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

Abstract

The Italian National Reference Center for Echinococcosis (CeNRE, Sassari, Italy) set up a diagnostic protocol of "one-step-PCR" useful for the detection of E. granulosus sensu stricto (E.g.s.s.) and the identification of its genotype (G1-G3). The purpose of this work was to perform the validation of the "PCR E.g.s.s." method. The procedures were performed employing the criteria of the World Organization for Animal Health as well as of the Italian Accreditation Body (ACCREDIA) based on the Regulation UNI CEI EN ISO/IEC 17025. Positive DNA samples belonging to E. granulosus, E. ortleppi, E. multilocularis, E. canadensis species were used for the experiments. Analytical specificity evidenced primer pairs Cal (Calreticulin l gene of 1001 bp) with an specificity higher respect to Ef1 (Elongation-Factor 1 Alpha gene of 706 bp) and NAD (Dehydrogenase-subunit 1 gene of 219 bp). The analytical sensitivity presented the capability to detect a very low amount of parasite DNA corresponding to a concentration of 12.5 pg/µl; accuracy and precision related to the operator performance, along with repeatability and reproducibility, evidenced high concordance among results and demonstrated an excellent κ values of Cohen. According to the good performance related to the evaluated parameters, the method "PCR E.g.s.s." was suitable for the validation procedure, and consequently, to be undergone to the accreditation process. In conclusion, the results demonstrated an elevated robustness and reliable features of the "PCR E.g.s.s." able to perform a rapid diagnosis of E. granulosus in only "one step", hence, it is likely to avoid the sequencing step.

Cystic Echinococcosis: Clinical, Immunological, and Biomolecular Evaluation of Patients from Sardinia (Italy)

“…The PCR for E.g.s.s. [59] method was able to determine the species and the genotype of E. granulosus s.s. G1 or G3 in only one step, avoiding the sequencing step. This assay was performed by using the primers pairs that amplified the Cal gene of 1001 bp (F5 : CAATTTACGGTAAAGCAT-3 -R5 : CCTCATCTCCACTCTCT-3 ) (Table 6).…”

Natural Products as Therapeutic Option for Echinococcossis

“…Other serological methods that have been used in the diagnosis of human CE include immunofluorescence assay, indirect hemagglutination assay, immunochromatographic test and dot immunogold filtration assay. However, these tests are associated with lower sensitivity and specificity, thus are used less [12].…”

“…DNA probes for Southern hybridization tests and polymerase chain reactions are very helpful in confirming diagnosis of CE. Moreover, PCR has high sensitivity and specificity for the pathogen's DNA, thus allows for precise determination of infection status and identification of genus, species, and genotype [12]. As such, PCR is the foremost molecular analysis in the diagnosis of human CE.…”

Echinococcus granulosus İnfeksiyonu Tanısında Moleküler Uygulamalar ve Yeni Yaklaşımlar

“…Mitochondrial DNA (mtDNA) markers are ideal for evolutionary studies such as phylogeography, population genetics, phylogeny, etc. Therefore, for the analysis of the mitochondrial genetic structure in CE genotypes, mitochondrial cytochrome c oxidase I (cox1) and NADH dehydrogenase subunit I (nad1) genes were also evaluated in the serum of CE to determine the source of DNA and compared to cyst tissue samples (Altintas et al, 2013;Jafari et al, 2018;Moradi et al, 2019;Santucciu et al, 2019;.…”