Diagnosis of diseases and proof of treatment through DNA quantification using qPCR: The case of Haemonchosis

Background: Haemonchus contortus is globally distributed nematodes and causative agents of haemonchosis, via ingestion of Haemonchus eggs. This disease is one of the most important gastrointestinal nematodes (GINs) infecting sheep, goats, and cattle worldwide. Control of Haemonchus infections is constrained by a lack of sensitive methods for screening of animal faeces. Methodology: In this study, we investigated the yield of DNA extracted from known quantities (10,000, 5,000, 1,000, 500, 100, 50 and 10) of Haemonchus contortus eggs, as well as directly from samples of single eggs and eggs from faeces, using a commercial DNA extraction kits or bead-beating and phenol chloroform isoamyalcohol. The amount of DNA extracted was quantified with NanoDrop, PCR was performed and Ct values was determined by developping a SYBR Green real-time PCR (qPCR) assay for detection and quantification of H. contortus by using specific primers based on a conserved region of the ribosomal internal transcribed spacer 2 (rRNA ITS2) gene. Results: The most effective DNA extraction method for Haemonchus eggs in faeces samples or in isolated sample consisted in the combination of mechanical lysis of eggs using beads. The Eggshell was well destroyed with the method involving the bead-beating and phenol chloroform isoamyalcohol with the consequence of a better extraction of DNA and a high amount of DNA. Agarose gel electrophoresis after PCR shows the presence of ITS2 gene of Haemonchus cortortus at 265 bp from the isolated genomic DNA. For the accurate quantification of the extracted DNA, the use of Ct values from qPCR gives better results compared to the NanoDrop readings and the PCR. The quantity of DNA revealed by qPCR was proportional to the number of eggs contained in the faeces or in the isolated eggs. Conclusion: The method of DNA isolation, the choice of the molecular marker developed here combined with qPCR make it possible to diagnose Haemonchosis and represent an improved potentially automatable, and cost-effective method for the diagnosis and the proof of treatment of Haemonchus infections in goat.
Keywords: Diagnosis, qPCR, DNA extraction, DNA quantification, Haemonchus contortus eggs, Treatment.