New multiplex PCR method for the simultaneous diagnosis of the three known species of equine tapeworm.

The research article presents a new method to detect the three known species of equine tapeworms using a multiplex PCR method. This technique amplifies specific gene regions in the tapeworms and accurately identifies them, even when present in very low amounts.

Justification and Methodology of the Research

• The article starts by noting the existing methods for diagnosing tapeworm infections in horses, which typically involve postmortem examinations or identifying eggs in feces. However, these techniques may not provide a differential diagnosis, leading to a gap that this study aims to address.
• To meet this objective, the researchers developed a multiplex polymerase chain reaction (PCR) method. This method involves amplifying specific, highly-variable gene regions (called SSUrRNA) in three species of tapeworms: Anoplocephala magna, Anoplocephala perfoliata, and Anoplocephaloides mamillana.
• The technique uses three sets of primers to facilitate the amplification process. These primers exclusively amplify a section known as the internal transcribed spacer 2 (ITS-2) in the target genes of these tapeworm species.

Testing and Experimentation of the Method

• The researchers tested their method on three types of samples: genomic DNA of the tapeworms, DNA extracted from negative control fecal samples with added eggs, and DNA extracted from eggs set in a PCR reaction mix.
• To ensure specificity and prevent false-positives, they also tested the method against the DNA of three other organisms (Gasterophilus intestinalis, Parascaris equorum and Strongylus vulgaris) that could potentially be present in horses. The method did not result in any amplification against these organisms.

Results and Conclusiveness of the Research

• The newly developed PCR method was successful in specifically identifying the tapeworm species. More impressively, it was able to detect their presence with extreme sensitivity, identifying as few as 50 eggs in a fecal sample or as low as 0.7ng of control genomic DNA from the species.
• The standardization and successful validation of this method can help identify infections pitched by either Anoplocephala magna or A. perfoliata, two of the most common tapeworms. Additionally, this method can also detect mixed infections by the three tapeworm species.