Development of real-time PCR assay for simultaneous detection and genotyping of cystic echinococcosis in humans and livestock

Abstract

Objective: To develop and evaluate a single-tube one-step real-time quantitative PCR (qPCR) assay for simultaneous diagnosis and genotyping of cystic echinococcosis (CE) in humans and livestock in the Sudan, and to compare it with conventional PCR assay. Methods: Hydatid cysts were obtained from slaughtered animals and from humans after surgical interventions. DNA from the hydatid cysts and associated germ layers was extracted using a commercially available kit. The mitochondrial NADH dehydrogenase subunit 1 (NAD1) was used as a target for PCR amplifications. qPCR and conventional nested PCR assays were compared in this study. Results: The qPCR assay amplified the NAD 1 gene of hydatid cysts on melting temperature generated at 80 °C. Ten-folds serial dilutions of DNA with known dilution of 1 × 106 to 1 × 101 (1 ng-1 fg) resulted in detection of as little as 1 fg of DNA with an R2 value equivalent to 0.997. Similar sensitivities were encountered from both qPCR and the conventional nested PCR. The two assays did not amplify DNAs from Fasciola gigantica, Taenia saginata, Schistosoma bovis and DNA-free samples (negative controls). The PCR amplified products were purified for subsequent sequencing. The sequence data were analysed to insure the specificity of the amplified PCR products and to identify the genotype(s) of hydatid cysts. All cysts were identified as E. canadensis genotype 6 (G6). Conclusions: The developed qPCR should be used as a rapid and reliable assay for diagnosis and genotyping of CE. The assay is highly recommended for the epidemiological surveillance in humans and livestock in endemic countries.