Rapid DNA extraction protocol from soil for polymerase chain reaction‐mediated amplification

Abstract

A simple and rapid method of DNA extraction from soil was developed and DNA was made suitable for subsequent efficient amplification by the polymerase chain reaction (PCR). Key features of the extraction and purification were cold lysozyme-and SDS-assisted lysis with either freezing-thawing or bead beating, cold phenol extraction of the resulting soil suspension, CsCl and KAc precipitation and, finally, spermine-HC1 or glass milk purification of DNA. Crude DNA preparations contained 4-20 p g DNA per g of soil extracted, and at least 50% of this was recovered in the final purified DNA preparations. The resulting DNA was pure enough to be restricted by various enzymes, and was amplifiable at concentrations of up to 20 ng of soil-derived DNA per 50 p1 reaction mix.Amplification of a 683 bp target sequence, put, was performed with different Taq DNA polymerases. Application of the protocol enabled us to detect target DNA derived from roughly lo3 introduced Pseudomonaspuorescens (RP4 : : put) cfu per g of soil. The fate of an introduced population in the soil could be followed to this limit with PCR-assisted detection of target DNA. In addition, target DNA was detected in soil 5 months after release, when the introduced organism was no longer detectable on selective agar plates.The extraction and purification protocol applied to various different soil types resulted in DNA of sufficient purity to permit amplification by PCR.

Abattoir sources of psychrophilic clostridia causing blown pack spoilage of vacuum-packed chilled meats determined by culture-based and molecular detection procedures

“…It is possible that the failure to amplify Cl. gasigenes-specific 16S rDNA fragment during the initial test was due to interference of PCR-inhibiting soil components, such as humic acids (Ogram et al 1987;Smalla et al 1993).…”

Diversity of Fungi, Bacteria, and Actinomycetes on Leaves Decomposing in a Stream

“…in plant litter in soil, while Wohl and McArthur (73) documented the presence of a suite of actinomycete genera from submersed macrophytes. This is not surprising, considering the importance of this bacterial group in the transformation of organic matter (58,59).…”

“…Actinomycetes are a group of gram-positive bacteria (Class Actinobacteria) characterized by a high GϩC content (12) that perform a wide array of important functions in various habitats (58), including contributing significantly to organic matter processing (59). Actinomycetes, like Actinokineospora (48), Streptomyces, Nocardiodes, Pseudonocardia, Nocardia, and Micromonospora (73), have been found on decomposing plant litter.…”

Sobrevivência de Xanthomonas axonopodis pv. manihotis em manipueira sob condições ambientais

“…Morgan et al (1993), analisando a sobrevivência de A. salmonicida em água doce, também verificaram a presença de células não cultivá-veis em meio de cultura, mas detectáveis através da técnica de PCR. Smalla et al (1993), apesar de observarem a presença de DNA de Pseudomonas fluorescens em solo, após cinco meses da infestação, não conseguiram recuperar células cultiváveis em meio, o que demonstra a presença no meio de células bacterianas não cultiváveis e de DNA intacto e passível de detecção. Estas evidências demonstram a necessidade do emprego de um conjunto de méto-dos convencionais de isolamento com a detecção através de técnicas moleculares.…”