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Title: SENSITIVE AND SPECIFIC PCR ASSAY FOR BIOTYPING TRICHINELLA ISOLATES

Author
item APPLEYARD, G - CAP, AA, CANADA
item Zarlenga, Dante
item POZIO, E - INST SUPER. DI SANITA
item GAJADHAR, A - CAP,AA CANADA

Submitted to: Trichinellosis International Conference Proceedings
Publication Type: Proceedings
Publication Acceptance Date: 10/21/1996
Publication Date: N/A
Citation: N/A

Interpretive Summary: Morphological criteria alone are not sufficient to distinguish between the 5 species and 3 other genotypes of Trichinella (the helminth parasite known because of foodborne infections of humans). Consequently, confusing nomenclature has arisen and understanding of systematics and parasite ecology has been impeded. Herein, we identified a region with the large subunit ribosomal DNA that is post-transcriptionally excised from the RNA, exhibits variability in length among the various species and can be utilized as a diagnostic character for Trichinella species in a standard polymerase chain reaction (PCR). This work will greatly facilitate the elucidation of parasites within this genus and easily enable epidemiological studies as well as permit species diagnosis from single larvae and therefore from host biopsies.

Technical Abstract: Ribosomal DNA gap-sequences within the genus Trichinella are variable. A previously published assay for differentiating isolates using PCR amplification of ribosomal gap sequences was modified and evaluated. The assay was tested on extracted DNA from Trichinella T1 to T8 reference isolates, crude DNA from boiled Trichinella larvae and genomic DNA from pig and dog. Modification of primer sequences increased the detection limit as well as specificity for DNA from boiled larvae. The addition of 2% glycerol or 1% dimethylsulfoxide, but not 1% formamide, improved the amplified yield for one primer set. One primer set consistently differentiated T1, T4, and T7 and from all other sylvatic genotypes. The other primer set further differentiated T3, T5, T8 and T2/T6. DNA from as little as a single larva can be detected and biotyped by this method.