Detection of Echinococcus granulosus and Echinococcus multilocularis in Cyst Samples Using a Novel Single Tube Multiplex Real-Time Polymerase Chain Reaction

• DOI: 10.5578/mb.21005
• Publication Date: 2016-05-16T13:55:35Z
• Authors: Hüseyin CAN; Tonay İNCEBOZ; Ayşe CANER; Esra ATALAY ŞAHAR; Muhammet KARAKAVUK; Mert DÖŞKAYA; Fehmi ÇELEBİ; Aysu DEĞİRMENCİ D...; Sultan GÜLÇE İZ; Yüksel GÜRÜZ; Metin KORKMAZ
• Keywords: Echinococcus multilocularis; Taenia; Echinococcus; Cystic echinococcosis; Taenia hydatigena

Cystic echinococcosis (CE) and alveolar (AE) caused by Echinococcus granulosus multilocularis, respectively, are important helminthic diseases worldwide as well in our country. Epidemiological studies conducted Turkey showed that the prevalence of CE is 291-585/100.000. It has also been seroprevalence AE 3.5%. For diagnosis AE, radiological (ultrasonography, computed tomography, magnetic resonance) serological methods, addition to clinical findings, being used. The definitive relies on pathological examination When hydatid cysts sterile or does not contain protoscolex, problems may occur during discrimination E.granulosus E.multilocularis species. In this study, we aimed develop a novel multiplex real-time polymerase chain reaction (M-RT-PCR) targeting mitochondrial 12S rRNA gene using Echi S (5'-TTTATGAATATTGTGACCCTGAGAT-3') A (5'-GGTCTTAACTCAACTCATGGAG-3') primers three different probes; Anchor Ech (5'-GTTTGCCACCTCGATGTTGACTTAG-fluoroscein-3'), Granulosus (5'-LC640-CTAAGGTTTTGGTGTAGTAATTGATATTTT-phosphate-3') Multilocularis (5'-LC705-CTGTGATCTTGGTGTAGTAGTTGAGATT-phosphate-3') will enable same assay. During M-RTR-PCR, plasmids containing (GenBank: AF297617.1) NC_000928.2) regions were used positive controls. Cysts samples patients which pathologically confirmed be (n: 10) 15) healthy human DNA 25) negative control 12 parasites (Taenia saginata, Hymenolepis nana, Trichuris trichiura, Fasciola hepatica, Enterobius vermicularis, Toxoplasma gondii, Pneumocystis jirovecii, Trichomonas vaginalis, Cryptosporidium hominis, Strongyloides stercoralis, Plasmodium falciparum, vivax) M-RT-PCR. constructed detect analytic sensitivity test TOPO cloning. Positive diluted determine analytical specificity distilled water at 10(6)-10(5)-10(4)-10(3)-10(2)-10(1)-1 plasmid copy dilution each reaction. According results, assay for was 1 plasmid/µl non-existence cross reactivity with parasites' Displaying cyst among 25 species 100%. As result, M-RT-PCR developed present study provided sensitive, specific, rapid, reliable method from samples.