A multiplex PCR for unequivocal differentiation of all encapsulated and non-encapsulated genotypes of Trichinella

Abstract

We have developed a single PCR test for the simple and unequivocal differentiation of all currently recognised genotypes of Trichilnella. Partial DNA sequence data were generated from internal transcribed spacers ITS1 and ITS2, and from the expansion segment V region of the ribosomal DNA repeat from five species of Trichinella and two additional genotypes, designated T5 and T6. Five different PCR primer sets were identified which, when used simultaneously in a multiplex PCR, produce a unique electrophoretic DNA banding pattern for each species and genotype including three distinct genotypes of Trichinella pseudospiralis. The banding patterns for each parasite genotype consist of no more than two well-defined DNA fragments, except isolates of T. pseudospiralis which generate multiple, closely migrating bands. The expansion segment V-derived primer set contributes at least one fragment to each genotypic pattern and, therefore, functions both as a means for differentiation as well as an internal control for the PCR. The reliability and reproducibility of each DNA banding pattern were verified using multiple geographical isolates of each Trichinella genotype. The technique was developed further to distinguish genotypes at the level of single muscle larvae using a nested, multiplex PCR, whereby the entire internal transcribed spacer region as well as the gap region of the expansion segment V of the large subunit ribosomal DNA are amplified concurrently in a first-round PCR using primer sets specific for each region, followed by the multiplex PCR for final diagnosis.