A multiplex PCR for unequivocal dierentiation of all encapsulated and non-encapsulated genotypes of Trichinella Dante S. Zarlenga a, *, M. Barry Chute a , Anthony Martin b , Christian M.O. Kapel b a US Department of Agriculture, ARS, Immunology and Disease Resistance Laboratory, Beltsville, MD 20705, USA b The Royal Veterinary and Agricultural University, Bu Èlowsvej 17, DK-1870 Frederiksberg C, Denmark Received 16 April 1999; received in revised form 21 June 1999; accepted 21 June 1999 Abstract We have developed a single PCR test for the simple and unequivocal dierentiation of all currently recognised genotypes of Trichinella. 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 ®ve species of Trichinella and two additional genotypes, designated T5 and T6. Five dierent PCR primer sets were identi®ed 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-de®ned 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 dierentiation as well as an internal control for the PCR. The reliability and reproducibility of each DNA banding pattern were veri®ed 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 ampli®ed concurrently in a ®rst-round PCR using primer sets speci®c for each region, followed by the multiplex PCR for ®nal diagnosis. 1999 Published by Elsevier Science Ltd. Keywords: Diagnosis; Multiplex; Polymerase chain reaction; Ribosomal DNA; Trichinella 1. Introduction Trichinellosis is a globally distributed zoonotic disease caused by the ingestion of raw or under- cooked meats harbouring parasites of the genus Trichinella. Disease eradication is compounded by the limitless host speci®city of Trichinella lar- vae, which are capable of infecting a broad range of carnivores and omnivores. Although the inci- dence of human trichinellosis originating from swine has been on the decline in recent years in the United States and in countries of the European Union, the occurrence of this disease International Journal for Parasitology 29 (1999) 1859±1867 0020-7519/99/$20.00 1999 Published by Elsevier Science Ltd. PII: S0020-7519(99)00107-1 * Corresponding author. Tel.: 301-504-8754; fax: 301-504- 8979. E-mail address: Zarlenga@lpsi.barc.usda.gov (D.S. Zarlenga)