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AutoPLP: A Padlock Probe Design Pipeline for Zoonotic Pathogens
Sowmya Ramaswamy Krishnan1,2, Ruben R G Soares3, Narayanan Madaboosi4
1Protein Bioinformatics Lab, Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai 600036, India.
Abstract:
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.
Insights
A new pipeline, AutoPLP, automates the design of padlock probes (PLPs) for pathogen detection. This innovation enhances molecular diagnostics, enabling rapid identification of infectious agents like rabies virus and drug-resistant tuberculosis.
Area of Science:
- Molecular biology
- Bioinformatics
- Infectious disease diagnostics
Background:
- Novel zoonotic infections pose a growing threat to global health.
- Existing diagnostic methods require enhancement for rapid, molecular-level pathogen detection.
- Padlock probes (PLPs) combined with isothermal amplification offer high sensitivity and specificity but face design complexities.
Purpose of the Study:
- To develop an automated pipeline, AutoPLP, for designing padlock probes (PLPs).
- To overcome limitations in current PLP design for isothermal nucleic acid amplification techniques (NAAT).
- To facilitate the application of PLP-based diagnostics in point-of-care testing.
Main Methods:
- Developed AutoPLP, an integrated pipeline with three modules for automated PLP design.
- Pipeline modules include sequence curation, multiple sequence alignment, and conservation analysis.
- Incorporated experimental parameter filtration (Tm, GC content, secondary structure) and in silico validation against host genomes.
Main Results:
- AutoPLP automates the design of complete PLP sequences, including backbone and restriction site information.
- The pipeline successfully designed PLPs for detecting rabies virus.
- Demonstrated utility in designing probes for drug-resistant Mycobacterium tuberculosis strains.
Conclusions:
- AutoPLP streamlines the design of highly specific and sensitive padlock probes.
- The automated pipeline addresses key challenges in applying NAAT for pathogen detection.
- AutoPLP has significant potential for advancing molecular diagnostics in infectious disease surveillance.
