A single, multiplex PCR for differentiating all species of Trichinella

The genus Trichinella is currently divided into seven species and at least three additional, unclassified genotypes, Trichinella T6, T8 and T9, where both T8 and T9 have been deemed very similar to T. britovi. Other than for the non-encapsulated species, the absence of distinguishing morphological characters and the overlapping nature of the biological characters within this genus make these traits unsuitable for diagnosis. Consequently, we have developed a simple PCR test for the unequivocal differentiation of all currently recognized species of Trichinella including Trichinella T6. DNA sequence data from each Trichinella genotype were generated from internal transcribed spacers, ITS1 and ITS2, and from expansion segment V (ESV) of the rDNA repeat, from which five different PCR primer sets were chosen. When used simultaneously, this primer mix generates a simple and unique electrophoretic DNA banding pattern for each species and genotype. The ESV-derived primer set contributes at least one band to each agarose gel-derived genotypic pattern and therefore functions as an internal control for PCR integrity. Geographical isolates of each Trichinella genotype were used to verify the reliability and reproducibility of respective DNA banding patterns using single muscle larvae.