TropD-detector a CRISPR/LbCas12a-based system for rapid screening of Trypanosoma cruzi in Chagas vectors and reservoirs

Categories: Disease & Virus

May 31, 2025

Bioinformatic analysis

To achieve molecular detection of T. cruzi, we targeted conserved DNA regions to enable specific detection of the parasite. A thorough literature review led to selection of three genes: SR18S, H2 A, and Cytb^29,30,31. Representative sequences from the NCBI database of each gene were downloaded and aligned using the SNAPGENE software (Fig. 1A). Consequently, we designed primers for the SR18 s and H2 A gene, while primers for the Cytb gene were adapted from previously reported for T. cruzi detection³¹. We designed guide RNAs (gRNAs) for each gene with 24-pb length and a PAM sequence TTTV in the CHOPCHOP v2 software³². These gRNAs were chosen based on high specificity for T. cruzi, efficiency scores, and zero off-target effects in other genome regions. Then, we filtered the designs in BLAST and selected the most optimal for each gene. In like manner, designed primers were analyzed in BLAST to select the most specific design with 100% homology for T. cruzi. Each primer was compared and aligned against negative controls with Rhodnius prolixus, Leishmania infantum, Trypanosoma theileri, and Lutzomyia sp as shown in Annex Table 1.

The three analyzed genes were tested on species used as non-target controls. Ten possible designs were obtained within the amplified region for each gene, with potential binding sites containing PAM (TTTV) motifs. One gRNA was selected for each gene, prioritizing high specificity and 0% off-target score, as shown in Table 1.

Table 1 gRNAs designed for each gene

DNA isolation from T. cruzi from R. pallescens intestine

The collection of R. pallescens specimens was conducted between November 2021 and February 2022 by personnel from the Medical Entomology Laboratory in local neighborhoods of Bucaramanga, Colombia. The sampling was carried out with Angulo trap³³, in three neighborhoods with forest fragments. A total of 26 specimens were collected: twelve from the “Pan de Azúcar” neighborhood, eight from “Limoncito,” and six from “Bucarica.” DNA extraction was done with Qiagen DNeasy Blood and Tissue protocol (Qiagen, Valencia, CA, USA) from dissected hindgut samples and stored at − 20 °C.

Blood sample collection and DNA extraction of D. marsupialis

Collected samples from D. marsupialis were obtained from specimens treated at the Wildlife Rescue and Care Center of the metropolitan area of Bucaramanga. Initially, these animals were weighed employing a digital scale with a 20 kg capacity (Vibra, Terrace, USA^®). A veterinarian anesthetized each specimen using xylazine (doses of 2.2 mg/kg) and ketamine hydrochloride (doses of 5–7.5 mg/kg). Ultimately, 0.5–1 mL blood samples were isolated from the caudal vein for each animal, employing 3 mL syringes and 23G x 1” needles, and stored in microtubes with EDTA. DNA extraction from D. marsupialis blood samples was conducted with the Corpogen extraction Kit, following the manufacturer’s protocol (CorpoGen, Bogotá, Colombia^®). Briefly, 250 µL of blood with EDTA was extracted, with the final elution carried out in 100 µL of elution buffer. Finally, the quality and quantity of the extracted DNA were assessed using a NanoDrop 2000 (Thermo Fisher Scientific, Massachusetts, USA) The collection of blood samples from D. marsupialis was conducted as part of the research project titled “Epidemiological characterization of Trypanosoma cruzi and Leishmania spp. infection in D. marsupialis (common opossum) from the Metropolitan Area of Bucaramanga, Colombia,” led by the Universidad Cooperativa de Colombia. This study was approved by the Scientific Research Ethics Committee, which issued Bioethical Concept No. BIO563, as recorded in Minutes No. 2/2023, dated October 25, 2023.

For wild insects, Decree 1376 of June 27, 2013, was taken into account, which regulates the collection permit for specimens of wild species of biological diversity for non-commercial scientific research purposes. The Medical Entomology Laboratory, part of CINTROP-UIS, operates under Framework Permit for Specimen Collection No. IDB0398, issued by the National Authority of Environmental Licenses. The collection of R. pallescens was conducted following laboratory animal handling protocols and approved by the Scientific Research Ethics Committee (CEINCI) Comité de ética en investigación científica CEINCI” of the Industrial University of Santander (acronym in Spanish CEINCI), as recorded in Minutes No. 15, dated August 27, 2021. Nucleotides used in this study were sourced from the project “Producción de nucleótidos a partir de biomasa residual de la agroindustria para diagnóstico por biología molecular en el departamento de Santander,” also approved by CEINCI under Minutes No. 20, dated May 20, 2022. Further- more, the study was carried out in compliance with the arrive guidelines and following the 1989 Colombian Law 84 (Chapter IV, Art. 23–26) and Resolution 8430 (1993, Title IV, Art. 83–93) that regulates animal research in Colombia.

Determination of parasite DNA concentration by qPCR

To estimate the DNA concentration in our samples, we used a reference sample with a previously determined parasite DNA concentration³⁴. Based on this reference, we performed a relative quantification using TaqMan qPCR, following the standard protocol for this method. qPCR reactions were carried out in a QuantStudio 1 thermocycler, using a total reaction volume of 20 µL, containing 10 µL of Luna Universal Probe qPCR Master Mix (New England Biolabs, NEB), 0.8 µL of each primer (10 µM) and 0.4 µL of TaqMan probe (10 µM). Quantification was performed using the cycle threshold (Ct) method, interpolating the values against a standard curve generated from serial dilutions of DNA with a known concentration (Annex Fig. 2).

PCR and RPA amplification

Designed primers were validated through PCR using DNA isolated from the intestine of R. pallescens infected with T. cruzi. We employed the Q5^® Hot Start High-Fidelity 2× Master Mix (New England Biolabs, American), in a T100 thermocycler (Bio-RAD Inc). After validation, we amplified DNA samples of T. cruzi from the Silvio X10 strain culture and blood samples of D. marsupialis naturally infected with the parasite (Fig. 1B). The amplified products were visualized on a 2% agarose gel stained with SYBR^® Safe DNA Gel Stain (Invitrogen, #S33102). Designed primers were also tested against various DNA samples used as negative controls, including Rhodnius prolixus, Leishmania infantum, Trypanosoma theileri, and Lutzomyia sp (Annex Fig. 1). RPA amplification, we employed the RPA TwistAmpbasic KIT from TwistDX Co. (Cambridge, UK). For RPA amplification, the reaction was incubated at 37 °C for 20 min with the addition of 2.5 µL of 280 mM Magnesium Acetate (MgOAc) and 2.4 µL of each primer at a concentration of 10 µM (Fig. 1C). During the incubation, the reaction was briefly agitated at the 4-minute mark. The same concentrations recommended by the supplier were used. The primers used for PCR amplification of Cytb gene were also utilized for RPA amplification in samples obtained from culture, vector, and reservoir of T. cruzi.

gRNAs validation

Designed gRNAs were synthesized commercially by IDT (Integrated DNA Technologies, Inc.) and validated through in vitro cleavage of PCR products, which were observed on agarose gel using amplified T. cruzi DNA extracted from R. pallescens. We adapted the methodology from the in vitro digestion protocol by New England Biolabs (M0653), with concentrations modified during the standardization. CRISPR reactions were performed in a 30 µL reaction mixture consisting of 6 µL of 300 nM LbCAS12a protein, 6 µL of 300 nM gRNAs, and 6 µL of NEB reaction buffer, incubated at 37° C for ten minutes. To visualize the DNA cleavage, 15 µL of the reaction mix was run on a 2% agarose gel stained with SYBR^® Safe DNA Gel Stain (Invitrogen, #S33102). Full-length agarose gel images, including additional experiments that were not relevant to the main study, are provided in the Supplementary Information (Annex Fig. 3). Lanes irrelevant to the study (e.g., genomic DNA tests) are not discussed in the main text.

Target gene sequencing

After gRNA validation, the DNA amplified was sequenced from T. cruzi genes SR18 s, H2 A, and Cytb extracted from R. pallescens intestine. Bidirectional sequencing was conducted through Macrogen’s Sanger sequencing service. The obtained sequences were aligned and compared with reference sequences obtained from the NCBI database. The results were analyzed using the SnapGene software.

Visualization of fluorescent reporter cleavage

Once it is verified that LbCas12a protein produces the cleavage in the amplified product, the ssDNA reporter is added (Fig. 1D). The fluorescent system methodology was based on Chen et al. 2018. We selected a single-stranded DNA (ssDNA) reporter probe labeled with a fluorophore and quencher (FQ) (56-FAM/TTATT/3IABkFQ), which was synthesized commercially by IDT³². It is excited about a wavelength of 480 nm and emits detectable fluorescence at 530 nm. The ssDNA reporter, gRNA, and LbCas12a protein concentrations were adapted as previously described³⁵. The fluorescence reaction mixture was adjusted to 100 nm of ssDNA reporter, while the final concentration of gRNA and LbCas12 was set at 60 nm. An equivalent volume of protein was used in the NEB buffer; molecular water was added to achieve a final volume of 50 µL.

We quantified the emitted fluorescence using dark plates in a SYNERGY H1 spectrophotometer, taking measurements every three minutes at 37° C for 2 h, with 40-s shaking intervals between readings (Fig. 1E). The samples were subsequently visualized on a UV transilluminator, allowing visual identification of positive samples (Fig. 1F). To evaluate the sensitivity of the CRISPR/LbCas12a system, we diluted the amplified DNA obtained from PCR to final concentrations of 40 ng/uL, 20 ng/uL, 10 ng/uL, 5 ng/uL, 1 ng/uL. Those dilutions were tested through RPA amplification in the designed device. The same approach was applied to assess the sensitivity of PCR combined with CRISPR/LbCas12 system by performing serial dilutions from 1:2 to 1: 32 using T. cruzi DNA extracted from cultures of the parasite with an initial concentration of 172.8 parasite equivalents/mL. The DNA diluted was amplified through PCR using the Cytb gene and observed on a 2% agarose gel. RPA amplification was conducted in the dilution 1:32 and compared with the results obtained through PCR. Cleavage by the CRISPR system was then evaluated using the amplified DNA from these dilutions with the designed guide. Each assay was performed in triplicate.

Design an LED reading device

We designed a portable device using affordable materials (Fig. 1G). It features a darkened cavity to prevent external light. At one end, a blue LED emits light in the range of 480–485 nm is powered by a conventional 9-volt battery. A lens with a focal length of f = 5 cm focused the light onto a PCR tube sample. The emitted fluorescence from the sample is observed at a 90˚ angle relative to the incident light. A high-pass filter was used with a cutoff wavelength of 500 nm. A camera was positioned behind the filter to capture the image of the excited sample. A light trap was positioned adjacent to the sample to reduce the intensity of reflected light. Fluorescence visualization of the samples was achieved using a standard mobile phone. We validated the samples obtained from the culture, insects, and reservoir. Subsequently, we confirmed the system’s sensitivity using samples derived from the dilution of the amplified product and the dilution of the DNA provided to the PCR. All the assays were compared with the results obtained under UV light visualization, and in the spectrophotometric quantification.

Statistical analysis

Fluorescence readings from the spectrophotometer were normalized as reported³⁶. We first employed a Kolmorgorov-Smirnov test to evaluate the normality of the replicates. Kruskal-Wallis test was used to evaluate differences in non-normal data, followed by a post hoc Dunn’s test, while a one-way ANOVA was conducted in normal data, followed by a Tukey post hoc test. The homogeneity of the replicas was applied to the normalized data in the sensitivity tests (Annex Table 2). All analyses were executed using Statistica Version 10.

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