Rapid molecular identification of human taeniid cestodes by pyrosequencing approach

Abstract

Taenia saginata, T. solium, and T. asiatica are causative agents of taeniasis in humans. The difficulty of morphological identification of human taeniids can lead to misdiagnosis or confusion. To overcome this problem, several molecular methods have been developed, but use of these tends to be time-consuming. Here, a rapid and high-throughput pyrosequencing approach was developed for the identification of three human taeniids originating from various countries. Primers targeting the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of the three Taenia species were designed. Variations in a 26-nucleotide target region were used for identification. The reproducibility and accuracy of the pyrosequencing technology was confirmed by Sanger sequencing. This technique will be a valuable tool to distinguish between sympatric human taeniids that occur in Thailand, Asia and Pacific countries. This method could potentially be used for the molecular identification of the taeniid species that might be associated with suspicious cysts and lesions, or cyst residues in humans or livestock at the slaughterhouse.

Figure 1. Alignment of the mitochondrial cox1 gene and design of primers.

Alignment of the mitochondrial cox1 gene derived from T. solium (AB524785), T. saginata (AB645845) and T. asiatica (AB608739). The broken arrows indicate the position of Cest_F (forward primer) and the biotinylated Cest_R (reverse primer) for template amplification. The solid arrow indicates Cest_S (sequencing primer), and the rectangular box shows the position (992–1017) of the target region used for species level identification. The dots indicate identical nucleotides between the sequences.

Figure 2. Sequence analysis pyrograms of T. solium T. saginata and T. asiatica cox1 genes.

Pyrograms showing the sequence analysis of 26-base fragments of the cox1 gene of T. solium (a, d) T. saginata (b, e) and T. asiatica (c, f) using pyrosequencing. The theoretical pyrogram patterns (top of each panel) and representative raw data from the control plasmids (a, b, c) and DNA extracted from taeniid proglottids (d, e, f) from pyrosequencing (bottom of each panel) are shown. The pyrosequencing was performed by the addition of enzyme (E), substrate (S), and four different nucleotides dispensed sequentially. The letters under the black bars show the dispensation (Disp:) order. The actual sequence detected by the pyrosequencing is indicated below the panels after “Seq:”. The Y-axis represents the level of fluorescence emitted by the incorporation of a nucleotide base, and the X-axis represents the total number of bases added at that point in time; A, C, G, T, nucleotide bases. The light gray bars show the regions that can be used to distinguish between T. solium, T. saginata and T. asiatica.