A SYBR green I real-time polymerase chain reaction (PCR) assay for detection and quantification of Trichomonas gallinae

Abstract

Trichomonas gallinae are parasitic flagellates of importance in wild and domestic birds. The parasite is worldwide distributed, and Columbine birds are its main host. Current research focuses mostly on epidemiological and phylogenetic studies. However, there is still a lack of knowledge regarding parasite-host interaction or therapy development. Real-time PCR is a useful tool for diagnostic and quantification of gene copies in a determined sample. By amplification of a 113-bp region of the 18S small subunit ribosomal RNA gene, a SYBR green-based real-time PCR assay was developed. A standard curve was prepared for quantification analysis. Assay efficiency, linearity, and dissociation analysis were successfully performed. Specificity, sensibility, and reproducibility analysis were tested. This assay could be a useful tool not only for diagnostic purposes but also for future in vivo and in vitro T. gallinae studies.

Adaptation of the classical end-point ITS-PCR for the diagnosis of avian trichomonosis to a real-time PCR reveals Bonelli’s eagle as a new host for Trichomonas gypaetinii

“…Another approach in the detection of trichomonads using qPCR was the one designed to differentiate T. gallinae from Tetratrichomonas gallinarum, but those species are more distant and probably with higher differences in GC content, and also, the last species is not common in the oropharynx of birds (Sigrist et al 2022). An advantage of the use of ITS-PCR instead of 18S PCR could be a higher sensitivity, although this point should be further tested (Rentería-Solís et al 2020;Martínez-Herrero et al 2021).…”

“…In this study, the differences in the composition of base pairs between the three genotypes of T. gallinae and T. gypaetinii sequences are not enough to allow the differentiation of the species/genotypes by comparison of the melting temperatures since they presented very similar values (82.73–83.48 °C in T. gallinae and 82.74 °C in T. gypaetinii ). Similarly, in a recent assay using a qPCR method for the diagnosis of T. gallinae , in which a conserved region (18S rRNA) was selected for amplification, the technique was not able to discriminate between T. gallinae and Tritrichomonas foetus (Rentería-Solís et al 2020 ). Another approach in the detection of trichomonads using qPCR was the one designed to differentiate T. gallinae from Tetratrichomonas gallinarum , but those species are more distant and probably with higher differences in GC content, and also, the last species is not common in the oropharynx of birds (Sigrist et al 2022 ).…”