Improved methods for isolating DNA from Ostertagia ostertagi eggs in cattle feces.
A multiplex PCR assay for differentiating strongyle eggs from cattle has recently been described; however, the egg disruption and DNA extraction procedures, though effective, are inadequate for large studies or clinical application. The purpose of this research was to evaluate methods for disrupting trichostrongyle eggs, then assess commercial kits for extracting egg DNA using Ostertagia ostertagi as a model species. Egg disruption procedures tested included probe sonication, bath sonication, bead beating, boiling, microwaving, proteinase K/SDS digestion, freezing, and various combinations of the above with the incorporation of sodium dodecyl sulfate. These procedures were evaluated in conjunction with four commercial DNA extraction kits: DNA Stool mini kit and DNeasy Plant kit (Qiagen), Fast DNA kit (QBiogene), and the MAP extraction kit (Tetracore). Results showed that egg disruption was best accomplished with the bead beater and ceramic beads, resulting in 100% disruption within 1min. When DNA extraction was preceded by the isolation of eggs from feces, all procedures except the Fast DNA kit produced PCR-ready DNA from at least two eggs. The DNeasy Plant kit allowed consistent detection of DNA released from one egg. Due to the morphological similarities among trichostrongyle eggs in ruminants, strongyle eggs in equids, and hookworm eggs, the methods described herein may have broad application to other nematodes.
Harmon
Aaron F
AF
Department of Biology and Microbiology, South Dakota State University, Brookings, SD 57007, USA.
Zarlenga
Dante S
DS
Hildreth
Michael B
MB
D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis* / Q000469:parasitology; D018509:DNA, Helminth / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D003937:Diagnosis, Differential; D005243:Feces / Q000469:parasitology; D010028:Ostertagia / Q000235:genetics / Q000302:isolation & purification*; D010270:Parasite Egg Count / Q000662:veterinary; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary; D012680:Sensitivity and Specificity; D013045:Species Specificity; D014251:Trichostrongyloidea / Q000302:isolation & purification
D018509:DNA, Helminth