Assessment of Real-Time Polymerase Chain Reaction for the Detection of Trichostrongylus spp. DNA from Human Fecal Samples

Abstract

Sporadic cases of Tricostrongylosis are reported in humans. Diagnosis of enteric Trichostrongylus relies primarily on coproscopic analysis but morphological identification is difficult because of similarity among nematode species. The method is time consuming and requires some expertise. To overcome these limitations, we developed a molecular approach by real-time polymerase chain reaction (PCR) to provide a rapid, specific, and sensitive tool to detect Trichostrongylus spp. in human feces. We designed primers and probe specific for Trichostrongylus rDNA region 5.8S and internal transcribed spacer 2. Three Italian family clusters were analyzed and DNA sequencing was performed to confirm real-time PCR results comparing with known GenBank sequence data. Sequence analysis showed ≥ 99% identity to Trichostrongylus colubriformis and Trichostrongylus axei. This study provides a molecular methodology suitable for fast and specific detection of Trichostrongylus in fecal specimens and to distinguish the zoonotic species.

Figure 1.

Schematic representation of part of the rDNA transcriptional unit and relative locations of primers (Tricho-F, Tricho-R and JhTsp) used in this study. The region harboring 5.8S and internal transcribed spacer 2 region (ITS2) rDNA was amplified with Tricho-F and Tricho-R by real-time polymerase chain reaction and with JhTsp and Tricho-R for DNA sequencing.

Figure 2.

Nucleotide sequences alignment of 5.8S-ITS2 region using ClustalW. The locations of forward primer JhTsp and reverse primer Tricho-R are underlined in each line. Asterisks indicate identical nucleotides. ITS2 = internal transcribed spacer 2 region.