Identification of Trypanosomatids by detecting Single Nucleotide Fingerprints using DNA analysis by dynamic chemistry with MALDI-ToF.

Protozoan parasites of the Trypanosomatidae family can cause devastating diseases in humans and animals, such as Human African Trypanosomiasis or Sleeping Sickness, Chagas disease and Leishmaniasis. Currently, there are molecular assays for detecting parasitic infections and their post-treatment monitoring based on nucleic acid amplification, but there are still certain limitations which limit the development of assays that can detect and discriminate between parasite infections with a single test. Here, we present the development of a novel molecular assay for the rapid identification of Trypanosomatids, integrating DNA analysis by dynamic chemistry in conjunction with Matrix-Assisted Laser Desorption Ionization - Time-of-Flight Mass Spectrometry (MALDI-ToF). Differentiation of Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp. is now possible using a single reaction tube, and enables rapid identification of Trypanosomatids. The test is based on a singleplex PCR, using a specific primer pair that amplifies a 155 base pair segment of the 28S ribosomal RNA gene, within a conserved homology region of Trypanosomatidae species. Amplified fragments are analysed by dynamic chemistry using two abasic PNA probes and the four reactive nucleobases - containing an aldehyde functional group - with MALDI-ToF to identify unique molecular patterns created by each specie due to their single base differences (Single Nucleotide Fingerprint 'SNF') in this highly homologous region. This novel assay offers the possibility to expand routine diagnostic testing for Trypanosomatids, and monitoring of therapeutic responses to these infectious diseases.