DNA extraction methods and multiple sampling to improve molecular diagnosis of Sarcocystis spp. in cattle hearts.
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.
Laboratório de Doenças Parasitárias (Ladopar), Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria (UFSM), Av. Roraima 1000, prédio 44, sala 5139, 97105-900, Santa Maria, RS, Brazil. pbraunig@gmail.com.
Patrícia
P
Bräunig
Laboratório de Doenças Parasitárias (Ladopar), Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria (UFSM), Av. Roraima 1000, prédio 44, sala 5139, 97105-900, Santa Maria, RS, Brazil.
Luiza Pires
LP
Portella
Programa de Pós-Graduação em Economia e Desenvolvimento, UFSM, Rua Mal. Floriano Peixoto 1750, 97015-372, Santa Maria, RS, Brazil.
Alfredo Skrebsky
AS
Cezar
Laboratório de Microbiologia, Departamento de Estudos Agrários, Universidade Regional do Noroeste do Estado do Rio Grande do Sul (UNIJUÍ), Rua do Comércio 3000, 98700-000, Ijuí, RS, Brazil.
Felipe
F
Libardoni
Laboratório de Doenças Parasitárias (Ladopar), Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria (UFSM), Av. Roraima 1000, prédio 44, sala 5139, 97105-900, Santa Maria, RS, Brazil.
Luis Antonio
LA
Sangioni
Laboratório de Doenças Parasitárias (Ladopar), Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria (UFSM), Av. Roraima 1000, prédio 44, sala 5139, 97105-900, Santa Maria, RS, Brazil.
Fernanda Silveira Flores
FS
Vogel
Laboratório de Biotecnologia e Reprodução Animal (BioRep), Departamento de Clínica de Grandes Animais, UFSM, Av. Roraima 1000, Hospital Veterinário, 97105-900, Santa Maria, RS, Brazil.
Paulo Bayard Dias
PB
Gonçalves
D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000469:parasitology*; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification*; D004275:DNA, Ribosomal / Q000235:genetics / Q000302:isolation & purification; D006321:Heart / Q000469:parasitology; D016133:Polymerase Chain Reaction / Q000662:veterinary; D015203:Reproducibility of Results; D012522:Sarcocystis / Q000235:genetics / Q000302:isolation & purification*; D012523:Sarcocystosis / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*; D013048:Specimen Handling / Q000662:veterinary*
D016054:DNA, Protozoan; D004275:DNA, Ribosomal
18S rDNA; Genomic DNA; Isolation protocols; PCR; Sarcocystis cysts