Trypanosoma cruzi detection

Torhorst, Carson W., Ledger, Kimberly J., White, Zoe S., Milleson, Michael P., Corral, Catalina C., Beatty, Norman L. & Wisely, Samantha M., 2023, Trypanosoma cruzi infection in mammals in Florida: New insight into the transmission of T. cruzi in the southeastern United States, International Journal for Parasitology: Parasites and Wildlife 21, pp. 237-245 : 240

2.4. Trypanosoma cruzi detection and DTU determination

Trypanosoma cruzi infection was identified using a qPCR (quantitative polymerase chain reaction) assay which detected the nucleic acid of the parasite following previously described methods ( Duffy et al., 2013; Ramírez et al., 2017). We inferred T. cruzi infection based on the identification of T. cruzi nucleic acid in peripheral blood ( Curtis-Robles et al., 2018a; Torhorst et al., 2022; Zecca et al., 2020). Detection was based on a 166bp segment of T. cruzi satellite DNA (satDNA) amplified using a 2x Roche Fast Start universal master mix with the forward primer Cruzi1, reverse primer Cruzi2, and TaqMan probe Cruzi3 PrimeTime® 5′ 6-FAMTM/ZENTM/3′ IB®FQ (IDT, Coralville, Iowa). The 166bp satDNA segment was from the 195bp tandem repeating satDNA unit that has been used as a molecular diagnostic tool to identify T. cruzi in infected domestic dogs, wildlife, and triatomines ( Curtis-Robles et al., 2016, 2017, 2018b; Torhorst et al., 2022; Zecca et al., 2020). Each reaction (20μL) contained master mix (18μL) and host template DNA (2uL) (20ng /uL). The prepared master mix also contained a separate exogenous amplification control (10x Exo IPC Mix, 50 IPC Exo) which contained primers and a probe, labeled with fluorophore VIC (5’ - VIC™/TAMRA™ Quencher - 3′) (Thermo Fisher Scientific, Waltham, MA). An exogenous amplification control was used in this reaction to identify host template DNA amplification inhibition, which could have led to false positives. A negative control (blanked with molecular grade water), an exogenous amplification positive control (containing molecular grade water and exogenous amplification control), and a positive control dilutions series from a cell culture isolate of TcI were used to verify each assay. To exclude contamination by the positive control, the positive controls and master mix reagents were stored separate from the tested gDNA samples. Each rtPCR T. cruzi detection assay was performed using an ABI 7500 FAST Real Time PCR machine (Thermo Fisher Scientific, Waltham, MA).

A sample was considered T. cruzi infected when the Ct value was ≤38 ( Beatty et al., 2021; Torhorst et al., 2022). Any sample that amplified between the Ct values of 38–40 was labeled as suspect positive, and these samples were rescreened. If amplification occurred at the second attempt, these samples were then considered T. cruzi infected ( Beatty et al., 2021; Torhorst et al., 2022). Samples that did not amplify the exogenous control were rescreened at a template DNA dilution series of 1:5 and 1:10 to eliminate amplification inhibition within the sample.

The samples which screened positive for T. cruzi were then subject to DTU determination via a multi-stepped, multiplexed qPCR ( Beatty et al., 2021; Cura et al., 2015; Torhorst et al., 2022). Each DTU determination assay was performed using an ABI 7500 FAST Real Time PCR machine (Thermo Fisher Scientific, Waltham, MA). For each reaction, 2uL of template (18μL) (20ng /uL) was added to each well with the prepared master mix. The following reactions were performed to identify the infecting DTU: SL-MTq intended to identify TcI, the TcII/TcV/TcVI complex, and the TcIII/TcIV complex, while the 24Sα- III/IV MTq was used to differentiate TcIII from TcIV ( Cura et al., 2015). The 18S-COII MTq assay was not used in this study, as there were no florescent indications of this DTU complex ( Cura et al., 2015). DTU identification was discontinued after 3 unsuccessful amplification attempts. Each assay was verified with a water negative control and the positive controls of the DTU intended to be identified in the specified assays described in Cura et al. (2015).