Optimization of DNA Extraction from Field-Collected Mammalian Whole Blood on Filter Paper for Trypanosoma cruzi (Chagas Disease) Detection

Abstract

Blood filter paper strips are cost-effective materials used to store body fluid specimens under challenging field conditions, extending the reach of zoonotic pathogen surveillance and research. We describe an optimized procedure for the extraction of parasite DNA from whole blood (WB) stored on Type I Advantec Nobuto strips from both experimentally spiked and field-collected specimens from canine and skunks, respectively. When comparing two commercial kits for extraction, Qiagen’s DNeasy Blood & Tissue Kit performed best for the detection of parasite DNA by PCR from Trypanosoma cruzi-spiked canine WB samples on Nobuto strips. To further optimize recovery of β-actin from field-collected skunk WB archived on Nobuto strips, we modified the extraction procedures for the Qiagen kit with a 90 °C incubation step and extended incubation post-addition of proteinase K, a method subsequently employed to identify a T. cruzi infection in one of the skunks. Using this optimized extraction method can efficaciously increase the accuracy and precision of future molecular epidemiologic investigations targeting neglected tropical diseases in field-collected WB specimens on filter strips.

Dried Blood Spots technology for veterinary applications and biological investigations: technical aspects, retrospective analysis, ongoing status and future perspectives

“…For example, Cardona-Ospina et al ( 2019 ) summarized RNA extraction from FTA cards used for detection and characterization of viral RNA pathogens from fieldwork. Commercially available DNA extraction kits can be optimized to provide the most accurate and precise recovery for molecular epidemiological studies as it was shown for Trypanosoma cruzi DNA extracted from canine and skunk DBS (Gulas-Wroblewski et al 2021 ). The issue of the best practice for preparation of samples from FTA cards for the molecular diagnosis under large-scale epidemiological studies was addressed in the work of Ahmed et al ( 2011 ) using African trypanosomes as a model object.…”

Utilizing blood filter paper and ear punch samples for the detection of rabbit hemorrhagic disease virus 2 by RT-rtPCR

“…We evaluated 2 sample types: blood filter paper and ear punch samples. Filter paper has been validated as a suitable sample type for disease detection (e.g., avian influenza, Chagas disease), 11,14 and allows for sample collection in the field with little training, equipment, and without the need for climate-controlled transport. 6 Samples must be eluted from filter paper before analysis, leading to lower sensitivity in some assays.…”

Translational Research of Zoonotic Parasites: Toward Improved Tools for Diagnosis, Treatment and Control

“…Hong et al [8] prioritised the development of an accurate and cost-effective diagnostic test, as well as the use of animals as sentinels, as effective tools for the epidemiological surveillance of leishmaniases. Gulas-Wroblewski et al [9] developed cost-effective DNA storage and DNA extraction methods for the molecular detection of Trypanosoma cruzi in field-based investigations. Furthermore, Zheoat et al [10] discovered that 2 ,4 -dimethoxy-6 -hydroxychalcone from a plant extract of Polygonum salicifolium had anti-leishmanial and anti-trypanosomal effects in the absence of toxicity against a human cell line, suggesting its potential use against Leishmania mexicana, Trypanosoma brucei and T. congolense.…”