| Autor: |
Devyatov AA; Federal State Budgetary Institution 'Centre for Strategic Planning and Management of Biomedical Health Risks' of the Federal Medical Biological Agency, 10 bld 1, Pogodinskaya Str., 119121 Moscow, Russia., Davydova EE; Federal State Budgetary Institution 'Centre for Strategic Planning and Management of Biomedical Health Risks' of the Federal Medical Biological Agency, 10 bld 1, Pogodinskaya Str., 119121 Moscow, Russia., Luparev AR; Federal State Budgetary Institution 'Centre for Strategic Planning and Management of Biomedical Health Risks' of the Federal Medical Biological Agency, 10 bld 1, Pogodinskaya Str., 119121 Moscow, Russia., Karseka SA; Federal State Budgetary Institution 'Centre for Strategic Planning and Management of Biomedical Health Risks' of the Federal Medical Biological Agency, 10 bld 1, Pogodinskaya Str., 119121 Moscow, Russia., Shuryaeva AK; Federal State Budgetary Institution 'Centre for Strategic Planning and Management of Biomedical Health Risks' of the Federal Medical Biological Agency, 10 bld 1, Pogodinskaya Str., 119121 Moscow, Russia., Zagainova AV; Federal State Budgetary Institution 'Centre for Strategic Planning and Management of Biomedical Health Risks' of the Federal Medical Biological Agency, 10 bld 1, Pogodinskaya Str., 119121 Moscow, Russia., Shipulin GA; Federal State Budgetary Institution 'Centre for Strategic Planning and Management of Biomedical Health Risks' of the Federal Medical Biological Agency, 10 bld 1, Pogodinskaya Str., 119121 Moscow, Russia. |
| Abstrakt: |
One of the main challenges for the mass introduction of the molecular diagnostics of soil-transmitted helminths (STHs) into clinical practice is the lack of a generally recognized effective method for isolating parasitic DNA from fecal samples. In the present study, we assessed the effects of various pretreatment procedures on the efficiency of removing PCR inhibitors and extracting Toxocara canis DNA from feces. We evaluated the effectiveness of four destructive methods (bead beating, the action of temperature-dependent enzymes, freeze-heat cycles, and incubation in a lysis buffer) on the integrity of T. canis eggs and the efficiency of DNA extraction. Also, we evaluated the effects of prewashes and the use of commercial concentrators on DNA extraction from fecal samples contaminated with T. canis eggs. A bead beating procedure was sufficient to destroy the T. canis eggs, while the effects of enzymes and freeze-heat cycles did not lead to a significant destruction of the eggs or the release of Toxocara DNA. Helminth DNA isolation protocols that do not include a bead beating step are not preferred. The preconcentration of STH eggs from feces using a commercial concentrator and subsequent washing can significantly increase the yield of DNA from STHs and reduce PCR inhibition. |
