DNA extraction methods and multiple sampling to improve molecular diagnosis of Sarcocystis spp. in cattle hearts

Abstract

Molecular detection of Sarcocystis spp. in tissue samples can be useful for experimental and diagnostic purposes. However, the parasite spreads unevenly through tissues, forming tissue cysts, and the cystic wall is an obstacle in DNA extraction protocols. Therefore, adequate sampling and effective disruption of the cysts are essential to improve the accuracy of DNA detection by PCR. The aims of this study were to evaluate the suitability of four protocols for DNA extraction from cysts of Sarcocystis spp. present in bovine myocardium samples or after their harvest in phosphate-buffered saline (PBS) solution as well as determine the effects of single or multiple sampling on the accuracy of molecular diagnosis of sarcocystosis in cattle hearts. Cysts and myocardium samples from nine bovine hearts were randomly distributed to four DNA extraction protocols: kit, kit with modification, DNAzol, and cetyl-trimethyl ammonium bromide (CTAB). Samples were submitted to DNA extraction and PCR as replicates of each heart (simplicate, duplicate, and triplicate), and the probability of a true positive diagnostic was calculated. Among the protocols tested, the kit with modification was determined to be the most suitable for DNA extraction from cysts in PBS solution (92.6 % of DNA detection by PCR); DNAzol resulted in higher DNA detection frequency from bovine myocardium samples (48.1 %). Multiple sampling improved the molecular diagnosis of Sarcocystis spp. infection in cattle hearts, increasing at 22.2 % the rate of true positive diagnostic.

Detection of Sarcocystis spp. and Toxoplasma gondii in swine and detection of DNA of these protozoa in tissues and sausages

“…The blood of each animal was identified and centrifuged to obtain serum. The sausages and tissues were macerated, randomly selected, and divided into two 50 µg aliquots, following the protocol described by Bräunig et al (2016). Subsequently, the materials were frozen at -20 °C until laboratory analyses.…”

“…To obtain total DNA from all pig samples, the Genomic DNA Purification Kit (Promega®, Madison, USA) was used, following the manufacturer's recommendations, with adaptation in the lysis step, according to Bräunig et al (2016).…”

Microscopic and Molecular Detection of Sarcocystis cruzi (Apicomplexa: Sarcocystidae) in the Heart Muscle of Cattle

“…Later, PCR was carried out to identify Sarcocystis species by amplification of partial sequence of small subunit ribosomal RNA (18S rRNA) gene of Sarcocystis . For amplification of ∼700 bp of 18s rRNA of Sarcocystis species, primers SarcoF 5′-CGCAAATTACCCAATCCTGA-3′ and SarcoR 5′-ATTTCTCATAAGGTGCAGGAG-3′were used ( 15 , 22 ). The 20 μl PCR mixture contained 1 μl of template DNA, 10 μl Taq DNA Polymerase 2x Master Mix RED (Ampliqon, 2mM MgCl2) and 1 μM of each primer.…”

Molecular detection of Toxoplasma gondii in placentas of women who received therapy during gestation in a toxoplasmosis outbreak

“…The placenta samples were subjected to DNA extraction through the Wizard® Genomic DNA purification kit (Promega, USA) according to the manufacturer's instructions, with modifications in the lysis step according to previous study (Moré et al, 2011;Bräunig et al, 2016). After RNase addition, the lysis step was carried out at a higher temperature (55°C) and kept overnight, covering a 16-h interval.…”