Abstract
The purpose of this study was to evaluate the practicality of using real-time PCR for quantifying feces-derived trichostrongyle eggs. Haemonchus contortus eggs were used to evaluate fecal contaminants, time after egg embryonation, and the presence of competing and non-competing DNAs as factors that might interfere with generating reproducible results during simplex and multiplex quantitative real-time PCR (QPCR). Real-time PCR results showed linear quantifiable amplification with DNA from five to 75 eggs. However, threshold cycle (C T) values obtained by amplification of DNA from egg numbers between 75 and 1,000 did not differ significantly. Inhibitors of QPCR were effectively removed during DNA extraction as exemplified by the absence of any improvement in C T values with bovine serum albumin or phytase treatments. Changes from egg embryonation could only be detected during the first 6 h. Noncompetitive DNA did not appear to impact amplification; however, in a multiplex reaction a competing trichostrongyle such as Cooperia oncophora can hinder amplification of H. contortus DNA, when present at tenfold greater amounts. This study demonstrates the usefulness of QPCR for amplification and quantification of trichostrongyle eggs, and identifies potential limitations, which may be addressed through multiplex assays or the addition of a standard: exogenous DNA target.
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Acknowledgements
This paper was approved for publication as Journal Series No.3552 by the Director, Agricultural Experiment Station, South Dakota State University. We would like to thank Dr. Larry Holler for his contribution with establishing our lab strain of H. contortus. Institutional animal care and use committee approval was obtained before this study. All experiments performed herein were in compliance with U.S. laws.
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This work was supported in parts by Pfizer Animal Health, South Dakota State University Research Support Grant, the SDSU Functional Genomics Core Facility, and the South Dakota State University Agricultural Experiment Station.
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Harmon, A.F., Williams, Z.B., Zarlenga, D.S. et al. Real-time PCR for quantifying Haemonchus contortus eggs and potential limiting factors. Parasitol Res 101, 71–76 (2007). https://doi.org/10.1007/s00436-006-0428-0
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DOI: https://doi.org/10.1007/s00436-006-0428-0