Multiplex PCR on single unembryonated Ascaris (roundworm) eggs.
A sensitive and inexpensive method for DNA isolation and amplification by polymerase chain reaction (PCR) from single unembryonated Ascaris sp. eggs is described. The resistant shell of single eggs was crushed mechanically and PCR applied to the crude egg contents without any further purification steps. The ITS1 region of the rDNA and three regions of the mtDNA could be successfully amplified. Using two primer sets, it was possible to amplify the rDNA and mtDNA simultaneously in one single reaction. The ability to perform PCR on single unembryonated eggs may result in better and more precise species identification of eggs recovered from faecal material, environmental samples and possibly archaeological samples. In addition, single egg PCR makes it possible to perform population genetic studies without having to recover adult worms by deworming or autopsy.
Carlsgart
Josefine
J
Danish Centre for Experimental Parasitology, Department of Veterinary Pathobiology, Faculty of Life Sciences, University of Copenhagen, Dyrlaegevej 100, 1870 Frederiksberg C, Copenhagen, Denmark.
Roepstorff
Allan
A
Nejsum
Peter
P
D000818:Animals; D001200:Ascaris / Q000145:classification* / Q000235:genetics* / Q000254:growth & development; D017931:DNA Primers; D018509:DNA, Helminth / Q000032:analysis*; D004272:DNA, Mitochondrial / Q000032:analysis; D021903:DNA, Ribosomal Spacer / Q000032:analysis; D005243:Feces / Q000469:parasitology; D010063:Ovum / Q000254:growth & development*; D010270:Parasite Egg Count; D016133:Polymerase Chain Reaction / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012680:Sensitivity and Specificity
D017931:DNA Primers; D018509:DNA, Helminth; D004272:DNA, Mitochondrial; D021903:DNA, Ribosomal Spacer; D012337:RNA, Ribosomal, 18S