Microbes responsible for some infections, such as SARS-CoV-2, can rapidly mutate into variants that evade detection and treatment.
Researchers have now developed a procedure called AutoPLP that they say enables the design of nucleic acid probes to detect new variants quickly, accurately, and easily.
They described the procedure in a paper released Wednesday in ACS Infectious Diseases, saying that AutoPLP, a computer program, was named for the padlock probes (PLPs) it designs.
“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,” the authors noted.
Rolling circle amplification (RCA) works in a similar way to PCR to detect pathogens by analyzing genetic material, but its advantage is that it doesn’t involve the complex temperature cycling that PCR does. Both techniques require nucleic acid probes with sequences matching those of the target pathogen in specific locations, but RCA uses highly specific padlock probes.
As a pathogen mutates, its genetic sequence changes as well, and researchers have to keep redesigning their probes.
As a result, researchers at the Indian Institute of Technology Madras wanted to develop a tool that could not only design these PLPs automatically, but also systematically consider all the necessary technical parameters at once to make the process easier and more robust.
“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,” they wrote.
AutoPLP, the computer program they developed, can take the genome sequences of similar pathogens as input and run a series of analyses and database searches, outputting a set of customized padlock probe sequences.
The researchers used the program to design probes against the rabies virus, which is transmitted between animals and people, and Mycobacterium tuberculosis, which is responsible for tuberculosis, and compared them to previously reported probes.
For the rabies virus, AutoPLP targeted three genes, yielding probes with a higher and narrower range of melting temperatures than those in the literature.
For M. tuberculosis, the group designed 13 probes specifically targeting two genes responsible for drug-resistant strains.
They believe their tool could help hasten the discovery of new pathogen variants, helping combat them rapidly and effectively by enabling the design of precise molecular diagnostic tests.