Rapid and highly specific detection of communicable pathogens using one-pot loop probe-mediated isothermal amplification (oLAMP)

Rapid nucleic acid isothermal amplification techniques with minimal demand for laboratory instruments are desirable for early screening and mitigation of communicable diseases (e.g., COVID-19). However, most of the current techniques available employ non-specific signal generation strategies which increase the chance of false positives. Others require a two-step amplification-detection process, in which the tube is opened after amplification, prior to the detection step. This promotes cross-contamination that would lead to unreliable results. Here, we developed a one-pot loop probe-mediated isothermal amplification scheme (oLAMP) coupled with a concurrent target-specific detection readout. We have shown that the said scheme is compatible with crude sample preparation methods and is readily integrated with either fluorescence or electrochemical readouts. The entire process would take 30–40 min, and we show that our scheme could detect 0.5 or 1 copy/μL of the SARS-CoV-2 N gene using fluorescence or electrochemical readouts, respectively. Moreover, we demonstrate that the scheme is robust across different biological matrices and other interfering substances. We further show that our method can detect the presence of Salmonella drug resistance gene in crude food samples. Thus, our platform offers a readily scalable and highly specific diagnostic pathway for rapid and decentralized detection of communicable diseases.