RESUMO
INTRODUCTION: The main objective of this study was to develop a One-tube nested MGB probe real-time PCR Assay for detecting Echinococcus multilocularis infection in human plasma cell free DNA (cfDNA). METHODS: cfDNA was extracted from 10 E.m.-infected patients using a NucleoSnap DNA Plasma Kit and characterized by genomic sequencing. We designed nested PCR primers and MGB probe for Echinococcus multilocularis detection. The specificity, sensitivity and reproducibility of this assay were analyzed, and its validity was confirmed in 13 early stage clinical samples. RESULTS: Several Echinococcus multilocularis-specific sequences were detected in the cfDNA of E.m.-infected patients, and CBLO020001206.1 was selected as the candidate sequence. We designed the primers and probe for the one tube nested real-time PCR. No cross-reactions with E.g. were observed. The detection limit was as low as 1 copy for Echinococcus multilocularis. The coefficients of variation were lower than 5% in intra- and inter-assays. 11 out of 13 patients were positive with nested MGB Probe PCR Assay and 3 patients were positive without outer primer in early stage Alveolar Echinococcosis pateints. CONCLUSION: The one-tube nested MGB probe real-time PCR assay is a simple, rapid, and cost-effective method for detection of Echinococcus multilocularis infection in patients' Plasma DNA.
