A single, multiplex PCR for differentiating all species ofTrichinella

Abstract

Summary: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.

Zoonotic helminth infections with particular emphasis on fasciolosis and other trematodiases

“…The definitive method for diagnosis of trichinellosis is microscopic analysis of muscle biopsies though this has limited use for detection of light and moderate infections. More recent alternatives include detection of antibodies against Trichinella spiralis excretory-secretory (E -S) proteins in human sera by enzyme-linked immunosorbent assay (ELISA; Moskwa et al 2006) and polymerase chain reaction (PCR)-based identification approaches (Wu et al 1999;Zarlenga et al 2001). The release of the T. spiralis genome sequence draft assembly (Washington University Genome Sequencing Center; http://genome.wustl.edu/home.cgi) together with recent advances in proteomics for the identification of individual Trichinella E-S proteins has expanded the panel of Trichinella antigens that may be used to detect trichinellosis earlier post-infection Robinson et al , 2007.…”

Biology and genome of Trichinella spiralis

“…Several other PCR-based assays evaluated specific genomic sequences (Appleyard et al, 1999;Soule et al, 1993;Wu et al, 1998;Zarlenga et al, 1999) and a multiplex method was developed that differentiates all Trichinella spp. (Zarlenga et al, 2001). Other discriminating methods have been described, such as overall length and cleavage fragment length polymorphisms in the 5S rRNA intergenic spacer region (Rombout et al, 2001), and a reverse line blot (RLB) assay (Gubbels et al, 1999;Rijpkema et al, 1995).…”

Parasitic nematodes—From genomes to control

“…Keeping sufficient parasites in refugia, can maintain the susceptible gene pool, but requires monitoring fecal egg counts to determine the level of pasture contamination and the FAMACHA # method for tracking anemia in small ruminants. Using PCR techniques, one could also decide to drug treat based upon the pathogenicity of the parasite species that populate the pastures (Zarlenga et al, 2001).…”