Article Title: Genome-wide locus sequence typing (GLST) of eukaryotic pathogens
Figure Lengend Snippet: Preliminary GLST (multiplex) trials on T. cruzi I mock infections. We created mock infections by mixing 10 4 , 10 5 and 10 6 RNAlater-preserved TcI-Sylvio epimastigote (epi) cells with uninfected Rhodnius prolixus vector gut (UVG). DNA extracted from these mock infections was subjected to the multiplexed, 203-target GLST reaction (using the same cycling conditions as for single-target reactions – see Methods or Supplementary Fig. 2 legend) and products were electrophoresed in 0.8% agarose gel. Fainter banding of GLST products from lower concentration mock infections encouraged follow-up on sensitivity thresholds using additional dilution curves and qPCR. Next to DNA ladder (L) and no-template control (NTC), the gel also contains TcZ primer product from pure TcI epimastigote DNA. TcZ primers provide a highly sensitive positive control (PC) as they target 195 bp satellite DNA repeats that make up ca. 5% of the T. cruzi genome.
Article Snippet: Insects were euthanized with CO2 and hindguts drawn into 5 volumes of RNAlater (Sigma-Aldrich) by pulling the abdominal apex toward the posterior with sterile watchmaker’s forceps.
Techniques: Multiplex Assay, Plasmid Preparation, Agarose Gel Electrophoresis, Concentration Assay, Real-time Polymerase Chain Reaction, Positive Control