DNA extraction in Echinococcus granulosus and Taenia spp. eggs in dogs stool samples applying thermal shock

• Published in: Experimental parasitology Vol. 186; pp. 10 - 16
• Main Authors: Hidalgo, Alejandro, Melo, Angélica, Romero, Fernando, Hidalgo, Víctor, Villanueva, José, Fonseca-Salamanca, Flery
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-03-2018
• ISSN: 0014-4894; 1090-2449
• DOI: 10.1016/j.exppara.2018.01.016

The extraction of DNA in taeniid eggs shows complications attached to the composition of stool samples and the high resistance of eggs to degradation. The objective of this study was to test a method of DNA extraction in taeniid eggs by applying a thermal shock to facilitate the chemical-enzymatic degradation of these elements. A group of six tubes containing 1 ml of dog stool sample was spiked with eggs of Echinococcus granulosus and another group of six with Taenia pisiformis. Samples were floated with supersaturated sugar solution and centrifuged. The upper portion of each tube (500 μl) was aspirated and deposited in 1.5 ml tubes. Three tubes from each group were incubated at −20 °C and then at 90 °C, the remaining three from each group, incubated at room temperature. Proteinase K and lysis buffer were added to each tube and incubated for 12 h at 58 °C. The lysis effect was evaluated by microscopy at 3, 6 and 12 h and integrity by electrophoresis in 1% agarose gels. With the same experimental scheme, the thermal shock effect was evaluated in extractions of 1, 2, 3 and 4 eggs of each species and the DNA was quantified. Additionally, the protocol was applied in samples of 4 dogs diagnosed with natural infection by Taeniidae worms. Finally, all the extractions were tested by PCR amplification. Both E. granulosus and T. pisiformis eggs showed a similar response in the tests. In samples without treatment, the lysis effect was poor and showed no differences over time, but in those subjected to thermal shock, eggs degradation increased with time. In both treatments, there was no DNA loss integrity. The protocol applied to limited amounts of eggs yielded PCR products in 100% of the samples exposed to thermal shock, allowing PCR amplifications up to 1 egg. In non-exposed samples, the results were not replicable. However, DNA quantification showed low values in both treatments. In turn, DNA extractions with thermal shock in infected dog samples finally yielded PCR amplifications in 100%. It was concluded that thermal shock facilitates the DNA extraction for molecular analysis in taeniid eggs. The technique is effective extracting DNA even from a single egg and also to analyze natural infections samples with a relatively simple implementation.
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•It was compared taeniid eggs samples exposed and non-exposed to thermal shock.•DNA was extracted using ammonium acetate-chloroform-isopropanol.•Thermal shock did not affect DNA integrity.•All PCR samples with thermal shock amplified the expected products.•Method is simple to carry and with DNA acceptable performance.