Molecular detection of Trichostrongylus species through PCR followed by high resolution melt analysis of ITS-2 rDNA sequences.

Mohsen Arbabi, Hossein Hooshyar, Majid Lotfinia, Mohamad Ali Bakhshi

Molecular and biochemical parasitology 2020 Mar

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Polymerase chain reaction followed by high resolution melting (PCR-HRM) analysis is a simple, rapid and accurate method for molecular detection of various nematode species. The objective of the present study was, for the first time, to develop a PCR-HRM assay for the detection of various animal Trichostrongylus spp. A pair of primers targeting the ITS-2 rDNA region of the Trichostrongylus spp. was designed for the development of the HRM assay. DNA samples were extracted from 30 adult worms of Trichostrongylus spp., the ITS-2-rDNA region was amplified using PCR, and the resultant products were sequenced and characterized. Afterwards, the PCR-HRM analysis was conducted to detect and discriminate Trichostrongylus spp. Molecular sequence analysis revealed that 24, 4, and 1 of the samples were T. colubriformis, T. vitrinus and T. capricola, respectively. Results from PCR-HRM indicated that complete agreement was relatively found between speciation by HRM analysis and DNA sequencing for the detection of Trichostrongylus species. The PCR-HRM analysis method developed in the present study is fast and low-cost; the method can be comparable with other molecular detection techniques, representing a reliable tool for the identification of various species within the Trichostrongylus genus. Copyright © 2020 Elsevier B.V. All rights reserved.

Citation

Mohsen Arbabi, Hossein Hooshyar, Majid Lotfinia, Mohamad Ali Bakhshi. Molecular detection of Trichostrongylus species through PCR followed by high resolution melt analysis of ITS-2 rDNA sequences. Molecular and biochemical parasitology. 2020 Mar;236:111260