Development of high resolution melt qPCR assays for detection of Toxoplasma gondii, Cryptosporidium and Trypanosoma species

Abstract

Protozoan parasites are amongst the leading diseases causing agents in both animals and humans. Trypanosomosis, cryptosporidiosis and toxoplasmosis are diseases of medical, veterinary and economic importance caused by Trypanosoma spp., Cryptosporidium spp. and Toxoplasma gondii respectively. Diagnosis is the first line of defence in combatting diseases hence the need for reliable, rapid, specific and sensitive diagnostic assays. Therefore, the aim of this study was to develop a high resolution melt real-time PCR (HRM-qPCR) assay for the detection of Trypanosoma spp., Toxoplasma gondii and Cryptosporidium spp. infections. The HRM-qPCR primers were designed from RIME, rP0, HSP70 and B1 genes of subgenus Trypanozoon species, T. congolense, Cryptosporidium spp. and Toxoplasma gondii respectively. The melt curve temperatures were 85°C for the RIME gene, ± 83°C for rP0 gene, 76°C for HSP70 gene and 81°C for B1 HRM-qPCR assays developed in this study. All the assays detected serially diluted DNA of the respective parasites down to 100 ag/ul which is equivalent to less than 1 Trypanosoma cell or Cryptosporidium or T. gondii oocyst per millilitre with all reaction conducted at 60°C for 40 cycles. The HRM-qPCR has showed higher detection sensitivity than conventional PCR for detection of Cryptosporidium spp., Trypanozoon spp, Trypanosoma congolense, Toxoplasma gondi from field derived samples. This newly developed HRM-qPCR assay requires validation with large samples from the field. Following standardization and validation the assays have potential to be used for diagnosis of subgenus Trypanozoon spp., Trypanosoma spp., Toxoplasma gondii and Cryptosporidium spp. infections.