Sensitive fluorescent detection of SARS-CoV-2 RNA using an enzymatic-based method

Rapid, quantitative, and sensitive detection of viral oligonucleotides can help to diagnose the infection before symptoms occur, monitor disease progression, and identify viral subtypes. A one-pot, simple, rapid hairpin-mediated nicking enzymatic signal amplification (HNESA) method was previously developed for nucleic acids detection. In the present work, this method was applied for the detection of SARS-CoV-2 RNA by designing an assistant probe (AP) that contains the complementary sequence for the target, the sequence of hybridization with the loop region of the molecular beacon (MB), and the recognition site of the nicking endonuclease Nt.BstNBI. MB sequences (MB1 and MB2) were also designed and optimized in length and nucleotide composition. MB2 significantly amplified the fluorescence signal of the target sequence. The linear range was from 0.1 to 1 nM with a detection limit of 170.6 pM. Results can be obtained within 45 min, considering that the cyclic amplification involves only one AP and one MB. Based on this, the use of HNESA for diagnosing viral diseases, such as SARS-CoV-2, could be a complementary approach to polymerase chain reaction (PCR), and it is a technique that is quick, efficient and has high sensitivity.