Comparison of commercial DNA extraction kits for isolation and purification of bacterial and eukaryotic DNA from PAH-contaminated soils

Molecular characterization of the microbial populations of soils and sediments contaminated with polycyclic aromatic hydrocarbons (PAHs) is often a first step in assessing intrinsic biodegradation potential. However, soils are problematic for molecular analysis owing to the presence of organic matter, such as humic acids. Furthermore, the presence of contaminants, such as PAHs, can cause further challenges to DNA extraction, quantification, and amplification. The goal of our study was to compare the effectiveness of four commercial soil DNA extraction kits (UltraClean Soil DNA Isolation kit, PowerSoil DNA Isolation kit, PowerMax Soil DNA Isolation kit, and FastDNA SPIN kit) to extract pure, high-quality bacterial and eukaryotic DNA from PAH-contaminated soils. Six different contaminated soils were used to determine if there were any biases among the kits due to soil properties or level of contamination. Extracted DNA was used as a template for bacterial 16S rDNA and eukaryotic 18S rDNA amplifications, and PCR products were subsequently analyzed using denaturing gel gradient electrophoresis (DGGE). We found that the FastDNA SPIN kit provided significantly higher DNA yields for all soils; however, it also resulted in the highest levels of humic acid contamination. Soil texture and organic carbon content of the soil did not affect the DNA yield of any kit. Moreover, a liquid-liquid extraction of the DNA extracts found no residual PAHs, indicating that all kits were effective at removing contaminants in the extraction process. Although the PowerSoil DNA Isolation kit gave relatively low DNA yields, it provided the highest quality DNA based on successful amplification of both bacterial and eukaryotic DNA for all six soils. DGGE fingerprints among the kits were dramatically different for both bacterial and eukaryotic DNA. The PowerSoil DNA Isolation kit revealed multiple bands for each soil and provided the most consistent DGGE profiles among replicates for both bacterial and eukaryotic DNA.     

Biases during DNA extraction of activated sludge samples revealed by high throughput sequencing

Standardization of DNA extraction is a fundamental issue of fidelity and comparability in investigations of environmental microbial communities. Commercial kits for soil or feces are often adopted for studies of activated sludge because of a lack of specific kits, but they have never been evaluated regarding their effectiveness and potential biases based on high throughput sequencing. In this study, seven common DNA extraction kits were evaluated, based on not only yield/purity but also sequencing results, using two activated sludge samples (two sub-samples each, i.e. ethanol-fixed and fresh, as-is). The results indicate that the bead-beating step is necessary for DNA extraction from activated sludge. The two kits without the bead-beating step yielded very low amounts of DNA, and the least abundant operational taxonomic units (OTUs), and significantly underestimated the Gram-positive Actinobacteria, Nitrospirae, Chloroflexi, and Alphaproteobacteria and overestimated Gammaproteobacteria, Deltaproteobacteria, Bacteroidetes, and the rare phyla whose cell walls might have been readily broken. Among the other five kits, FastDNA^@ SPIN Kit for Soil extracted the most and the purest DNA. Although the number of total OTUs obtained using this kit was not the highest, the abundant OTUs and abundance of Actinobacteria demonstrated its efficiency. The three MoBio kits and one ZR kit produced fair results, but had a relatively low DNA yield and/or less Actinobacteria-related sequences. Moreover, the 50 % ethanol fixation increased the DNA yield, but did not change the sequenced microbial community in a significant way. Based on the present study, the FastDNA SPIN kit for Soil is recommended for DNA extraction of activated sludge samples. More importantly, the selection of the DNA extraction kit must be done carefully if the samples contain dominant lysing-resistant groups, such as Actinobacteria and Nitrospirae.     

Evaluation and optimization of nucleic acid extraction methods for the molecular analysis of bacterial communities associated with corroded carbon steel

Different DNA and RNA extraction approaches were evaluated and protocols optimized on in situ corrosion products from carbon steel in marine environments. Protocols adapted from the PowerSoil DNA/RNA Isolation methods resulted in the best nucleic acid (NA) extraction performances (ie combining high NA yield, quality, purity, representativeness of microbial community and processing time efficiency). The PowerSoil RNA Isolation Kit was the only method which resulted in amplifiable RNA of good quality (ie intact 16S/23S rRNA). Sample homogenization and hot chemical (SDS) cell lysis combined with mechanical (bead-beating) lysis in presence of a DNA competitor (skim milk) contributed to improving substantially (around 23 times) the DNA yield of the PowerSoil DNA Isolation Kit. Apart from presenting NA extraction strategies for optimizing extraction parameters with corrosion samples from carbon steel, this study proposes DNA and RNA extraction procedures suited for comparative molecular analysis of total and active fractions of bacterial communities associated with carbon steel corrosion events, thereby contributing to improved MIC diagnosis and control.     

Evaluation of five commercial nucleic acid extraction kits for their ability to inactivate Bacillus anthracis spores and comparison of DNA yields from spores and spiked environmental samples

This study evaluated five commercial extraction kits for their ability to recover DNA from Bacillus anthracis spores and spiked environmental samples. The kits evaluated represent the major types of methodologies which are commercially available for DNA or total nucleic acid extraction, and included the ChargeSwitch gDNA Mini Bacteria Kit, NucliSens Isolation Kit, Puregene Genomic DNA Purification Kit, QIAamp DNA Blood Mini Kit, and the UltraClean Microbial DNA Isolation Kit. Extraction methods were performed using the spores of eight virulent strains of B. anthracis. Viability testing of nucleic acid extracts showed that the UltraClean kit was the most efficient at depleting samples of live B. anthracis spores. TaqMan real-time PCR analysis revealed that the NucliSens, QIAamp and UltraClean kits yielded the best level of detection from spore suspensions. Comparisons of processed samples from spiked swabs and three powder types indicated that DNA extraction using the UltraClean kit resulted in the most consistently positive results and the lowest limit of detection. This study demonstrated that different nucleic extraction methodologies, represented here by various commercial extraction kits, differ in their ability to inactivate live B. anthracis spores as well as DNA yield and purity. In addition, the extraction method used can influence the sensitivity of real-time PCR assays for B. anthracis.     

Evaluation and optimization of nucleic acid extraction methods for the molecular analysis of bacterial communities associated with corroded carbon steel

Different DNA and RNA extraction approaches were evaluated and protocols optimized on in situ corrosion products from carbon steel in marine environments. Protocols adapted from the PowerSoil DNA/RNA Isolation methods resulted in the best nucleic acid (NA) extraction performances (ie combining high NA yield, quality, purity, representativeness of microbial community and processing time efficiency). The PowerSoil RNA Isolation Kit was the only method which resulted in amplifiable RNA of good quality (ie intact 16S/23S rRNA). Sample homogenization and hot chemical (SDS) cell lysis combined with mechanical (bead-beating) lysis in presence of a DNA competitor (skim milk) contributed to improving substantially (around 23 times) the DNA yield of the PowerSoil DNA Isolation Kit. Apart from presenting NA extraction strategies for optimizing extraction parameters with corrosion samples from carbon steel, this study proposes DNA and RNA extraction procedures suited for comparative molecular analysis of total and active fractions of bacterial communities associated with carbon steel corrosion events, thereby contributing to improved MIC diagnosis and control.     

Comparison of five commercial DNA extraction kits for the recovery of Yersinia pestis DNA from bacterial suspensions and spiked environmental samples

Aim:  To evaluate commercial DNA extraction kits for their ability to isolate DNA from Yersinia pestis suspensions and spiked environmental samples.
Methods and Results:  Five commercially available DNA extraction kits were evaluated: the ChargeSwitch gDNA Mini Bacteria Kit, the IT 1-2-3 Sample DNA Purification Kit, the MasterPure Complete DNA and RNA Purification Kit, the QIAamp DNA Blood Mini Kit and the UltraClean Microbial DNA Isolation Kit. The extraction methods were performed upon six Y. pestis strains and spiked environmental specimens, including three swab types and one powder type. Taqman real-time PCR analysis revealed that the use of the MasterPure kit resulted in DNA with the most consistently positive results and the lowest limit of detection from Y. pestis suspensions and spiked environmental samples.
Conclusion:  Comparative evaluations of the five commercial DNA extraction methods indicated that the MasterPure kit was superior for the isolation of PCR-amplifiable DNA from Y. pestis suspensions and spiked environmental samples.
Significance and Impact of the Study:  The results of this study can assist diagnostic laboratories with selecting the best extraction method for processing environmental specimens for subsequent detection of Y. pestis by real-time PCR.     

Detecting an elusive invasive species: a diagnostic PCR to detect Burmese python in Florida waters and an assessment of persistence of environmental DNA

Recent studies have demonstrated that detection of environmental DNA (eDNA) from aquatic vertebrates in water bodies is possible. The Burmese python, Python bivittatus, is a semi‐aquatic, invasive species in Florida where its elusive nature and cryptic coloration make its detection difficult. Our goal was to develop a diagnostic PCR to detect P. bivittatus from water‐borne eDNA, which could assist managers in monitoring this invasive species. First, we used captive P. bivittatus to determine whether reptilian DNA could be isolated and amplified from water samples. We also evaluated the efficacy of two DNA isolation methods and two DNA extraction kits commonly used in eDNA preparation. A fragment of the mitochondrial cytochrome b gene from P. bivittatus was detected in all water samples isolated with the sodium acetate precipitate and the QIAamp DNA Micro Kit. Next, we designed P. bivittatus‐specific primers and assessed the degradation rate of eDNA in water. Our primers did not amplify DNA from closely related species, and we found that P. bivittatus DNA was consistently detectable up to 96 h. Finally, we sampled water from six field sites in south Florida. Samples from five sites, where P. bivittatus has been observed, tested positive for eDNA. The final site was negative and had no prior documented evidence of P. bivittatus. This study shows P. bivittatus eDNA can be isolated from water samples; thus, this method is a new and promising technique for the management of invasive reptiles.     

Evaluating the effects of laboratory protocols on eDNA detection probability for an endangered freshwater fish

The effectiveness and accuracy of detection using environmental DNA (eDNA) is dependent on understanding the influence laboratory methods such as DNA extraction and PCR strategies have on detection probability. Ideally choice of sampling and extraction method will maximize eDNA yield and detection probability. Determining the survey effort required to reach a satisfactory detection probability (via increased PCR replicates or more sampling) could compensate for a lower eDNA yield if the sampling and extraction method has other advantages for a study, species or system. I analysed the effect of three different sampling and extraction methods on eDNA yield, detection probability and PCR replication for detecting the endangered freshwater fish Macquaria australasica from water samples. The impact of eDNA concentration, PCR strategy, target amplicon size and two marker regions: 12S (a mitochondrial gene) and 18S (a nuclear gene) was also assessed. The choice of sampling and extraction method and PCR strategy, rather than amplicon size and marker region, had the biggest effect on detection probability and PCR replication. The PCR replication effort required to achieve a detection probability of 0.95, ranged from 2 to 6 PCR replicates depending on the laboratory method used. As all methods yielded eDNA from which M. australasica was detected using the three target amplicons, differences in eDNA yield and detection probability between the three methods could be mitigated by determining the appropriate PCR replication effort. Evaluating the effect sampling and extraction methods will have on the detection probability and determining the laboratory protocols and PCR replication required to maximize detection and minimize false positives and negatives is a useful first step for eDNA occupancy studies.     

Convenient determination of DNA extraction efficiency using an external DNA recovery standard and quantitative-competitive PCR

Molecular biology techniques have advanced the field of microbial ecology through the analysis of nucleic acids. Most techniques that use DNA or RNA require their extraction from environmental matrices, which can be tedious and inefficient. While a number of extraction methods, both laboratory-based and commercially available, have been developed, none of these include a convenient method to determine extraction efficiency. We have developed an external DNA recovery standard, Lambda DNA (target DNA) contained within pBR322, allowing routine determinations of DNA recovery efficiency. Target DNA was added to sediments as whole cells, total DNA extracted using commercial DNA extraction/purification kits and the amount of target DNA recovered quantified by quantitative-competitive PCR (QC-PCR). Three commercially available kits (UltraClean Soil DNA, FastDNA SPIN and Soil Master DNA Extraction) were evaluated for recovery efficiency. Recoveries for the three kits ranged from undetectable to 43.3% with average recoveries of 14.9+/-16.0%, 28.3+/-10.5% and 2.4+/-0.1% (UltraClean, FastDNA and Soil Master, respectively). Quantification of target DNA proved robust in sediments heavily polluted with polycyclic aromatic hydrocarbons and the external recovery standard could be detected following extraction and amplification from as few as 1 x 10(3) cells added to 0.5 g sediment (wet weight). The external DNA recovery standard was also added directly to the sediment as purified plasmid DNA prior to extraction. It was recovered with similar efficiency as when added as whole cells, suggesting its usefulness in estimating DNA recovery in ribosomal DNA studies. These results show that, while the commercial kits offer expedited sample processing, the extraction efficiencies vary on a sample-by-sample basis and were <100%. Therefore, quantitative DNA studies require an estimation of DNA recovery.     

Comparison of eight methods for the extraction of Bacillus atrophaeus spore DNA from eleven common interferents and a common swab

Eight DNA extraction products or methods (Applied Biosystems PrepFiler Forensic DNA Extraction Kit; Bio-Rad Instagene Only, Bio-Rad Instagene & Spin Column Purification; EpiCentre MasterPure DNA & RNA Kit; FujiFilm QuickGene Mini80; Idaho Technologies 1-2-3 Q-Flow Kit; MoBio UltraClean Microbial DNA Isolation Kit; Sigma Extract-N-Amp Plant and Seed Kit) were adapted to facilitate extraction of DNA under BSL3 containment conditions. DNA was extracted from 12 common interferents or sample types, spiked with spores of Bacillus atropheaus. Resulting extracts were tested by real-time PCR. No one method was the best, in terms of DNA extraction, across all sample types. Statistical analysis indicated that the PrepFiler method was the best method from six dry powders (baking, biological washing, milk, plain flour, filler and talcum) and one solid (Underarm deodorant), the UltraClean method was the best from four liquids (aftershave, cola, nutrient broth, vinegar), and the MasterPure method was the best from the swab sample type. The best overall method, in terms of DNA extraction, across all sample types evaluated was the UltraClean method.     

Effects of water pH and proteinase K treatment on the yield of environmental DNA from water samples

Environmental DNA (eDNA) analysis has recently been applied to the study of aquatic macroorganisms. In most studies, sample water was filtered and the extracted DNA from the residues on the filter used for the following molecular analysis to detect species of interest. This quick, new biomonitoring method has received broad attention, but some unknowns remain, such as the eDNA yield in relation to water quality. Previous studies suggest that eDNA is composed of various forms, such as the free-floating naked form and in organelles and cells. Therefore, the eDNA yield in the filtration and extraction steps might change depending on the composition of eDNA. Especially the filtration efficiency of free-floating DNA would be affected by the electrical effect of water pH. In this study, not only the free-floating naked DNA, but also all DNA fragments released from the organisms and contained in the water were defined as eDNA, including cells and organelles. We examined (1) the effect of water pH on the eDNA yield at filtration and (2) the effect of proteinase K treatment on the extraction efficiency of DNA from filter samples, with consideration of the variety of the eDNA forms in water. In a laboratory experiment using the purified DNA of common carp (Cyprinus carpio carpio) spiked into ultrapure water, the water pH and DNA yield showed a negative relationship within the pH range of 5–9, that is, the DNA yield was higher in acidic conditions, plausibly because of pH-dependent adsorption onto the glass fiber filter at the filtration step. In case the field water contained eDNA derived from the inhabiting common carp and the purified DNA of ayu (Plecoglossus altivelis altivelis) spiked in the sample as an internal standard, adjustment of the pH to 5 prior to filtration did not increase the eDNA yield of common carp, and the spiked ayu DNA was not detected at all. During the DNA extraction step, a standard protocol including proteinase K treatment marked higher DNA yield than that without proteinase K treatment. Overall, the present results indicate successful collection of eDNA using filters without any special attention to the pH of the sample water, and a conventional protocol with proteinase K treatment is appropriate for eDNA recovery.     

Development of Procedures for Direct Extraction of Cryptosporidium DNA from Water Concentrates and for Relief of PCR Inhibitors

Extraction of high-quality DNA is a key step in PCR detection of Cryptosporidium and other pathogens in environmental samples. Currently, Cryptosporidium oocysts in water samples have to be purified from water concentrates before DNA is extracted. This study compared the effectiveness of six DNA extraction methods (DNA extraction with the QIAamp DNA minikit after oocyst purification with immunomagnetic separation and direct DNA extraction methods using the FastDNA SPIN kit for soil, QIAamp DNA stool minikit, UltraClean soil kit, or QIAamp DNA minikit and the traditional phenol-chloroform technique) for the detection of Cryptosporidium with oocyst-seeded samples, DNA-spiked samples, and field water samples. The study also evaluated the effects of different PCR facilitators (nonacetylated bovine serum albumin, the T4 gene 32 protein, and polyvinylpyrrolidone) and treatments (the use of GeneReleaser or ultrafiltration) for the relief from or removal of inhibitors of PCR amplification. The results of seeding and spiking studies showed that PCR inhibitors were presented in all DNA solutions extracted by the six methods. However, the effect of PCR inhibitors could be relieved significantly by the addition of 400 ng of bovine serum albumin/μl or 25 ng of T4 gene 32 protein/μl to the PCR mixture. With the inclusion of bovine serum albumin in the PCR mixture, DNA extracted with the FastDNA SPIN kit for soil without oocyst isolation resulted in PCR performance similar to that produced by the QIAamp DNA minikit after oocysts were purified by immunomagnetic separation.     

An evaluation of commercial DNA extraction kits for the isolation of bacterial spore DNA from soil

Aims: To evaluate six commercial DNA extraction kits for their ability to isolate PCR‐quality DNA from Bacillus spores in various soil samples. Methods and Results: Three soils were inoculated with various amounts of Bacillus cereus spores to simulate an outbreak or intentional release of the threat agent Bacillus anthracis. DNA was isolated from soil samples using six commercial DNA extraction kits. Extraction and purification efficiencies were assessed using a duplex real‐time PCR assay that included an internal positive control. The FastDNA^® SPIN kit for Soil showed the highest DNA extraction yield, while the E.Z.N.A.^® Soil DNA and PowerSoil^® DNA Isolation kits showed the highest efficiencies in removing PCR inhibitors from loam soil extracts. Conclusions: The results of this study suggest that commercially available extraction kits can be used to extract PCR‐quality DNA from bacterial spores in soil. The selection of an appropriate extraction kit should depend on the characteristics of the soil sample and the intended downstream application. Significance and Impact of the Study: The results of this study aid in the selection of an appropriate DNA extraction kit for a given soil sample. Its application could expedite sample processing for real‐time PCR detection of a pathogen in soil.     

Efficient isolation method for high‐quality genomic DNA from cicada exuviae

In recent years, animal ethics issues have led researchers to explore nondestructive methods to access materials for genetic studies. Cicada exuviae are among those materials because they are cast skins that individuals left after molt and are easily collected. In this study, we aim to identify the most efficient extraction method to obtain high quantity and quality of DNA from cicada exuviae. We compared relative DNA yield and purity of six extraction protocols, including both manual protocols and available commercial kits, extracting from four different exoskeleton parts. Furthermore, amplification and sequencing of genomic DNA were evaluated in terms of availability of sequencing sequence at the expected genomic size. Both the choice of protocol and exuvia part significantly affected DNA yield and purity. Only samples that were extracted using the PowerSoil DNA Isolation kit generated gel bands of expected size as well as successful sequencing results. The failed attempts to extract DNA using other protocols could be partially explained by a low DNA yield from cicada exuviae and partly by contamination with humic acids that exist in the soil where cicada nymphs reside before emergence, as shown by spectroscopic measurements. Genomic DNA extracted from cicada exuviae could provide valuable information for species identification, allowing the investigation of genetic diversity across consecutive broods, or spatiotemporal variation among various populations. Consequently, we hope to provide a simple method to acquire pure genomic DNA applicable for multiple research purposes.     

以川陕哲罗鲑为目标物种的水样环境DNA分析流程的优化

水样环境DNA分析包括水样采集、DNA提取和分析等流程,已成为监测濒危水生生物种群分布调查的重要手段.为减少在监测目标物种尤其濒危物种中的不确定性,对水环境DNA分析流程的优化至关重要.本研究以川陕哲罗鲑为目标物种,采用滤膜法采集养殖池中的水样,设计了 250 mL、500 mL、1 L和2 L等4种水样采集量,分别采用 PoweWater DNA Isolation kit和DNeasy Tissue and Blood DNA extraction kit 提取水样环境DNA(eDNA),使用物种mtDNA D_loop区特异性引物进行PCR扩增,通过研究滤膜法、水样采集量和水样DNA提取方法对水样eDNA中目标基因检出率的影响,探索适宜的eDNA分析操作方案.结果表明: 使用DNeasy Tissue and Blood DNA extraction kit提取的水样DNA中目的基因的检出率为100%,效果明显优于PoweWater DNA Isolation kit(目标基因的检出率为0);目标基因扩增条带的亮度随水样采样量的增加而增加,其中2 L水样目标基因的扩增效果较理想;序列比对结果显示,本试验从水样DNA中成功扩增得到了川陕哲罗鲑mtDNA Dloop区部分序列.表明DNA提取方法和水样采集量对目标物种的检出率有显著的影响,滤膜法、2 L水样采集量、DNeasy Tissue and Blood DNA extraction kit更适宜进行水样的DNA分析,mtDNA D-loop区可作为川陕哲罗鲑识别的特异性分子标记.     

A practical guide to sample preservation and pre‐PCR processing of aquatic environmental DNA

Environmental DNA (eDNA) is rapidly growing in popularity as a tool for community assessments and species detection. While eDNA approaches are now widely applied, there is not yet agreement on best practices for sample collection and processing. Investigators looking to integrate eDNA approaches into their research programme are required to examine a growing collection of disparate studies to make an often uncertain decision about which protocols best fit their needs. To promote the application of eDNA approaches and to encourage the generation of high‐quality data, here we review the most common techniques for the collection, preservation and extraction of metazoan eDNA from water samples. Specifically, we focus on experimental studies that compare various methods and outline the numerous challenges associated with eDNA. While the diverse applications of eDNA do not lend themselves to a one‐size‐fits‐all recommendation, in most cases, capture/concentration of eDNA on cellulose nitrate filters (with pore size determined by water turbidity), followed by storage of filters in Longmire's buffer and extraction with a DNeasy Blood & Tissue Kit (or similar) has been shown to provide sufficient, high‐quality DNA. However, we also emphasize the importance of testing and optimizing protocols for the system of interest.     

Comparison of Manual and Automatic (MagNA Pure) Isolation Methods of Total RNA from Adipose Tissue

Aim Comparison of manual and automatic (MagNA Pure) isolation methods of total RNA from adipose tissue with respect to its quality and recovery factor. Material 120 human subcutaneous adipose tissue samples (about 100 mg/sample) were collected from patients during surgical operations. The tissue sample was stabilized in RNAlater (QIAGEN GmbH, Germany). Methods Total RNA was extracted by the following kits: Rneasy Protect Mini, Rneasy Lipid Tissue (QIAGEN GmbH, Germany) and MagNA Pure Compact RNA Isolation (Tissue) for MagNA Pure Compact Instrument (Roche Diagnostics GmbH, Germany). Results The average RNA yields with Rneasy Lipid Tissue kits were about two-fold higher in comparison with the Rneasy Protect Mini kit. When the MagNA Pure Compact System was used, RNA yields from the same sample were more uniform compared with manual systems. It was also more convenient and less time-consuming than the manual approach. No DNA contamination of total RNA samples was detected except for samples isolated by Rneasy Protect Mini Kit. Conclusion Rneasy Lipid Tissue Kit and MagNA Pure Compact RNA Isolation Kit (Tissue) provide RNA samples of high quantity, purity and PCR amplificability. RNA samples are suitable for further processing using methods of molecular biology.     

Comparative analysis of microbial DNA extraction protocols for groundwater samples

A comparative analysis of four different DNA extraction protocols was performed to determine the best choice for groundwater microbial diversity studies using temperature gradient gel electrophoresis (TGGE) analysis. The methods used were a chelex-based method, a modified salting out procedure (MSOP), and the commercial kits Epicentre and FastDNA. Both commercial kits exhibited the greatest reproducibility in their methods; however, their band patterns were very different. The protocol that showed the highest diversity was the chelex-based method, and the one that showed the lowest diversity was the FastDNA kit.     

Evaluation of DNA extraction methods for Bacillus anthracis spores isolated from spiked food samples

Aims

Nine commercial DNA extraction kits were evaluated for the isolation of DNA from 10‐fold serial dilutions of Bacillus anthracis spores using quantitative real‐time PCR (qPCR). The three kits determined by qPCR to yield the most sensitive and consistent detection (Epicenter MasterPure Gram Positive; MoBio PowerFood; ABI PrepSeq) were subsequently tested for their ability to isolate DNA from trace amounts of B. anthracis spores (approx. 6·5 × 10¹ and 1·3 × 10² CFU in 25 ml or 50 g of food sample) spiked into complex food samples including apple juice, ham, whole milk and bagged salad and recovered with immunomagnetic separation (IMS).

Methods and Results

The MasterPure kit effectively and consistently isolated DNA from low amounts of B. anthracis spores captured from food samples. Detection was achieved from apple juice, ham, whole milk and bagged salad from as few as 65 ± 14, 68 ± 8, 66 ± 4 and 52 ± 16 CFU, respectively, and IMS samples were demonstrated to be free of PCR inhibitors.

Conclusions

Detection of B. anthracis spores isolated from food by IMS differs substantially between commercial DNA extraction kits; however, sensitive results can be obtained with the MasterPure Gram Positive kit.

Significance and Impact of the Study

The extraction protocol identified herein combined with IMS is novel for B. anthracis and allows detection of low levels of B. anthracis spores from contaminated food samples.     

Development of procedures for direct extraction of Cryptosporidium DNA from water concentrates and for relief of PCR inhibitors

Extraction of high-quality DNA is a key step in PCR detection of Cryptosporidium and other pathogens in environmental samples. Currently, Cryptosporidium oocysts in water samples have to be purified from water concentrates before DNA is extracted. This study compared the effectiveness of six DNA extraction methods (DNA extraction with the QIAamp DNA minikit after oocyst purification with immunomagnetic separation and direct DNA extraction methods using the FastDNA SPIN kit for soil, QIAamp DNA stool minikit, UltraClean soil kit, or QIAamp DNA minikit and the traditional phenol-chloroform technique) for the detection of Cryptosporidium with oocyst-seeded samples, DNA-spiked samples, and field water samples. The study also evaluated the effects of different PCR facilitators (nonacetylated bovine serum albumin, the T4 gene 32 protein, and polyvinylpyrrolidone) and treatments (the use of GeneReleaser or ultrafiltration) for the relief from or removal of inhibitors of PCR amplification. The results of seeding and spiking studies showed that PCR inhibitors were presented in all DNA solutions extracted by the six methods. However, the effect of PCR inhibitors could be relieved significantly by the addition of 400 ng of bovine serum albumin/mul or 25 ng of T4 gene 32 protein/mul to the PCR mixture. With the inclusion of bovine serum albumin in the PCR mixture, DNA extracted with the FastDNA SPIN kit for soil without oocyst isolation resulted in PCR performance similar to that produced by the QIAamp DNA minikit after oocysts were purified by immunomagnetic separation.