Molecular identification of cestodes and nematodes by cox1 gene real-time PCR and sequencing

Abstract

Unlike bacteria and fungi, identification of helminths by gene sequencing is not well-standardized. No "pan-cestode" or "pan-nematode" PCR primers are available. In this study, we designed 2 pairs of PCR primers for amplifying the cox1 genes of cestodes and nematodes respectively and validated their usefulness for real-time PCR and sequencing identification using clinical samples with cestodes and nematodes collected from a variety of animals and human in 7 countries in Asia, Europe and Africa. The detection limits of the cox1 real-time PCR assays for cestodes and nematodes were 10 copies/reaction of extracted DNA, corresponding to C_T values of 33 and 31 respectively. Real-time PCR using the 2 pairs of primers and probes showed positive results for all 20 clinical samples of cestodes and nematodes. Using phenotypic identification results as the reference standard, DNA sequencing successfully identified all the 5 cestodes and 7 nematodes with cox1 gene sequences available in GenBank, with all these names appearing as the best match of the cox1 gene sequences of the corresponding clinical samples. The percentage nucleotide identities between the cox1 gene sequences of the samples and those of the corresponding best match sequences in GenBank were 98-100%. For the remaining 5 cestodes and 3 nematodes, the corresponding cox1 gene sequences were not available in GenBank. cox1 gene sequencing is discriminative enough for accurately identifying most of the cestodes and nematodes in the present study. Further expansion of the cox1 gene sequence database will enable accurate identification of more cestodes and nematodes.

Keywords: Cestodes; Nematodes; Real-time PCR; Sequencing; cox1 gene.