Use of Pressure Cycling Technology for Cell Lysis and Recovery of Bacterial and Fungal Communities from Soil

Abstract

Selection of cell lysis methodology is critical to microbial community analyses due to the inability of any single extraction technology to recover the absolute genetic structure from environmental samples. Numerous methodologies are currently applied to interrogate soil communities, each with its own inherent bias. Here we compared the efficacy and bias of three physical cell lysis methods in conjunction with the PowerLyzer PowerSoil DNA Isolation Kit (MO BIO) for direct DNA extraction from soil: bead-beating, vortex disruption, and hydrostatic pressure cycling technology (PCT). PCT lysis, which is relatively new to soil DNA extraction, was optimized for soils of two different textures prior to comparison with traditional bead-beating and vortex disruption lysis. All cell lysis methods successfully recovered DNA. Although the two traditional mechanical lysis methods yielded greater genomic, bacterial, and fungal DNA per gram soil than the PCT method, the latter resulted in a greater number of unique terminal restriction fragments by terminal RFLP (T-RFLP) analysis. These findings indicate the importance of diversity and quantity measures when assessing DNA extraction bias, as soil DNA retrieved by PCT lysis represented populations not found using traditional mechanical lysis methods.

New horizons in developing cell lysis methods: A review

“…They compared the results to other conventional DNA extraction methods. It was shown that conventional methods yielded more DNA compared to PCT, but when it comes to bacterial diversity detection, PCT resulted in more diversity of extracted DNA (Bruner et al, 2015).…”

“…Rapid changes in pressure lead to the destabilization of the molecular interactions inside the membrane's lipid bilayer which results in cell lysis (Kalchayanand et al, 2002). Some studies have (Bruner et al, 2015). heat-insulated high-pressure line.…”

The Requirement of Genetic Diagnostic Technologies for Environmental Surveillance of Antimicrobial Resistance

“…Significant efforts have focused on developing unbiased general procedures [ 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 ], and associated devices [ 87 , 88 ]. The International Organization for Standardization has developed a standard (ISO standard 11063) for DNA extraction and analysis [ 89 , 90 , 91 , 92 ], that has been revised in 2020, to facilitate the comparison of results from geographically widespread measurements across continents and borders.…”

Aportes Y Dificultades De La Metagenómica De Suelos Y Su Impacto en La Agricultura.

“…En cuanto a las mezclas utilizadas en lisis química, se pueden categorizar en mezclas que contienen detergentes y cloruro de sodio (NaCl) y mezclas que contienen diversos tampones. En el caso de lisis enzimática se ha utilizado lizosima, proteinasa K, acromopeptidasa y pronasa E (Bruner et al, 2015;Wagner et al, 2015;Kashi, 2016;Mazziotti et al, 2018). Otro punto importante es el pH del suelo, ya que en suelos arcillosos que poseen un pH bajo, durante el paso de lisis celular se favorece la absorción del ADN en la superficie de la arcilla provocando bajos rendimientos de ADN, ahora se sabe que en algunos casos se pueden obtener mejores rendimientos de ADN utilizando nuevas formulaciones de buffer (solución tampón) (Narayan et al, 2016).…”