe to reverse-transcription LAMP (RT-Proofman-LAMP), can specifically detect the SARS-CoV-2 RNA with a detection limit of 100 copies. In addition to the real-time analysis, the assay is capable of endpoint visualization under a transilluminator within 50 min, providing a convenient reporting manner under the setting of point-of-care testing (POCT). In combination with different fluorophores, the one-pot multiplex assay was successfully achieved to detect multiple targets of SARS-CoV-2 and inner control simultaneously. In summary, the development of RT-Proofman-LAMP offers a versatile and highly-specific method for fast field screening and laboratory testing of SARS-CoV-2, making it a promising platform in COVID-19 diagnosis.

Keywords: COVID-19; LAMP; Proofman; SARS-CoV-2; Sequence-specific.

Fig. 1

The schematic of specific detection of LAMP products using the Proofman probe. (A) LAMP reaction process including the initial amplification phase and exponential amplification phase. Primer design was based on the target sequence: FIP and BIP were the inner primers; F3 and B3 were the outer primers; LF was the loop primer. (B) The principle of sequence-specific detection using the Proofman probe. The Proofman probe was designed based on the target sequence and a deliberate mismatch at the 3′ end of the probe was needed to trigger the cleavage activity of the proofreading enzyme (Pfu). Once the Proofman probe binds to the target loop of LAMP products, the Pfu can cleave the probe at the mismatching nucleotide, releasing the fluorophore. Then, the cleaved probe serves as a loop primer to enhance the amplification efficiency.

Fig. 2

Establishment of Proofman-LAMP for SARS-CoV-2 detection. (A) Design of LAMP primer sets and the Proofman probe based on the conservative sequence of SARS-CoV-2 gene N. (B) LAMP assay coupled with or without the Proofman to amplify the pUC57-N plasmid containing SARS-CoV-2 gene N cDNA. (C) Agarose gel image of the LAMP reaction (with/without Proofman probe) products (bottom) and endpoint image under 475 nm blue light (upper). PC, positive reaction with the template; NC, negative control reaction without the template; M, DNA marker. (D) Detection of SARS-CoV-2 gene N RNA using RT-Proofman-LAMP. (E) Endpoint image of RT-Proofman-LAMP after 50 min incubation. The reactions contained 1 × 10⁶ copies of DNA/RNA templates except for negative control (NC). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Sensitivity and specificity of RT-Proofman-LAMP. (A) The real-time RT-Proofman-LAMP using serially diluted SARS-CoV-2 gene N synthetic RNA (10⁷ copies to 10⁰ copies). (B) The linear relationship between the Tq value and the logarithm of the copy number in the range of 10⁷ copies to 10² copies of SARS-CoV-2 gene N synthetic RNA. The error bars are the standard deviation of three repetitive measurements. (C) The specificity of RT-Proofman-LAMP in the detection of SARS-CoV-2 gene N synthetic RNA. (D) The visual image of specific detection of SARS-CoV-2 gene N synthetic RNA after 50 min incubation. The amount of Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Candida tropicalis and SARS-CoV-gene N was 26 ng, 14 ng, 42 ng, 12 ng, 20 ng and 1 × 10⁶ copies, respectively. The SARS-CoV gene N homologous domain RNA was obtained by in vitro transcription (Figure S7).

Fig. 4

Duplex RT-Proofman-LAMP. (A) The real-time fluorescence in the FAM channel of duplex assay using different templates. (B) The real-time fluorescence in the HEX channel of duplex assay using different templates. The mixed template comprised an equal amount (1 × 10⁶ copies) of SARS-CoV-2 gene N and gene Orf1ab RNA. The gene Orf1ab RNA template was obtained by in vitro transcription and verified through RT-PCR (Figure S8).

Fig. 5

Evaluation of RT-Proofman-LAMP using spiked saliva samples. (A) The general workflow to detect saliva samples using Proofman-coupled LAMP. (B) Visual detection of singleplex RT-Proofman-LAMP. The spiked sample contained 50 copies of pseudovirus. (C) Duplex assay of RT-Proofman-LAMP using spiked saliva sample (containing 50 copies of pseudovirus). (D) Triplex assay of RT-Proofman-LAMP using spiked saliva sample (containing 50 copies of pseudovirus).