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Title: A SINGLE MULTIPLEX PCR FOR UNEQUIVOCAL DIFFRENTIATION OF SEVEN DISTINCT GENOTYPES OF TRICHINELLA

Author
item Zarlenga, Dante
item Chute, M
item MARTIN, ANTHONY - DENMARK
item KAPEL, CHRISTIAN - DENMARK

Submitted to: International Journal for Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/1/1999
Publication Date: N/A
Citation: N/A

Interpretive Summary: Trichinellosis is a globally distributed zoonotic disease caused by the ingestion of raw or undercooked meats harboring parasites of the genus Trichinella. Disease eradication is compounded by the limitless host specificity of Trichinella larvae which are capable of infecting nearly all carnivores and omnivores. Though the incidence 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 emanating from sylvatic genotypes and hosts remains a major source of human infection in these countries. Within most parasite genera, distinct morphological and/or biological characters exist among the species that permit differentiation and proper classification. However, the absence of distinguishing morphological characters and the overlapping nature of the biological characters within the genus Trichinella, make these traits unsuitable for proper diagnosis. Consequently, identification and classification of species within this genus must rely heavily upon molecular and biochemical data. Herein we develop a multiplex PCR test based upon rDNA repeat sequences that is capable of 1) differentiating all well-defined genotypes of Trichinella including three genotypic isolates of T. pseudospiralis; 2) circumventing the need for external PCR controls, and; 3) distinguishing single larvae using nested PCR. This work will greatly facilitate the elucidation of parasites within this genus for epidemiological studies as well as permit species diagnosis from host biopsies.

Technical Abstract: We have developed a single PCR test for the simple and unequivocal differentiation of all currently recognized genotypes of Trichinella. Partial DNA sequence data was generated from internal transcribed spacers ITS1 and ITS2, and drom the expansion segment V (ESV) region of the ribosomal DNA (rDNA) repeat from five species of Trichinella and two addiional 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 nnd genotype including three distinct genotypes of Trichinella pseudospiralis. The banding patterns for each parasite group consist of no more than two well-defined DNA fragments except for the Tasmanian isolate of T. pseudospiralis which generates three closely migrating bands. The ESV- derived primer set contributes at least one fragment to each genotypic pattern and therefore functions both as a means for differentiation as wel as an internal control for PCR integrity. The reliability and reproducibility of each DNA banding pattern was verified using multiple geographical isolates of each Trichinella genotype. The technique was further developed to distinguish single muscle larvae using a nested, multiplex PCR whereby the entire ITS region as well as the gap region of the ESV of the large subunit rDNA are amplified concurrenty in a first round PCR using primer sets specific for each region, followed by the multiplex PCR for final diagnosis. PCR profiles using DNA quantities derived from a single parasite, however, can be affected substantially by DNA integrity.