Emergence of novel zoonotic infections among the human population has increased the burden on global healthcare systems to curb their spread. To meet the evolutionary agility of pathogens, it is essential to revamp the existing diagnostic methods for early detection and characterization of the pathogens at the molecular level. Padlock probes (PLPs), which can leverage the power of isothermal nucleic acid amplification techniques (NAAT) such as rolling circle amplification (RCA), are known for their high sensitivity and specificity in detecting a diverse pathogen panel of interest. However, due to the complexity involved in deciding the target regions for PLP design and the need for optimization of multiple experimental parameters, the applicability of RCA has been limited in point-of-care testing for pathogen detection. To address this gap, we have developed a novel and integrated PLP design pipeline named AutoPLP, which can automate the probe design process for a diverse pathogen panel of interest. The pipeline is composed of three modules which can perform sequence data curation, multiple sequence alignment, conservation analysis, filtration based on experimental parameters (Tm, GC content, and secondary structure formation), and in silico probe validation via potential cross-hybridization check with host genome. The modules can also take into account the backbone and restriction site information, appropriate combinations of which are incorporated along with the probe arms to design a complete probe sequence. The potential applications of AutoPLP are showcased through the design of PLPs for the detection of rabies virus and drug-resistant strains of Mycobacterium tuberculosis.
Keywords: Mycobacterium tuberculosis; RCA; autoPLP; padlock probe; rabies; zoonotic pathogens.