A novel procedure for total nucleic acid extraction from small numbers of Eimeria species oocysts.
A series of experiments were performed in an attempt to extract genomic DNA from a small number of Eimerian oocysts. Sonication, ammonia, ethanol and lysozyme were all found to be unsuitable for the digestion of Eimeria oocysts. The chemicals and enzyme given were not capable of either disruption or digestion of oocysts for nucleic acid extraction. They had the capability of penetrating the oocyst wall but could not break-up the oocyst wall. It is impossible to obtain nucleic acid from Eimeria oocysts if the wall is not broken-up. In this study oocyst disruption was achieved using a simple but highly effective treatment regime involving sodium hypochlorite treatment, osmotic shock and proteinase K digestion. Following the disruption of the oocyst walls, a commercially available nucleic acid purification kit (Wizard DNA Purification Kit, Promega) can be used to prepare high quality nucleic acid.
Kaya
Galip
G
University of Bristol, School of Veterinary Medicine, Clinical Veterinary Science, Langford, Bristol, UK. gkaya@mku.edu.tr
Dale
Colin
C
Maudlin
Ian
I
Morgan
Kenton
K
D000818:Animals; D003048:Coccidiosis / Q000175:diagnosis / Q000662:veterinary; D016054:DNA, Protozoan / Q000302:isolation & purification*; D004539:Eimeria / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D005243:Feces / Q000469:parasitology; D033661:Oocysts / Q000737:chemistry; D010447:Peptide Hydrolases / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012756:Sheep
D016054:DNA, Protozoan; D010447:Peptide Hydrolases