Sequencing and Characterization of an Echinococcus Multilocularis DNA Probe and Its Use in the Polymerase Chain Reaction

The nucleotide sequence of the cloned Echinococcus multilocularis DNA probe pAL1 was determined in order to simplify and improve the sensitivity of a diagnostic assay through the application of the polymerase chain reaction (PCR). The insert-specific oligonucleotides BG1 and BG2 define a 2.6-kb fragment in the genomic DNA of E. multilocularis, while BG1 and BG3 define a 0.3 kb fragment. A PCR study including 14 independent E. multilocularis isolates in addition to Echinococcus granulosus. Echinococcus vogeli, Taenia spp. and other cestodes revealed that the 2.6-kb fragment was amplified from genomic DNA of all E. multilocularis isolates tested (originating from Switzerland, Alaska, Canada, France, Germany and Japan), but from genomic DNA of none of the other cestode species. PCR with BG1 and BG2 furthermore uniquely resulted in the synthesis of a 0.55-kb fragment specific for Taenia saginata and a 0.6-kb fragment specific for T. taeniaeformis. In contrast to the species specificity of the 2.6-kb BG1/BG2 product, the 0.3 kb (BG1/BG3) product demonstrated genus specificity: the 0.3-kb product was amplified from genomic DNA of all E. multilocularis, E. granulosus and E. vogeli isolates tested, but from genomic DNA of none of the other cestode species. The diagnostic sensitivity of PCR using both primer sets was determined to be 50 pg parasite DNA, suggesting the practical utility of this simple assay in demonstrating parasite DNA in specimens from a variety of sources. At the basic level, the pAL1-derived oligonucleotides may also prove useful in assessing strain variation, RFLPs or other manifestations of genetic variation in E. multilocularis.