Duplex Real-time PCR Assay for the Detection of Two Intestinal Parasites, Heterakis isolonche and Glaphyrostomum sp., in Okinawa Rail (Gallirallus okinawae)

Abstract

A duplex real-time polymerase chain reaction (PCR) assay was developed to decrease expended time for diagnosis as well as to improve on the sensitivity and specificity of current molecular diagnostic methods for the detection and differentiation of Heterakis isolonche and Glaphyrostomum sp. in Okinawa rail (Gallirallus okinawae). Novel PCR primers and probes were designed based on cytochrome c oxidase subunit I gene. Our results indicated that the assay could simultaneously detect both species without cross-amplification of Okinawa rail DNA. The assay detected DNA from a single parasite egg for both species and successfully amplified DNA extracted from feces. Thus, this new assay is an useful technique for monitoring the levels of parasite infection in Okinawa rail captive breeding program.