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Methods

MW 1 2 3 4 5 6. 500 bp. Development and Evaluation of SYBR Green-Based Real-time PCR for Detection of Echinococcus granulosus Hydatid cysts Recovered from Humans and Livestock in Sudan.

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Methods

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  1. MW 1 2 3 4 5 6 500 bp Development and Evaluation of SYBR Green-Based Real-time PCR for Detection of Echinococcusgranulosus Hydatid cysts Recovered from Humans and Livestock in Sudan Mohamed E. Ahmed1,2, Rihab M. Elageb1, Kamal H. Eltom2;Nasreen O. Musa2, Ibisam A. Ali3, Martin G. Gorbusch4, Imadeldin E. Aradaib1 Molecular Biology Laboratory, Department of Clinical Medicine, Faculty of Veterinary Medicine, University of Khartoum, Shambat, 13314, P. O. Box 32, Khartoum North, Sudan Unit of Animal Health and Safety of Animal Products, Institute for Studies and Promotions of Animal Exports, University of Khartoum, Department of Internal Medicine, Faculty of Medicine, International University of Africa, Khartoum, Sudan Department of Infectious Diseases, Faculty of Medicine, Amsterdam Medical College, the Netherlands Cystic echinococcosis (CE), caused by larval stage of Echinococcusgranulosus (EG) complex affects livestock and humans and the disease is of major public health importance in many areas in the Sudan Ten distinct genotypes designated as (G1-G10) are known worldwide, based on nucleotide sequence analysis of the mitochondrial cytochrome C oxidase subunit 1 (CO1) and NADH dehydrogenase 1 genes Methods A total of fifty hydatid cysts were obtained from the one humped camel (Camelus dromedaries) in Tamboul area, Central Sudan. Genomic DNA were extracted from the fertile sysys and subjected to conventional and real-time RT-PCR targeting the NADH 1 genes. 2% agarose electrophoesis of PCR amplifications products of 10-1 serially diluted DNA extracted from Echinococusgranulosus complex (EG) cyst. Primers targeted the CO1 gene of EG. Amplification profile generated from of different DNA extracted from hydatid cyst samples using SYBR green-based one-step quantitative PCR. Non-template negative control is illustrated in a green color. The standard curve generated from the amplification profile of the one-step SYBR green-based qPCR of serially diluted EG DNA with known dilution of 1X10-7 to 1X10-1 of DNA (1.0 ng to 10 fg ) showed a linear range of 7 logs of dilution with a R2 value equivalent to 0.99759 The melting curve analysis of the amplified DNA products from hydatid cysts was obtained with a distinct melting peak (Tm value) at 80°C. None specific amplifications or primer-dimers were observed from the non-template control (NTC) with Tm value below 70°C00 Conclusions The one-step real-time qPCR based on SYBR green chemistry afforded rapid detection of EG DNA. Amplification profile of serially diluted EG DNA with known dilution of 1X10-7 to 1X10-1 (1.0 ng – 1.0 fg ) of parasite DNA showed detection limit of as little as 1.0 fg of DNA. Presented at the Icophi Conference, Brazil, August 2013

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