AutoPLP: A Padlock Probe Design Pipeline for Zoonotic Pathogens

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.