Devyatov Alexander A, Davydova Ekaterina E, Luparev Andrey R, Karseka Sofia A, Shuryaeva Anna K, Zagainova Angelica V, Shipulin German A
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.
Diagnostics (Basel). 2023 Jun 24;13(13):2156. doi: 10.3390/diagnostics13132156.
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 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 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 eggs. A bead beating procedure was sufficient to destroy the eggs, while the effects of enzymes and freeze-heat cycles did not lead to a significant destruction of the eggs or the release of 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.
将土壤传播的蠕虫（STH）分子诊断方法大规模引入临床实践的主要挑战之一是缺乏一种普遍认可的从粪便样本中分离寄生虫DNA的有效方法。在本研究中，我们评估了各种预处理程序对去除PCR抑制剂和从粪便中提取DNA效率的影响。我们评估了四种破坏方法（珠磨法、温度依赖性酶的作用、冻融循环和在裂解缓冲液中孵育）对虫卵完整性和DNA提取效率的影响。此外，我们评估了预洗涤和使用商业浓缩器对从受虫卵污染的粪便样本中提取DNA的影响。珠磨法足以破坏虫卵，而酶和冻融循环的作用并未导致虫卵的显著破坏或DNA的释放。不包括珠磨步骤的蠕虫DNA分离方案并非首选。使用商业浓缩器对粪便中的STH虫卵进行预浓缩并随后洗涤，可以显著提高STH DNA的产量并减少PCR抑制。
