Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) for the identification of Trichinella isolates

Abstract

In the present study, polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis was developed to identify 5 species (Trichinella spiralis, Trichinella britovi, Trichinella nativa, Trichinella nelsoni and Trichinella pseudospiralis) and 3 phenotypes of uncertain taxonomic status (Trichinella T5, T6, and T8). Eleven restriction endonucleases were used to restrict 3 DNA fragments (1) a 2800 bp fragment of the 43 kDa excretory–secretory (E–S) protein gene, (2) a 1250 bp fragment amplified with the primer pair SB147A and (3) a 372 bp fragment amplified with the primer pair SB372A. This RFLP method allows the identification of the 8 Trichinella phenotypes as follows: T. spiralis by the HinfI or DdeI endonuclease restriction of the 2800 bp fragment; T. nativa by the RsaI restriction of the 2800 bp fragment, or by the AluI restriction of the 1250 bp fragment; T. britovi and Trichinella T8 by the AluI restriction of the 1250 bp fragments, and can be discriminated between them by the SspI restriction of the 2800 bp fragment; T. pseudospiralis by the MspI restriction of the 372 bp fragment; T. nelsoni by the HhaI or AluI restriction of the 2800 bp fragment; Trichinella T5 by the HhaI restriction of the 2800 bp fragment; Trichinella T6 by the AluI restriction of the 1250 bp fragment; and Trichinella T8 by the SspI or RsaI restriction of the 2800 bp fragment. This study reveals also an intraspecifies polymorphism in the 2800 bp and 1250 bp fragments for T. britovi, Trichinella T5 and T6.