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复杂环境样品中细菌、真菌和古菌DNA回收方法的评估

Assessment of methods to recover DNA from bacteria, fungi and archaea in complex environmental samples.

作者信息

Guillén-Navarro Karina, Herrera-López David, López-Chávez Mariana Y, Cancino-Gómez Máximo, Reyes-Reyes Ana L

机构信息

Laboratorio de Biotecnología Ambiental y Agroecológica, El Colegio de la Frontera Sur, CONACYT, Carretera Antiguo Aeropuerto Km 2.5, C.P. 30700, Tapachula, Chiapas, Mexico.

Programa de Bioenergía, Instituto Nacional de Investigaciones Forestales Agrícolas y Pecuarias, Campo Experimental Rosario Izapa, Km 18 Carretera Tapachula-Cacahoatán, Tuxtla Chico, Chiapas, Mexico.

出版信息

Folia Microbiol (Praha). 2015 Nov;60(6):551-8. doi: 10.1007/s12223-015-0403-1. Epub 2015 May 28.

Abstract

DNA extraction from environmental samples is a critical step for metagenomic analysis to study microbial communities, including those considered uncultivable. Nevertheless, obtaining good quality DNA in sufficient quantities for downstream methodologies is not always possible, and it depends on the complexity and stability of each ecosystem, which could be more problematic for samples from tropical regions because those ecosystems are less stable and more complex. Three laboratory methods for the extraction of nucleic acids from samples representing unstable (decaying coffee pulp and mangrove sediments) and relatively stable (compost and soil) environments were tested. The results were compared with those obtained using two commercial DNA extraction kits. The quality of the extracted DNA was evaluated by PCR amplification to verify the recovery of bacterial, archaeal, and fungal genetic material. The laboratory method that gave the best results used a lysis procedure combining physical, chemical, and enzymatic steps.

摘要

从环境样本中提取DNA是宏基因组分析研究微生物群落(包括那些被认为不可培养的群落)的关键步骤。然而,要获得足够数量的高质量DNA用于下游方法并不总是可行的,这取决于每个生态系统的复杂性和稳定性,对于来自热带地区的样本来说可能更成问题,因为这些生态系统不太稳定且更复杂。测试了三种从代表不稳定环境(腐烂咖啡果肉和红树林沉积物)和相对稳定环境(堆肥和土壤)的样本中提取核酸的实验室方法。将结果与使用两种商业DNA提取试剂盒获得的结果进行了比较。通过PCR扩增评估提取的DNA质量,以验证细菌、古菌和真菌遗传物质的回收率。效果最佳的实验室方法采用了结合物理、化学和酶促步骤的裂解程序。

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