Abstract
BackgroundHaemonchus contortus is one of the most pathogenic gastrointestinal nematodes of small ruminants. The current diagnostic approach for the detection of this species relies on coproscopic methods, which both have low sensitivity and are time consuming. Methods employing detection through DNA amplification, such as droplet digital polymerase chain reaction (ddPCR), offer an advantageous approach to the diagnosis of H. contortus. However, DNA extraction protocols need to be constantly updated for the optimal retrieval of diagnostically usable template. Here, we describe the evaluation of three genomic DNA extraction kits for the detection and quantification of H. contortus ITS2 amplicon DNA from faecal samples, using droplet digital PCR.ResultsDNA samples, extracted from faecal material with the Nucleospin DNA Stool kit, produced the highest amounts of ITS2 amplicon copies and had the lowest coefficient of variation across different dilutions and sample types (fresh or frozen) out of the tested kits (Nucleospin DNA Stool, E.Z.N.A.® Stool DNA Kit and QIAamp Fast DNA Stool Mini Kit). Furthermore, the protocol of this kit has the fewest number of steps and the price of DNA extraction per sample is reasonable (2.77 €).ConclusionsThe Nucleospin DNA Stool kit is an attractive option for the detection and quantification of H. contortus DNA in faecal samples of small ruminants in a diagnostic setting.
Highlights
Haemonchus contortus is one of the most pathogenic gastrointestinal nematodes of small ruminants
Sample preparations and experimental design The material used in this study originated from a single, fresh faecal sample from a sheep monospecifically infected with H. contortus, and was obtained from a routine veterinary diagnostic laboratory
We evaluated three commercial genomic DNA extraction kits for the detection and quantification of H. contortus ITS2 amplicon DNA faecal slurry samples, which were prepared either fresh or after they had been frozen at − 20 °C for 3 months
Summary
Haemonchus contortus is one of the most pathogenic gastrointestinal nematodes of small ruminants. The current diagnostic approach for the detection of this species relies on coproscopic methods, which both have low sensitivity and are time consuming. Current field diagnostics rely on coproscopic methods and the subsequent visual identification of the infective third stage larvae (L3) to confirm the presence (or absence) of H. contortus on any particular farm. This approach requires expertise in the form of trained technical staff as well as being laborious and time consuming as the culturing of larvae for more than a week is usually required for the identification of morphologically similar strongylid eggs [8]. For some applications, such as the declaration of freedom from
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