Submitted to: American Association of Veterinary Parasitologists Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: June 23, 2006
Publication Date: July 15, 2006
Citation: Harmon, A.F., Zarlenga, D.S., Hildreth, M.B. 2006. Development of a multiplex real-time PCR for differentiating and quantifying eggs of Haemonchus contortus, Ostertagia ostertagi, and Cooperia oncophora using fecal eggs [abstract]. American Association of Veterinary Parasitologists, p. 42
Despite recent assay development for both egg and larval stages of trichostrongyles nematodes, there is still a need for a quantitative assay which can differentiate the eggs of economically important trichostrongyles in a rapid manner. These morphologically identical eggs can be differentiated using PCR, but only qualitative results could be obtained. This study describes progress towards the development of a real-time quantitative PCR (QPCR) assay. This assay would allow for amplification of Ostertagia ostertagi, Cooperia oncophora, and Haemonchus contortus simultaneously in a quantitative manner. This was accomplished by first modifying previously described primers and probes to facilitate use with cattle trichostrongyles and increase specificity, and thoroughly testing them to insure that no cross reactivity occurred during QPCR. Plasmids were then created that contained the QPCR product and were used to assess the linearity of amplification and efficiency, so that any possible inhibition or variation could be measured. QPCR was then performed in conjunction with an optimized DNA extraction protocol to allow for the quantification of eggs. Eggs from a H. contortus mono-infection were tested. From this work, no cross reactions were found, even when primers and probes were tested against other common cattle nematodes. Plasmid based QPCR resulted in linear amplification with an R-square value between 0.98 and 0.99. Efficiency ranged from 92% to 115%. When H. contortus eggs were tested, the R-square remained at 0.98; however, efficiency was reduced to 28%. This reduction in efficiency indicates that fecal inhibitors are still interfering with PCR, but they do not effect linear amplification when evaluated in a multiplex format. These results suggest that quantitative differentiation is possible at the egg stage with QPCR, but more work is needed to demonstrate this unequivocally.