A simple and sensitive method to extract bacterial, yeast and fungal DNA from blood culture material

This study investigated the various commercially available kits and 'in-house' methods to extract DNA from Gram-negative and Gram-positive bacteria, yeast and fungal agents in commonly employed blood culture material. The main methods investigated were as follows; Qiagen QIAmp Blood kit, Roche high PCR template preparation kit, Puregene DNA extraction kit, boiling, glass beads/sonication and wash/alkali/heat lysis. The results indicated that a simple wash/alkali/heat lysis method was the most sensitive, reproducible, simple and cost-effective extraction method. This was the only method which removed any PCR inhibitors and inherent DNA which existed in virgin BacT/Alert aerobic, anaerobic and paediatric blood culture material. Contaminating microbial DNA from Lactococcus lactis or Bacillus coagulans was identified in all batches of BacT/Alert FAN aerobic blood culture material examined.     

Method evaluation of Fusarium DNA extraction from mycelia and wheat for down-stream real-time PCR quantification and correlation to mycotoxin levels

Identification of Fusarium species by traditional methods requires specific skill and experience and there is an increased interest for new molecular methods for identification and quantification of Fusarium from food and feed samples. Real-time PCR with probe technology (Taqman) can be used for the identification and quantification of several species of Fusarium from cereal grain samples. There are several critical steps that need to be considered when establishing a real-time PCR-based method for DNA quantification, including extraction of DNA from the samples. In this study, several DNA extraction methods were evaluated, including the DNeasy Plant Mini Spin Columns (Qiagen), the Bio robot EZ1 (Qiagen) with the DNeasy Blood and Tissue Kit (Qiagen), and the Fast-DNA Spin Kit for Soil (Qbiogene). Parameters such as DNA quality and stability, PCR inhibitors, and PCR efficiency were investigated. Our results showed that all methods gave good PCR efficiency (above 90%) and DNA stability whereas the DNeasy Plant Mini Spin Columns in combination with sonication gave the best results with respect to Fusarium DNA yield. The modified DNeasy Plant Mini Spin protocol was used to analyse 31 wheat samples for the presence of F. graminearum and F. culmorum. The DNA level of F. graminearum could be correlated to the level of DON (r(2) = 0.9) and ZEN (r(2) = 0.6) whereas no correlation was found between F. culmorum and DON/ZEA. This shows that F. graminearum and not F. culmorum, was the main producer of DON in Swedish wheat during 2006.     

Evaluation of methods for extracting Xylella fastidiosa DNA from the glassy-winged sharpshooter

The recent spread of the plant pathogenic bacterium Xylclla fastidiosa Wells et al. by an invasive vector species, Homalodisca coagulata Say, in southern California has resulted in new epidemics of Pierce's disease of grapevine. Our goal is to develop an efficient method to detect low titers of X. fastidiosa in H. coagulata that is amenable to large sample sizes for epidemiological studies. Detection of the plant pathogenic bacterium X. fastidiosa in its insect vector is complicated by low titers of bacteria, difficulty in releasing it from the insect mouthparts and foregut, and the presence of substances in the insect that inhibit polymerase chain reaction (PCr). To select the optimal protocol for DNA extraction to be used with PCR, we compared three standard methods and 11 commercially available kits for relative efficiency of X. fastidiosa DNA extraction in the presence of insect tissue. All of the protocols tested were proficient at extracting DNA from pure bacterial culture (1 x 10(5) cells), and all but one protocol successfully extracted sufficient bacterial DNA in the presence of insect tissue. Three DNA extraction techniques, immunomagnetic separation, the DNeasy Tissue kit (Qiagen, Hercules, CA), and Genomic DNA Purification kit (Fermentus, Hanover, MD), were compared more closely using a dilution series of X. fastidiosa (5000-0 cells) with and without insect tissue present. The DNeasy Tissue kit was the best kit tested, allowing detection of 5 x 10(3) X. fastidiosa cells with an insect head background.     

基于微滴式数字PCR方法的鱼类环境DNA样本处理与保存技术优化

以实验室内的鲫(Carassius auratus)为研究对象,利用微滴式数字PCR(Droplet Digital PCR,ddPCR)定量技术,优化了鱼类环境DNA(Environmental DNA,eDNA)样本的捕获、提取和保存方法,并对免DNA提取的PCR直扩技术进行了探索.研究结果如下:(1)在同一孔径、...     

Detection of Helicobacter pylori DNA in human feces by PCR: DNA stability and removal of inhibitors

In this study, the stability of Helicobacter pylori DNA in human feces and the effect of a diet lacking in plant material, the suspected source of PCR inhibitors in human feces, were investigated. In addition, a method to remove these inhibitors was developed. Stools inoculated with H. pylori were used as a model. For this purpose, a H. pylori suspension (10(8) CFU/ml) was used to spike stool samples obtained from four healthy adults known to be H. pylori negative. The evaluation of the stability of H. pylori DNA in feces showed that DNA was degraded after 3 days of contact with fecal material at 37 degrees C. A 2-day diet completely free of plant material was sufficient to eliminate PCR inhibitors from human feces. However, inhibitors were detected 48 h after a normal diet was resumed. A new technique consisting of agarose blocks containing embedded DNA as a template for PCR amplification was used for removal of inhibitors, following DNA extraction by a modified QIAamp tissue method (Qiagen, Hilden, Germany). When this method was applied to inhibiting stool samples known to have an inhibitory effect and spiked with H. pylori (5.10(8) CFU/g), a positive PCR was obtained showing that inhibitors present in the original DNA samples were completely removed. The agarose embedded DNA block method is highly efficient and provides clean, high quality template DNA for PCR purposes avoiding long and fastidious conventional extraction methods. In conclusion, this study confirms that H. pylori DNA degrades with time in stools. A diet free of plant material or a special DNA preparation can be used to remove inhibitors and to allow the detection of H. pylori.     

Simple and efficient cell disruption of extremely small quantities of mycelium of phytopathogenic mycotoxin-producing moulds for quantitative extraction of genomic DNA

DNA was efficiently and quantitatively isolated from extremely small quantities of mycelia (0.1–10 mg) of different phytopathogenic moulds by grinding freeze-dried mycelia with glass beads and then using a commercial DNA extraction kit. The efficiency of disruption of the mycelia and the quantitative DNA extraction was proved by microscopy and the quantification of isolated DNA by real time PCR. Presented at the 27^th Mykotoxin-Workshop, Dortmund, Germany, June 13–15, 2005 Financial support: German Research Foundation (DFG grant Pr 708/2). J.M. thanks the Cusanuswerk for a doctoral scholarship     

A rapid method for high throughput DNA extraction from plant material for PCR amplification

A rapid and reliable method is described for high throughput extraction of DNA from plant material using glass beads in a flat-bottomed microtitre plate. This procedure is quick, inexpensive, and allows up to 96 samples to be processed in parallel. PCR products produced by the recovered DNA are consistently equivalent to those produced through traditional extraction methods.     

Comparison of three methods of DNA extraction from cold-smoked salmon and impact of physical treatments

AIMS: To compare three bacterial DNA extraction procedures on cold-smoked salmon (CSS) and assess the impact on their efficiency of two physical treatments of the food matrix, ionizing irradiation and freezing. METHODS AND RESULTS: As molecular methods for bacterial detection have become an important analytical tool, we compared bacterial DNA extraction procedures on CSS. Working with frozen and irradiated CSS, we obtained negative responses from samples known to be highly contaminated. Thus, we decided to study the impact of these two physical treatments on bacterial DNA extraction procedures. The efficiency of bacterial DNA extraction directly from the fish matrix suspension was measured by an rpoB PCR-based reaction. The results demonstrated that the DNeasy tissue extraction kit (Qiagen, Courtaboeuf, France) was the most efficient and reproducible method. We also showed that freezing and ionizing irradiation have a negative impact on DNA extraction. This was found probably not to be due to inhibition as the PCR reaction remained negative after adding BSA to the PCR mix reaction. CONCLUSIONS: The extraction kit was the most efficient method. Physical treatments were shown to hamper bacterial DNA extraction. SIGNIFICANCE AND IMPACT OF THE STUDY: Attention must be paid to molecular bacterial detection on food products subject to freezing or to ionizing irradiation.     

A comparison of five methods for extraction of bacterial DNA from human faecal samples

The purity of DNA extracted from faecal samples is a key issue in the sensitivity and usefulness of biological analyses such as PCR for infectious pathogens and non-pathogens. We have compared the relative efficacy of extraction of bacterial DNA (both Gram negative and positive origin) from faeces using four commercial kits (FastDNA kit, Bio 101; Nucleospin C+T kit, Macherey-Nagal; Quantum Prep Aquapure Genomic DNA isolation kit, Bio-Rad; QIAamp DNA stool mini kit, Qiagen) and a non-commercial guanidium isothiocyanate/silica matrix method. Human faecal samples were spiked with additional known concentrations of Lactobacillus acidophilus or Bacteroides uniformis, the DNA was then extracted by each of the five methods, and tested in genus-specific PCRs. The Nucleospin method was the most sensitive procedure for the extraction of DNA from a pure bacterial culture of Gram-positive L. acidophilus (10(4) bacteria/PCR), and QIAamp and the guanidium method were most sensitive for cultures of Gram-negative B. uniformis (10(3) bacteria/PCR). However, for faecal samples, the QIAamp kit was the most effective extraction method and led to the detection of bacterial DNA over the greatest range of spike concentrations for both B. uniformis and L. acidophilus in primary PCR reactions. A difference in extraction efficacy was observed between faecal samples from different individuals. The use of appropriate DNA extraction kits or methods is critical for successful and valid PCR studies on clinical, experimental or environmental samples and we recommend that DNA extraction techniques are carefully selected with particular regard to the specimen type.     

Giardia intestinalis: DNA extraction approaches to improve PCR results

Difficulty in disrupting cysts of Giardia intestinalis, a cosmopolitan protozoan parasite, decreases the yield of DNA extracted and reduces the effectiveness of the polymerase chain reaction (PCR). To improve the detection of the Giardia Glutamate Dehydrogenase (gdh) gene, we re-evaluated the effects of deoxyribonucleic acid (DNA) extraction methods. Purified and concentrated cysts from 33 fecal samples were disrupted using conventional methods, and DNA extraction was conducted using two protocols: the QIAamp Stool Mini Kit and phenol/chloroform/isoamyl alcohol (PCI). PCR amplification was successful for 12 extracted DNA samples (36%) using PCI following a glass bead and freeze/thaw pretreatment and for all 33 samples (100%) using the QIAamp Stool Mini Kit following the aforementioned pretreatment. Consequently, the pretreatment of cysts with glass beads and freeze/thaw cycles followed by extraction of DNA with the QIAamp Stool Mini kit was the more effective protocol.     

Bryophyte DNA sequences from faeces of an arctic herbivore, barnacle goose (Branta leucopsis)

We tested DNA extraction methods and PCR conditions for the amplification of bryophyte DNA from barnacle goose (Branta leucopsis) faeces collected from Spitsbergen (Svalbard). Both the Qiagen stool kit and a silica-based extraction method received sufficient DNA from fresh and older droppings, as indicated by successful amplification of the plastid psbA-trnH spacer. Standard Taq polymerase outperformed two hot start polymerases. Sequencing of cloned PCR products revealed at least ten moss and two angiosperm sequences. This first example of identifying bryophyte DNA from faeces will allow analysing moss diets of arctic herbivores with a DNA barcoding approach.     

新生隐球菌总RNA抽提方法的实验研究

目的探讨快速获取高质量的新生隐球菌总RNA的实验方法。方法选取新生隐球菌的荚膜株、荚膜缺陷株,分别设计采用4种方法提取总RNA:酸洗玻璃珠法、液氮研磨法、异硫氰酸胍一步法、冷酸洗玻璃珠联合Yeast RNA kit法。用紫外线分光光度计测量其OD260、OD280的值,并且进行琼脂糖凝胶电泳,同时应用定量PCR法鉴定RNA质量。结果酸洗玻璃珠法、液氮研磨法、异硫氰酸胍一步法、冷酸洗玻璃珠联合Yeast RNA kit法的RNA产量分别为0.2μg/105细胞、0.4μg/105细胞、0.1μg/105细胞、0.6μg/105细胞。结论冷酸洗玻璃珠联合Yeast RNA kit法提取的RNA均一性和完整性最好,是简便、快捷地提取具有荚膜和细胞壁双重屏障的新生隐球菌RNA的理想方法。     

快速、高效的羊绒羊毛织品DNA提取方法的建立

目的：建立一种快速、高效的羊绒羊毛纺织品DNA提取的方法。方法：采用chelex-100法的3种处理、试剂盒法分别提取羊绒羊毛织品的DNA,用18S rDNA片段、山羊和绵羊源性成分PCR扩增结果来比较提取效果。结果：试剂盒法提取DNA的效果优于chelex-100法,整个提取过程约需2h。9种供试材料均提取到DNA,且含有山羊和/或绵羊源性成分,与显微镜观察结果的符合率为100%。结论：建立的试剂盒法是一种快速、高效的适用于羊绒羊毛织品DNA提取的方法,为应用分子生物学方法鉴别山羊绒和绵羊毛奠定了基础。     

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.     

Bacterial community analysis of activated sludge: an evaluation of four commonly used DNA extraction methods

The effectiveness of three commercially available direct DNA isolation kits (Mobio, Fast, Qiagen) and one published direct DNA extraction protocol (Bead) for extracting bacterial DNA from different types of activated sludge was investigated and mutually compared. The DNA quantity and purity were determined using real-time PCR targeting the bacterial 16S rDNA gene. Microbial community fingerprints were assessed by automated ribosomal intergenic spacer analysis. The resulting community profiles were analyzed with canonical correspondence analysis. Our results clearly demonstrate that direct DNA extraction methods can significantly influence the DNA quantity, purity, and observed community patterns of microbiota in activated sludge. Fast and Mobio generated high amounts of good quality DNA compared to Bead and Qiagen. Mobio also resulted in the detection of the highest number of species while Fast scored the best in discriminating between the community patterns of different activated sludge types. With respect to the characterization of community profiles, our analyses demonstrated a strong sludge type dependent variability among methods. Taking into account our results, we recommend Fast as the most suitable DNA extraction method for activated sludge samples used for bacterial community studies.     

Rapid and economic DNA extraction from a single salmon egg for real-time PCR amplification

Salmon eggs are common in Japanese sushi and other seafood products; however, certain fish eggs are used as counterfeit salmon eggs which are found in foods and processed products. This study develops a simple, rapid, and cost-effective method for DNA extraction, filtration (FT) and dilution (DL) protocols from a single salmon egg with good DNA quality for real-time PCR amplification. The DNA amount, DNA quality, and real-time PCR performance for different dilutions and different lengths of PCR amplicons were evaluated and compared with the common Qiagen tissue kit (QTK) and Chelex-100-based (CX) protocols. The extracted DNA from a single salmon egg using the FT or DL protocol can be applied in phylogenic research, food authentication and post-marketing monitoring of genetically modified (GM) food products.     

Selection and optimization of PCR-based methods for the detection of Histoplasma capsulatum var. capsulatum

BackgroundCurrent methods for the laboratory diagnosis of histoplasmosis are problematic in terms of their sensitivity, specificity and runtime.ObjectivesThus, in this study, we sought to select and optimize methods for the detection of Histoplasma capsulatum var. capsulatum by polymerase chain reaction (PCR).MethodsThree DNA extraction methods and three PCR methods were evaluated. We optimised the concentration of the components of this PCR reaction and determined its sensitivity and specificity using blood samples to which H. capsulatum had been added.ResultsThe DNA extraction method that yielded the highest-quality DNA used silica membranes (DNeasy Blood &; Tissue Kit, Qiagen, Hilden, Germany), and the amplification method with the best detection capacity used a target gene encoding a 100-kDa protein. Our optimisation of the PCR conditions indicated that the reaction works over a significant range of component concentrations; in addition, it was able to detect H. capsulatum better than traditional culture techniques, with a detection limit of only 10 pg of DNA.ConclusionsIn our experimental conditions, the PCR method selected in this work (instead of nested-PCR) is a tool sensitive enough for the diagnosis of histoplasmosis.     

Detection and typing of Helicobacter pylori cagA/vacA genes by radioactive,one-step polymerase chain reaction in stool samples from children

The detection and molecular typing of Helicobacter pylori virulence genes in human stool specimens by polymerase chain reaction (PCR) require an adequate amount of bacterial DNA and an appropriately adjusted PCR protocol. DNA was isolated from stool samples of 39 H. pylori-infected and nine uninfected Colombian children using the QIAamp Kit following the manufacturer's instructions but with modifications. DNA templates were amplified for the vacA s and m regions and for the cagA gene by PCR using radioactively labeled (32P) primers. The modifications in the standard Qiagen protocol of stool DNA extraction increased the final concentration of eluted total stool DNA 4.7 times (117 +/- 17 versus 22 +/- 3 ng/microl; P < 0.0001). Nevertheless, its amplification by regular PCR programs (30-40 cycles) did not generate visible signals because of the very low ratio of H. pylori DNA to other DNA. PCR for 80 cycles successfully amplified vacA in 36/39 samples (sensitivity, 92.3%) and cagA fragments in 21/39 (53.8%) fecal DNA samples. Both s and m vacA regions were amplified in 33/36 (91.7%) DNA samples. The s1m1 genotype was the most commonly isolated variant, accounting for 17/36 or 47.2% of positive samples. The s2m2 genotype was ascertained to be frequent also (14/36 or 38.9%). Almost all (94.1%) s1m1 genotypes were cagA positive. The majority of s2m2 genotypes (78.6%) were not associated with the cagA gene. Neither cagA nor vacA fragments were amplified from DNA isolates of H. pylori-uninfected children nor from DNA isolated from six gastrointestinal bacterial strains (specificity, 100%). The data suggest that the proposed modified technique of DNA extraction and PCR assay of stool samples may be an effective and reliable noninvasive tool for the detection and typing of H. pylori cagA/vacA virulence genes in infected individuals.     

Rapid and Economic DNA Extraction from a Single Salmon Egg for Real-Time PCR Amplification

Salmon eggs are common in Japanese sushi and other seafood products; however, certain fish eggs are used as counterfeit salmon eggs which are found in foods and processed products. This study develops a simple, rapid, and cost-effective method for DNA extraction, filtration (FT) and dilution (DL) protocols from a single salmon egg with good DNA quality for real-time PCR amplification. The DNA amount, DNA quality, and real-time PCR performance for different dilutions and different lengths of PCR amplicons were evaluated and compared with the common Qiagen tissue kit (QTK) and Chelex-100-based (CX) protocols. The extracted DNA from a single salmon egg using the FT or DL protocol can be applied in phylogenic research, food authentication and post-marketing monitoring of genetically modified (GM) food products.     

Evaluation of 96-well high-throughput DNA extraction methods for 16S rRNA gene metabarcoding

Gaining meaningful insights into bacterial communities associated with animal hosts requires the provision of high-quality nucleic acids. Although many studies have compared DNA extraction methods for samples with low bacterial biomass (e.g. water) or specific PCR inhibitors (e.g. plants), DNA extraction bias in samples without inherent technical constraint (e.g. animal samples) has received little attention. Furthermore, there is an urgent need to identify a DNA extraction methods in a high-throughput format that decreases the cost and time for processing large numbers of samples. We here evaluated five DNA extraction protocols, using silica membrane-based spin columns and a 96-well microplate format and based on either mechanical or enzymatic lysis or a combination of both, using three bacterial mock communities and Illumina sequencing of the V4 region of the 16SrRNA gene. Our results showed that none of the DNA extraction methods fully eliminated bias associated with unequal lysis efficiencies. However, we identified a DNA extraction method with a lower bias for each mock community standard. Of these methods, those including an enzymatic lysis showed biases specific to some bacteria. Altogether, these results again demonstrate the importance of DNA extraction standardization to be able to compare the microbiome results of different samples. In this attempt, we advise for the use of the 96-well DNeasy Blood and Tissue kit (Qiagen) with a zirconia bead-beating procedure, which optimizes altogether the cost, handling time and bacteria-specific effects associated with enzymatic lysis.