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Background  Molecular-based surveys have indicated that  Ancylostoma ceylanicum , a zoonotic hookworm, is likely the second most prevalent hookworm species infecting humans in Asia. Most current PCR-based diagnostic options for the detection of  Ancylostoma  species target the Internal Transcribed Spacer (ITS) regions of the ribosomal gene cluster. These regions possess a considerable degree of conservation among the species of this genus and this conservation can lead to the misidentification of infecting species or require additional labor for accurate species-level determination. We have developed a novel, real-time PCR-based assay for the sensitive and species-specific detection of  A .  ceylanicum  that targets a non-coding, highly repetitive genomic DNA element. Comparative testing of this PCR assay with an assay that targets ITS sequences was conducted on field-collected samples from Argentina and Timor-Leste to provide further evidence of the sensitivity and species-specificity of this assay.    Methods/Principal findings  A previously described platform for the design of primers/probe targeting non-coding highly repetitive regions was used for the development of this novel assay. The assay’s limits of detection (sensitivity) and cross-reactivity with other soil-transmitted helminth species (specificity) were assessed with real-time PCR experiments. The assay was successfully used to identify infections caused by  A .  ceylanicum  that were previously only identified to the genus level as  Ancylostoma  spp. when analyzed using other published primer-probe pairings. Further proof of sensitive, species-specific detection was provided using a published, semi-nested restriction fragment length polymorphism-PCR assay that differentiates between  Ancylostoma  species.    Conclusions/Significance  Due to the close proximity of people and domestic/wild animals in many regions of the world, the potential for zoonotic infections is substantial. Sensitive tools enabling the screening for different soil-transmitted helminth infections are essential to the success of mass deworming efforts and facilitate the appropriate interpretation of data. This study describes a novel, species-specific, real-time PCR-based assay for the detection of  A .  ceylanicum  that will help to address the need for such tools in integrated STH deworming programs.    Trial registration  ANZCTR.org.au  ACTRN12614000680662

A novel, species-specific, real-time PCR assay for the detection of the emerging zoonotic parasite Ancylostoma ceylanicum in human stool