Discrimination between E. granulosus sensu stricto, E. multilocularis and E. shiquicus Using a Multiplex PCR Assay

Abstract

BackgroundInfections of Echinococcus granulosus sensu stricto (s.s), E. multilocularis and E. shiquicus are commonly found co-endemic on the Qinghai-Tibet plateau, China, and an efficient tool is needed to facilitate the detection of infected hosts and for species identification.Methodology/Principal FindingsA single-tube multiplex PCR assay was established to differentiate the Echinococcus species responsible for infections in intermediate and definitive hosts. Primers specific for E. granulosus, E. multilocularis and E. shiquicus were designed based on sequences of the mitochondrial NADH dehydrogenase subunit 1 (nad1), NADH dehydrogenase subunit 5 (nad5) and cytochrome c oxidase subunit 1 (cox1) genes, respectively. This multiplex PCR accurately detected Echinococcus DNA without generating nonspecific reaction products. PCR products were of the expected sizes of 219 (nad1), 584 (nad5) and 471 (cox1) bp. Furthermore, the multiplex PCR enabled diagnosis of multiple infections using DNA of protoscoleces and copro-DNA extracted from fecal samples of canine hosts. Specificity of the multiplex PCR was 100% when evaluated using DNA isolated from other cestodes. Sensitivity thresholds were determined for DNA from protoscoleces and from worm eggs, and were calculated as 20 pg of DNA for E. granulosus and E. shiquicus, 10 pg of DNA for E. multilocularis, 2 eggs for E. granulosus, and 1 egg for E. multilocularis. Positive results with copro-DNA could be obtained at day 17 and day 26 after experimental infection of dogs with larval E. multilocularis and E. granulosus, respectively.Conclusions/SignificanceThe multiplex PCR developed in this study is an efficient tool for discriminating E. granulosus, E. multilocularis and E. shiquicus from each other and from other taeniid cestodes. It can be used for the detection of canids infected with E. granulosus s.s. and E. multilocularis using feces collected from these definitive hosts. It can also be used for the identification of the Echinococcus metacestode larva in intermediate hosts, a stage that often cannot be identified to species on visual inspection.

A dual PCR-based sequencing approach for the identification and discrimination of Echinococcus and Taenia taxa

“…Although the previously described PCR [29] was inexpensive and easy to perform, the following limitations became apparent: it could not distinguish between E. granulosus s.s. genotypes (G1/3); it worked reliably only on DNA extracted from cysts; it was not sensitive enough to be used for the specific identification of eggs when these were present in low numbers [29]; extensive phylogenetic analysis was not possible because of limited informative nucleotide sites; and, finally, taeniids other than Echinococcus could not be detected. Similar issues might hamper other one-step or single species-specific detection systems [30][31][32].…”

“…Recently, we developed a multiplex PCR (Egc-mPCR) as a rapid and reliable test for the identification of all recognized species within the E. granulosus complex [29]. However, like other one-step techniques, such as high-resolution melting (HRM) [30], loop-mediated isothermal amplification (LAMP) [31] and multiplex PCR [32], the Egc-mPCR did not permit further extensive and deeper systematic analyses.…”

Validation of a one-step PCR assay for the molecular identification of Echinococcus granulosus sensu stricto G1–G3 genotype

“…The species and genotype were previously determined by the amplification with COX1 primer pairs [41] and sequencing, as described above. The primer pairs compared corresponded to Ef1a (F 5′: TCC TAA CAT GCC TTG GTA T-3′ R5′: GTT ACA GCC TTG ATC ACG -3′), that amplified the sequence of Elongation-Factor 1 Alpha of 706 bp [42], Cal (F5′: CAA TTT ACG GTA AAG CAT -3′ R5′: CCT CAT CTC CAC TCTCT-3′), that amplified the gene Calreticulin l of 1001 bp, [42], and NAD1 (F5′: GGT TTT ATC GGT ATG TTG GTG TTA GTG-3′-R5′: CAT TTC TTG AAG TTA ACA GCA TCA CG-3′), that amplified the amplicon NADH-dehydrogenase-subunit 1 of 219 bp [43].…”

Echinococcus spp.

“…Quantification of the parasite load in fecal samples collected in the environment is also made possible thanks to real-time PCR (Knapp et al, 2014). Techniques which combine the identification of several Echinococcus species and of the animal species of the collected feces (multiplex PCR) (Boubaker et al, 2013;Dinkel et al, 2011 ;Knapp et al, 2016;Liu et al, 2015;Trachsel et al, 2007) may provide more accurate data for epidemiological studies, especially in areas where several Echinococcus species and several definitive hosts co-exist and in the context of control. However, even though molecular techniques appear currently to be the most appropriate to study Echinococcus infection in the environment and their automation has been developed and evaluated , there are very few studies on the eggs in the environment and only one on-going study (unpublished yet) on wastewater (Conraths and Deplazes, 2015;Lass et al, 2015;Szostakowska et al, 2014).…”