Soil pretreatment and fast cell lysis for direct polymerase chain reaction from forest soils for terminal restriction fragment length polymorphism analysis of fungal communities

Abstract

Humic substances in soil DNA samples can influence the assessment of microbial diversity and community composition. Using multiple steps during or after cell lysis adds expenses, is time-consuming, and causes DNA loss. A pretreatment of soil samples and a single step DNA extraction may improve experimental results. In order to optimize a protocol for obtaining high purity DNA from soil microbiota, five prewashing agents were compared in terms of their efficiency and effectiveness in removing soil contaminants. Residual contaminants were precipitated by adding 0.6 mL of 0.5 M CaCl2. Four cell lysis methods were applied to test their compatibility with the pretreatment (prewashing + Ca2+ flocculation) and to ultimately identify the optimal cell lysis method for analyzing fungal communities in forest soils. The results showed that pretreatment with TNP + Triton X-100 + skim milk (100 mM Tris, 100 mM Na4P2O7, 1% polyvinylpyrrolidone, 100 mM NaCl, 0.05% Triton X-100, 4% skim milk, pH 10.0) removed most soil humic contaminants. When the pretreatment was combined with Ca2+ flocculation, the purity of all soil DNA samples was further improved. DNA samples obtained by the fast glass bead-beating method (MethodFGB) had the highest purity. The resulting DNA was successfully used, without further purification steps, as a template for polymerase chain reaction targeting fungal internal transcribed spacer regions. The results obtained by terminal restriction fragment length polymorphism analysis indicated that the MethodFGB revealed greater fungal diversity and more distinctive community structure compared with the other methods tested. Our study provides a protocol for fungal cell lysis in soil, which is fast, convenient, and effective for analyzing fungal communities in forest soils.

An improved method for extraction of microbial DNA from alkaline-saline soil

“…Therefore, A 260/230 ratios obtained using commercial kits are lower than those obtained using our proposed method. Pre-treatment of soil with phenol:chloroform-guanidine thiocynate or Triton X-100-skim milk during the DNA extraction has shown the improvement in DNA quality (Antony-Babu et al, 2013;Cheng et al, 2016). However, the implementation of any of those steps increase the time effort, reagents, and cost of the extraction technique.…”

Assessment of Common Factors Associated with Droplet Digital PCR (ddPCR) Quantification of Paratrichodorus allius in Soil

“…Therefore, the importance of purification in eliminating inhibitory substances from soil DNA samples is underscored [ 13 ]. Rapid DNA purification kits are available these days, but a significant portion of DNA is usually lost and DNA degradation or damage during the process is quite common during the process [ 48 ]. In our study as well, a decrease in DNA copy numbers was observed in purified samples compared to their respective crude samples, indicating a loss of DNA during the purification steps ( Figure 2 b).…”

Development of Loop-Mediated Isothermal Amplification Assay for Rapid Detection and Analysis of the Root-Knot Nematode Meloidogyne hapla in Soil

“…Humic acid, originating from clay particles, is one of the most important contaminants as it may co-purify with DNA. To overcome this problem, a pre-washing step of soil with specific buffers efficiently removes contaminants [38], but with a risk of DNA loss. Nonfat skim milk is included in several DNA extraction protocols for its known ability to compete with DNA for adsorbing sites on clay particles, which results in more efficient DNA extraction [37,39].…”