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

We have developed a single PCR test for the simple and unequivocal differen tiation of all currently recognised genotypes of Trichinella. Partial DNA s equence 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 simult aneously in a multiplex PCR, produce a unique electrophoretic DNA banding p attern 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 ex pansion segment V-derived primer set contributes at least one fragment to e ach genotypic pattern and, therefore, functions both as a means for differe ntiation as well as an internal control for the PCR. The reliability and re producibility of each DNA banding pattern were verified using multiple geog raphical isolates of each Trichinella genotype. The technique was developed further to distinguish genotypes at the level of single muscle larvae usin g a nested, multiplex PCR, whereby the entire internal transcribed spacer r egion as well as the gap region of the expansion segment V of the large sub unit ribosomal DNA are amplified concurrently in a first-round PCR using pr imer sets specific for each region, followed by the multiplex PCR for final diagnosis. 1999 Published by Elsevier Science Ltd.