Method for purification of bacterial endospores from soils: UV resistance of natural Sonoran desert soil populations of Bacillus spp. with reference to B. subtilis strain 168

Endospores of Bacillus spp. were purified from three Sonoran desert soil samples by Chelex extraction and NaBr density gradient centrifugation and their UV resistances compared with that of B. subtilis strain 168. Natural spore populations exhibited tight adherence to soil particles which was not readily overcome by the extraction and purification procedure. It was observed that spores purified from soil exhibited 2-3 fold higher resistance to UV (as measured by the 90% lethal dose, LD90) than did B. subtilis strain 168 grown on NSM, a standard laboratory sporulation medium, and purified by the same extraction procedure. Cultivation of spore-forming bacteria isolated from soil on NSM resulted in production of spores with essentially identical UV resistance as strain 168, suggesting that spore UV resistance is influenced by the environment in which spores are produced.     

Extraction of DNA from soil using nanoparticles by magnetic bioseparation

Aims: To develop a simple, rapid and inexpensive soil DNA extraction protocol. Methods and Results: The protocol relies on the use of superparamagnetic silica‐magnetite nanoparticles for the isolation and purification of DNA from soil samples. DNA suitable for use in molecular biology applications was obtained from a number of soil samples. Conclusions: The DNA extracted using the tested method successfully permitted the PCR amplification of a fragment of the bacterial 16S rDNA gene. The extracted DNA could also be restriction endonuclease digested. Significance and Impact of the Study: The protocol reported here is simple and permits rapid isolation of PCR‐ready soil DNA. The method requires only small quantities of soil sample, is scalable and suitable for automation.     

A simple and reliable method for SSU rRNA gene DNA extraction, amplification, and cloning from single AM fungal spores

 We describe a method that allows quick and easy PCR amplification and cloning of nearly complete SSU rRNA genes from arbuscular mycorrhizal fungi. The procedure tested on spores from 37 different glomalean isolates was based on magnetic separation with Dynabeads, followed by nested PCR with two primer pairs. All trials led to visible amplification products of the expected size. Thereafter, the PCR fragments could be quickly and efficiently cloned by means of a topoisomerase-activated vector (pCR2.1-TOPO). The technique is rapid, uncomplicated and comparatively inexpensive. The use of single spores for DNA extraction has some advantages over multispore-preparations, e.g. it is less susceptible to contamination with other organisms present in the cultures. The method can be used for the quick and reliable preparation of a large number of samples and is highly reproducible. It could also be used for genes other than the SSU rRNA gene. Accepted: 25 October 2000     

Comparison of methods of extracting Salmonella enterica serovar Enteritidis DNA from environmental substrates and quantification of organisms by using a general internal procedural control

This paper compares five commercially available DNA extraction methods with respect to DNA extraction efficiency of Salmonella enterica serovar Enteritidis from soil, manure, and compost and uses an Escherichia coli strain harboring a plasmid expressing green fluorescent protein as a general internal procedural control. Inclusion of this general internal procedural control permitted more accurate quantification of extraction and amplification of S. enterica serovar Enteritidis in these samples and reduced the possibility of false negatives. With this protocol it was found that the optimal extraction method differed for soil (Mobio soil DNA extraction kit), manure (Bio101 soil DNA extraction kit), and compost (Mobio fecal DNA extraction kit). With each method, as little as 1.2 x 10(3) to 1.8 x 10(3) CFU of added serovar Enteritidis per 100 mg of substrate could be detected by direct DNA extraction and subsequent S. enterica-specific TaqMan PCR. After bacterial enrichment, as little as 1 CFU/100 mg of original substrate was detected. Finally, the study presents a more accurate molecular analysis for quantification of serovar Enteritidis initially present in soil or manure using DNA extraction and TaqMan PCR.     

Spore-forming bacteria in soil cultivated with GM white poplars: Isolation and characterization

The impact of transgenic white poplars (Populus alba L. cv. ‘Villafranca’) was assessed on the soil aerobic spore-forming bacteria (SFB). The genetically modified poplars, expressing either the StSy gene for resveratrol production or the bar gene for herbicide tolerance, were cultivated in greenhouse. The occurrence of SFB was monitored in soil samples collected at eight different timepoints over a two-year period. The total culturable bacterial population of the StSy and bar trials underwent significant seasonal fluctuations in the range of 10⁶−2.5 × 10⁸ CFU/g dry soil and of 10⁴−5 × 10⁸ CFU/g dry soil, respectively. Changes occurred also within the culturable SFB population with size varying at 10³−5 × 10⁴ CFU/g dry soil and 10²−2 × 10⁵ CFU/g dry soil in the StSy and bar trials, respectively. No significant differences in the size of the total and SFB culturable populations were observed when comparing each transgenic line with the nontransformed control line while seasonal shifts of soil bacterial populations were evident in both trials. The culturable SFB fraction included three isolates (SFB-1, SFB-2 and SFB-3) classified by 16S rDNA sequence analysis as members of the Bacillus genus. According to the reported data, cultivation of both herbicide-resistant and resveratrol-producing GM white poplars did not affect the culturable SFB population at the soil level.     

Microscale detection of specific bacterial DNA in soil with a magnetic capture-hybridization and PCR amplification assay.

A magnetic capture-hybridization PCR technique (MCH-PCR) was developed to eliminate the inhibitory effect of humic acids and other contaminants in PCRs targeting specific soil DNA. A single-stranded DNA probe, which was complementary to an internal part of the target gene, was used to coat magnetic beads. After hybridization in a suspension of soil DNA, magnetic extraction of the beads separated the hybrid DNA from all other soil DNA, humic acids, and other interfering soil components. The MCH was followed by PCR amplification of the specific target DNA. In barley rhizosphere soil, detection of a lux gene inserted in a Pseudomonas fluorescens strain could be demonstrated in nonsterile soil samples (0.5 mg). This corresponded to a detection of fewer than 40 bacterial cells per cm of barley root. The MCH-PCR technique greatly improves the current protocols for PCR detection of specific microorganisms or genes in soil because specific target DNA sequences from very small soil samples can be extracted and determined.     

DNA extraction and PCR detection of <Emphasis Type="Italic">Paenibacillus larvae</Emphasis> spores from naturally contaminated honey and bees using spore-decoating and freeze-thawing techniques

Summary Paenibacillus larvae causes American foulbrood (AFB), a severe disease that affects the brood of honey bee Apis mellifera. AFB is worldwide distributed and causes great economic losses to beekeepers, but in many cases early diagnosis could help in its prevention and control. The aim of the present work was to design a reliable protocol for DNA extraction of P. larvae spores from naturally contaminated honey and adult bees. A novel method that includes a step of spore-decoating followed by an enzymatic spore disruption and DNA purification was developed. Also a freeze-thaw cycle protocol was tested and the results were compared. The DNA extracted was used as template for specific bacterial detection by amplification of a 16S rDNA fragment. Both methods allowed the direct detection by polymerase chain reaction (PCR) of P. larvae spores present in naturally contaminated material. The spore-decoating strategy was the most successful method for DNA extraction from spores, allowing specific and remarkably sensitive PCR detection of spores in all honey and bees tested samples. On the other hand freeze-thawing was only effective for detection of spores recovered from bees, and extensive damage to DNA affected detection by PCR. This work provides new strategies for spore DNA extraction and detection by PCR with high sensitivity, and brings an alternative tool for P. larvae detection in natural samples.     

Usefulness of strb1 and 16S rDNA-targeted PCR for detection of Streptomyces spp. in environmental samples

In this study, we revealed rapid detection of streptomycin-producing Streptomyces spp. by extraction of total soil DNA from 14 soil samples using a modified lysis method followed by PCR amplification ofa genus-specific sequence in the Streptomyces' 16S rDNA gene. DNA band of the expected size (438 bp) was seen with all the samples. Additionally, specific amplification of the streptomycin-coding gene (strb1) directly from soil revealed the presence of a single DNA band of 940 bp. These results indicate that PCR-amplification of Streptomyces specific genes could be used for direct detection of streptomycin-producing Streptomyces species from soil.     

Real-time loop-mediated isothermal amplification assay for rapid and sensitive detection of anthrax spores in spiked soil and talcum powder

Loop-mediated isothermal amplification (LAMP) assay is a powerful and innovative gene amplification technique that specifically amplifies the target gene under isothermal conditions with a high degree of sensitivity, rapidity and specificity. The major advantage of the LAMP assay is monitoring of amplified products without the requirement of any sophisticated equipment. In the present study a real time LAMP assay was employed for rapid and real time detection of Bacillus anthracis spores spiked in 0.1 g of soil and talcum powder ranging from 2 to 10⁷ spores. DNA was isolated from spiked soil and talcum powder using PBS containing 1% Triton X-100, and heat treatment. Isolated DNA was used as template for LAMP and PCR. LAMP amplification was obtained in 60 min under isothermal condition at 63°C by employing a set of six primers targeting the pag gene of B. anthracis. The detection limit of LAMP assay in soil and talcum powder was found to be as low as 5 spores, compared to 10³ spores and 10⁴ spores by PCR in talcum powder and soil, respectively. The findings suggest that LAMP is a more rapid and sensitive assay than PCR for detecting anthrax spores, additionally the methodology to prepare DNA from spiked samples is simple, rapid and cost effective.     

Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to process difficult to amplify samples and low copy number sequences from natural environments

Microbial assessment of natural biodiversity is usually achieved through polymerase chain reaction (PCR) amplification. Deoxyribonucleic acid (DNA) sequences from natural samples are often difficult to amplify because of the presence of PCR inhibitors or to the low number of copies of specific sequences. In this study, we propose a non-specific preamplification procedure to overcome the presence of inhibitors and to increase the number of copies prior to carrying out standard amplification by PCR. The pre-PCR step is carried out through a multiple displacement amplification (MDA) technique using random hexamers as priming oligonucleotides and phi 29 DNA polymerase in an isothermal, whole-genome amplification reaction. Polymerase chain reaction amplification using specific priming oligonucleotides allows the selection of the sequences of interest after a preamplification reaction from complex environmental samples. The procedure (MDA-PCR) has been tested on a natural microbial community from a hypogean environment and laboratory assemblages of known bacterial species, in both cases targeting the small subunit ribosomal RNA gene sequences. Results from the natural community showed successful amplifications using the two steps protocol proposed in this study while standard, direct PCR amplification resulted in no amplification product. Amplifications from a laboratory assemblage by the two-step proposed protocol were successful at bacterial concentrations >or= 10-fold lower than standard PCR. Amplifications carried out in the presence of different concentrations of fulvic acids (a soil humic fraction) by the MDA-PCR protocol generated PCR products at concentrations of fulvic acids over 10-fold higher than standard PCR amplifications. The proposed procedure (MDA-PCR) opens the possibility of detecting sequences represented at very low copy numbers, to work with minute samples, as well as to reduce the negative effects on PCR amplifications of some inhibitory substances commonly found in environmental samples.     

Molecular detection of anthrax spores on animal fibres

AIMS: To develop a rapid, specific and sensitive diagnostic test for the detection of the spores of Bacillus anthracis on commercial samples of animal fibres (e.g. wool and cashmere). METHODS AND RESULTS: Extraction of DNA from spores using a mechanical disruption method based on bead beating was evaluated but subsequently abandoned as it compromised the sensitivity of the overall protocol. A multiplex PCR and two nested amplification reactions designed for B. anthracis were developed during this study. CONCLUSIONS: A simple selective incubation step in combination with multiplex PCR was found to be more effective than generic DNA extraction coupled to a sensitive nested amplification reaction. SIGNIFICANCE AND IMPACT OF THE STUDY: The rapid diagnostic test could be applied to the analysis of commercial fibre samples for the detection of anthrax as required by health and safety legislation resulting in considerable savings in time and expense.     

DNA sexing of Nipponia nippon by duplex polymerase chain reaction

The aim of this study was to develop a rapid, simple, sensitive, and accurate duplex polymerase chain reaction (PCR) to sex Nipponia nippon, a monomorphic bird. Amplification by duplex PCR of a sex-related gene on the female chromosome and the 12S rRNA gene yielded good results using genomic DNA extracted from a feather follicle or the membranes of eggshell samples. To simplify the DNA extraction procedure, a simple boiling method was used. Our simple boiling DNA extraction method produced similar PCR amplification results as when using DNA extracted using TRIzol. Sex determination in the endangered Nipponia nippon is of crucial value to breeding programs. The duplex PCR protocol that we developed provides a simple sex identification method that is based on amplification of a sex-related gene, and we anticipate that it will facilitate effective conservation and management of Nipponia nippon.     

Rapid extraction of DNA from diverse soils by guanidine thiocyanate method

Molecular methods are being frequently used for the study of soil microbial communities as majority of naturally occurring microbial populations are non-culturable. In the present study, we describe a protocol of DNA extraction from diverse soils using a combination of heat, enzyme (lysozyme) and guanidine thiocyanate. The efficacy of the procedure was evaluated in terms of yield, purity and duration of extraction. The protocol was effective for neutral, acidic as well as alkaline soils (pH range 4.5-8.5). The extracted soil DNA was observed with negligible shearing on agarose gel and the time taken for restriction digestion was very less. Further, the DNA extracted was almost completely devoid of contaminants and pure enough which could be used for PCR amplification and Southern hybridization.     

Comparison of Methods of Extracting Salmonella enterica Serovar Enteritidis DNA from Environmental Substrates and Quantification of Organisms by Using a General Internal Procedural Control

This paper compares five commercially available DNA extraction methods with respect to DNA extraction efficiency of Salmonella enterica serovar Enteritidis from soil, manure, and compost and uses an Escherichia coli strain harboring a plasmid expressing green fluorescent protein as a general internal procedural control. Inclusion of this general internal procedural control permitted more accurate quantification of extraction and amplification of S. enterica serovar Enteritidis in these samples and reduced the possibility of false negatives. With this protocol it was found that the optimal extraction method differed for soil (Mobio soil DNA extraction kit), manure (Bio101 soil DNA extraction kit), and compost (Mobio fecal DNA extraction kit). With each method, as little as 1.2 × 10³ to 1.8 × 10³ CFU of added serovar Enteritidis per 100 mg of substrate could be detected by direct DNA extraction and subsequent S. enterica-specific TaqMan PCR. After bacterial enrichment, as little as 1 CFU/100 mg of original substrate was detected. Finally, the study presents a more accurate molecular analysis for quantification of serovar Enteritidis initially present in soil or manure using DNA extraction and TaqMan PCR.     

A simple and efficient method for PCR amplifiable DNA extraction from ancient bones

A simple and effective modified ethanol precipitation-based protocol is described for the preparation of DNA from ancient human bones. This method is fast and requires neither hazardous chemicals nor special devices. After the powdering and incubating of the bone samples Dextran Blue was added as a carrier for removing the PCR inhibitors with selective ethanol precipitation. This method could eliminate the time-consuming separate decalcification step, dialysis, application of centrifugation-driven microconcentrators and the second consecutive PCR amplification. The efficiency of this procedure was demonstrated on ten 500–1200-year-old human bones from four different Hungarian burial sites. A mitochondrial specific primer pair was used to obtain sequence information from the purified ancient DNA. The PCR amplification, after our DNA extraction protocol, was successful from each of the 10 bone samples investigated. The results demonstrate that extraction of DNA from ancient bone samples with this new approach increases the success rate of PCR amplification.     

Detection of polychlorinated biphenyl degradation genes in polluted sediments by direct DNA extraction and polymerase chain reaction.

It was the aim of this study to specifically detect the DNA sequences for the bphC gene, the meta-cleavage enzyme of the aerobic catabolic pathway for biphenyl and polychlorinated biphenyl degradation, in aquatic sediments without prior cultivation of microorganisms by using extraction of total DNA, PCR amplification of bphC sequences, and detection with specific gene probes. The direct DNA extraction protocol used was modified to enhance lysis efficiency. Crude extracts of DNA were further purified by gel filtration, which yielded DNA that could be used for the PCR. PCR primers were designed for conserved regions of the bphC gene from a sequence alignment of five known sequences. The specificity of PCR amplification was verified by using digoxigenin-labeled DNA probes which were located internal to the amplified gene sequence. The detection limit for the bphC gene of Pseudomonas paucimobilis Q1 and Pseudomonas sp. strain LB400 was 100 cells per g (wet weight) or approximately five copies of the target sequence per PCR reaction mixture. In total-DNA extracts of aerobic top layers of sediment samples obtained from three different sampling sites along the Elbe River, which has a long history of anthropogenic pollution, Pseudomonas sp. strain LB 400-like sequences for the bphC gene were detected, but P. paucimobilis Q1 sequences were not detected. No bphC sequences were detected in an unpolluted lake sediment. A restriction analysis did not reveal any heterogeneity in the PCR product, and the possibility that sequences highly related to the bphC gene (namely, nahC and todE) were present was excluded.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extraction of High Quality DNA from Seized Moroccan Cannabis Resin (Hashish)

The extraction and purification of nucleic acids is the first step in most molecular biology analysis techniques. The objective of this work is to obtain highly purified nucleic acids derived from Cannabis sativa resin seizure in order to conduct a DNA typing method for the individualization of cannabis resin samples. To obtain highly purified nucleic acids from cannabis resin (Hashish) free from contaminants that cause inhibition of PCR reaction, we have tested two protocols: the CTAB protocol of Wagner and a CTAB protocol described by Somma (2004) adapted for difficult matrix. We obtained high quality genomic DNA from 8 cannabis resin seizures using the adapted protocol. DNA extracted by the Wagner CTAB protocol failed to give polymerase chain reaction (PCR) amplification of tetrahydrocannabinolic acid (THCA) synthase coding gene. However, the extracted DNA by the second protocol permits amplification of THCA synthase coding gene using different sets of primers as assessed by PCR. We describe here for the first time the possibility of DNA extraction from (Hashish) resin derived from Cannabis sativa. This allows the use of DNA molecular tests under special forensic circumstances.     

Improved high-throughput sunflower and cotton genomic DNA extraction and PCR fidelity

The extraction of high-quality genomic DNA for PCR amplification from sunflower (Helianthus annuus) and cotton (Gossypium spp.) is challenging because of the presence of polysaccharides, secondary metabolites, and polyphenolics in the tissues. A high-throughput DNA extraction protocol was needed in our laboratory for simple sequence repeats (SSR)-marker screening and other molecular analyses that do not require organic extraction steps of phenol or chloroform. Here we describe 2 improved highthroughput protocols for DNA extraction and in-PCR modification that result in successful PCR amplification of sunflower and cotton. While the sunflower DNA extraction protocol uses reducing agents such as sodium metabisulfite and dithiothreitol (DTT), the cotton protocol uses polyvinylpyrrolidone (PVP) in PCR reactions and reducing agents in the DNA extraction procedure.     

Small-Scale DNA Sample Preparation Method for Field PCR Detection of Microbial Cells and Spores in Soil

Efficient, nonselective methods to obtain DNA from the environment are needed for rapid and thorough analysis of introduced microorganisms in environmental samples and for analysis of microbial community diversity in soil. A small-scale procedure to rapidly extract and purify DNA from soils was developed for in-the-field use. Amounts of DNA released from bacterial vegetative cells, bacterial endospores, and fungal conidia were compared by using hot-detergent treatment, freeze-thaw cycles, and bead mill homogenization. Combining a hot-detergent treatment with bead mill homogenization gave the highest DNA yields from all three microbial cell types and provided DNA from the broadest range of microbial groups in a natural soil community. Only the bead mill homogenization step was effective for DNA extraction from Bacillus globigii (B. subtilis subsp. niger) endospores or Fusarium moniliforme conidia. The hot-detergent–bead mill procedure was simplified and miniaturized. By using this procedure and small-scale, field-adapted purification and quantification procedures, DNA was prepared from four different soils seeded with Pseudomonas putida cells or B. globigii spores. In a New Mexico soil, seeded bacterial targets were detected with the same sensitivity as when assaying pure bacterial DNA (2 to 20 target gene copies in a PCR mixture). The detection limit of P. putida cells and B. globigii spores in different soils was affected by the amount of background DNA in the soil samples, the physical condition of the DNA, and the amount of DNA template used in the PCR.