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.

Keywords: Chagas disease; DNA extraction; Nobuto strip; PCR; Trypanosoma cruzi; blood filter paper; mammalian surveillance; neglected tropical diseases.

Figure 1

Trypanosoma cruzi DNA recovery from spiked canine whole blood specimens. Bars indicate one standard deviation. As detailed in the text for use with Qiagen DNeasy Blood & Tissue Kit: QIA A = extraction optimization method A; QIA B = extraction optimization method B. ZR Pathogen = Zymo Research Quick-DNA/RNA Pathogen Miniprep; ZR Duet = Zymo Research ZR-Duet DNA/RNA Miniprep Plus kit. Cq = quantification cycle; Nobuto = whole blood samples processed from Nobuto blood filter papers; Blood = whole blood samples processed directly; MED = medium spiking load; HI = high spiking load.

Figure 2

β-actin DNA recovered from skunk whole blood archived on Nobuto blood filter paper strips. DNA extracted according to protocols outlined in the text for Qiagen DNeasy Blood & Tissue Kit: Method A = extraction optimization method A; Method B = extraction optimization method B; Method C = extraction optimization method C. Cq = quantification cycle.