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.     

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.     

Quantification of mitochondrial DNA copy number: Pre-analytical factors

Mitochondrial DNA (mtDNA) content is important for understanding many cellular processes. Several pre-analytical factors, from sample collection to DNA extraction can affect measurement of mtDNA copy number. In the present study, whole blood samples yielded a higher mtDNA copy number than buffy coat samples. mtDNA content is affected by the cell separation method used and the time between blood withdrawal and cell separation. Thus, reference values must be established with the same type of sample. As to the DNA isolation and purification method, the manual phenol method can give randomly false high values. The QIAamp DNA Mini Kit provided the most highly reproducible mtDNA/nDNA yield.     

红豆杉属植物三种不同总DNA提取方法的分析比较

红豆杉属植物均为濒危物种,也是国家一级保护植物.以红豆杉属植物叶片为材料,利用三种不同的DNA提取方法提取总DNA,用分光光度计和琼脂糖凝胶电泳方法检测所得总DNA的得率和纯度,用PCR扩增的方法检测所得总DNA的质量,并对三种不同提取方法的结果进行了比较分析.结果表明:CTAB法提取的DNA纯度和得率均较高,可直接用...     

沼气池污泥微生物总DNA提取方法的比较

沼气发酵系统是一个复杂的生态系统,其污泥微生物超过99%是不可培养的。为了优化沼气池纤维素的转化效率、沼气的产率和开展污泥微生物多样性研究,本研究采用化学裂解法、溶菌酶裂解法和QIAampDNA Stool Mini Kit提取了沼气池污泥样品中微生物的总DNA,对三种方法的DNA得率、纯度、大片段提取效果以及是否含有PCR反应抑制剂进行了研究,最后对16S rRNA基因V3区的扩增产物进行了PCR变性梯度凝胶电泳(PCR-DGGE)分析。与化学裂解法和QIAamp DNA Stool Mini Kit法相比,溶菌酶裂解法得到的DNA量大、片段长、片段分布广、PCR扩增效率高;同时PCR-DGGE图谱显示,溶菌酶裂解法可更好地展示沼气池污泥中微生物的多样性。该结果为进一步提高沼气池中纤维素的转化效率和沼气生产优势菌种的质和量打下了一定的前期基础。     

A comparison of five methods for DNA isolation from liver and rumen flukes to perform ITS-2+ amplification

Five different DNA isolation methods (4 commercial kits and a modification of phenol-chloroform method) were compared for the discrimination of adults of Fasciola hepatica and Dicrocoelium dendriticum (liver flukes), and Calicophoron daubneyi (rumen fluke) collected from sheep in southern Italy. The second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA) plus flanking 5.8S and 28S sequence (ITS-2+) was amplified by polymerase chain reaction (PCR) from serial diluted DNA templates (6 ng - 60 fg) of each fluke species. Overall, in terms of efficiency in detection limit, the best results were obtained either with phenol-chloroform purification or with QIAamp DNA Mini Kit (Qiagen), but using this latter method, rapid, safe and not expensive, an increased level of sensitivity sufficient to detect small amounts of target-DNA was achieved. In addition, electrophoresis analysis following PCR also showed that ITS-2+ could be useful as a genetic marker for the molecular identification of F. hepatica, D. dendriticum and C. daubneyi in definitive and intermediate hosts. Furthermore, for the first time, the ITS-2 sequence of D. dendriticum was defined.     

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 DNA extraction methods from mouse stomachs for the quantification of H. pylori by real-time PCR

Real-time PCR methods have recently been developed for the quantification of Helicobacter pylori from infected mouse stomachs. However, the extent to which results is affected by the efficiency of different methods of DNA extraction and the degree of inhibition of the subsequent PCR have largely been ignored. In this study, mouse stomachs were processed using two homogenisation methods: complete disruption using a blender and homogenisation by vortexing with glass beads. Each procedure was followed by DNA purification by three different protocols-two commercially available kits-Qiagen DNA Mini Tissue kit and Qiagen Stool Kit and a phenol-chloroform extraction method. PCR inhibition was assessed by screening for mouse DNA and for H. pylori DNA after spiking stomach extracts with H. pylori 16S rDNA. PCR inhibition was found to be lower in DNA samples prepared by vortexing and processed by column kits. Validation of procedures was performed by quantification of H. pylori DNA and mouse DNA in infected mouse stomachs. Homogenisation with glass beads followed by the Qiagen Tissue kit was found to be the most suitable protocol combining high extraction and detection efficiency of 16S rDNA in the presence of a mouse DNA background.     

Evaluation of three methods for effective extraction of DNA from human hair

In this paper we evaluate three different methods for extracting DNA from human hair i.e. the Chelex method, the QIAamp DNA Mini Kit method and the ISOHAIR method. Analysis of DNA prepared from dyed hairs with the ISOHAIR method suggested that the DNA extracts contained PCR inhibitors. On the other hand, few inhibition was observed when DNA from dyed hairs were extracted using the Chelex method and the QIAamp DNA Mini Kit method. In conclusion, the Chelex method is recommended for PCR experiments in view of its simplicity and cost-effectiveness. To assess the reliability of the Chelex method for the extraction of genomic DNA from both natural and dyed hair samples, minisatellite variant repeat (MVR)-polymerase chain reaction (PCR) patterns of Chelex-extracted DNA were compared using hairs (three natural black hairs and three dyed hairs) with buccal swabs from six individuals. Complete agreement was observed between hair and swab samples in each individual, proving the utility of the Chelex method.     

Comparison of DNA Extraction Kits for Detection of Burkholderia pseudomallei in Spiked Human Whole Blood Using Real-Time PCR

Burkholderia pseudomallei, the etiologic agent of melioidosis, is endemic in northern Australia and Southeast Asia and can cause severe septicemia that may lead to death in 20% to 50% of cases. Rapid detection of B. pseudomallei infection is crucial for timely treatment of septic patients. This study evaluated seven commercially available DNA extraction kits to determine the relative recovery of B. pseudomallei DNA from spiked EDTA-containing human whole blood. The evaluation included three manual kits: the QIAamp DNA Mini kit, the QIAamp DNA Blood Mini kit, and the High Pure PCR Template Preparation kit; and four automated systems: the MagNAPure LC using the DNA Isolation Kit I, the MagNAPure Compact using the Nucleic Acid Isolation Kit I, and the QIAcube using the QIAamp DNA Mini kit and the QIAamp DNA Blood Mini kit. Detection of B. pseudomallei DNA extracted by each kit was performed using the B. pseudomallei specific type III secretion real-time PCR (TTS1) assay. Crossing threshold (C_T) values were used to compare the limit of detection and reproducibility of each kit. This study also compared the DNA concentrations and DNA purity yielded for each kit. The following kits consistently yielded DNA that produced a detectable signal from blood spiked with 5.5×10⁴ colony forming units per mL: the High Pure PCR Template Preparation, QIAamp DNA Mini, MagNA Pure Compact, and the QIAcube running the QIAamp DNA Mini and QIAamp DNA Blood Mini kits. The High Pure PCR Template Preparation kit yielded the lowest limit of detection with spiked blood, but when this kit was used with blood from patients with confirmed cases of melioidosis, the bacteria was not reliably detected indicating blood may not be an optimal specimen.     

An effective method for isolation of DNA from pig faeces and comparison of five different methods

Polymerase chain reaction (PCR) detection of microorganism in faecal specimens is hampered by poor recovery of DNA and by the presence of PCR inhibitors. In this paper, we describe a new modified method for extracting PCR-quality microbial community DNA from pig faecal samples, which combines the pretreatment with polyformaldehyde, and subsequent DNA lysis in the presence of CTAB, salt, PVP, and beta-mercaptoethanol, followed by isolation of nucleic acids using chloroform (no phenol) based protocol. The method resulted in a 1.3- to 11-fold increase in DNA yield when compared to four other widely used methods. Genomic DNA extracted from all five methods was assessed by both agarose gel electrophoresis and polymerase chain reaction for amplification of 16S rDNA specific fragments. The results showed that the improved method represented a reproducible, simple, and rapid technique for routine DNA extraction from faecal specimens and was notably better than using the QIAamp DNA Stool Mini Kit.     

Rapid,High Quality DNA Isolation from Cypress (Cupressus sempervirens L.) Needles and Optimization of the RAPD Marker Technique

A protocol for extracting high quality DNA from cypress (Cupressus sempervirens L.), a gymnosperm of economic and ecological importance to the Mediterranean, is presented using the commercially supplied DNeasy^TM Plant Mini Kit (QIAGEN GmbH, Germany). Additional steps were introduced in the QIAGEN protocol to significantly enhance DNA quantity and quality. For one sample, the procedure can be completed in less than one hour, and more than 10 samples can be processed in a day. DNA yield and purity were monitored by gel electrophoresis and by determining absorbance at UV (A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀). Both ratios were between 1.7 and 2.0, indicating that the presence of contaminating metabolites was minimal. The average DNA yield obtained from 100 mg starting material was around 22 g, which compares very favorably with numbers indicated by the manufacturer. Additionally, restriction and PCR analyses of the extracted DNA showed its compatibility with downstream applications. Using this DNA, the parameters for the randomly amplified polymorphic DNA (RAPD) protocol were optimized based on the use of (1) an AmpliTaq^® DNA Polymerase, Stoffel fragment (Perkin-Elmer), and (2) a high initial denaturation temperature (97.5 °C). Reproducible amplification products were achieved in all PCR reactions. Seven to eight bands per primer were obtained with most individuals. This represents a number at the high end of published results with other plant species.     

狗獾粪便DNA提取方法的初步探讨

在京杭大运河扬州段堤坝上采集狗獾的新鲜粪便,采用预处理-酚/氯仿抽提法、NaCl改良法、异硫氰酸胍裂解法、淀粉吸附法及QIAamp试剂盒法5种方法对粪便样品中狗獾DNA进行提取,探讨狗獾粪便样品中DNA提取方法和优化条件。结果表明,在5种提取方法中,淀粉吸附法的效果明显优于其它4种方法。采用粪便裂解液快速裂解细胞后,加入淀粉去除其中的大量PCR抑制物,然后用蛋白酶K裂解、酚/氯仿/异戊醇抽提,最后使用UNIQ-10柱纯化粪便DNA,对线粒体控制区和微卫星位点的PCR扩增反应及测序结果证实该方法的可行性。以上结果表明,通过该方法获得的粪便DNA能够用于更深入的分子遗传学等学科的研究。     

An effective method for isolation of DNA from pig faeces and comparison of five different methods

Polymerase chain reaction (PCR) detection of microorganism in faecal specimens is hampered by poor recovery of DNA and by the presence of PCR inhibitors. In this paper, we describe a new modified method for extracting PCR-quality microbial community DNA from pig faecal samples, which combines the pretreatment with polyformaldehyde, and subsequent DNA lysis in the presence of CTAB, salt, PVP, and β-mercaptoethanol, followed by isolation of nucleic acids using chloroform (no phenol) based protocol. The method resulted in a 1.3- to 11-fold increase in DNA yield when compared to four other widely used methods. Genomic DNA extracted from all five methods was assessed by both agarose gel electrophoresis and polymerase chain reaction for amplification of 16S rDNA specific fragments. The results showed that the improved method represented a reproducible, simple, and rapid technique for routine DNA extraction from faecal specimens and was notably better than using the QIAamp^® DNA Stool Mini Kit.     

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.     

Multiplex real-time PCR assay for simultaneous detection of Omphalotus guepiniformis and Lentinula edodes

A rapid multiplex real-time PCR assay was developed to achieve highly specific, simultaneous detection of two kinds of mushrooms, Omphalotus guepiniformis and Lentinula edodes. Primers and TaqMan minor groove binder probes were designed according to the internal transcribed spacers 1-5.8S region of rDNA and evaluated by the specificity for fruiting bodies of 17 O. guepiniformis, 16 L. edodes and samples from 57 other species. DNA extracts of all the target species had positive signals with no cross-reaction, the limit of detection being 0.00025 ng of DNA. Threshold cycle (Ct) values for raw and processed fruiting bodies and for fruiting bodies (1% (w/w)) mixed with foodstuffs or artificial gastric juice contents ranged from 17.16 to 26.60 for both examined species. This new assay proved specific to the target species, highly sensitive, and applicable to processed food samples and gastric juice contents, making it useful for rapidly identifying O. guepiniformis and L. edodes.     

Genotyping of Plant and Animal Samples without Prior DNA Purification

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors.PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination^{1, 2}. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)^3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion³. Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required^2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues^6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size.     

DNA isolation of mucus from Salmo trutta (Linnaeus, 1758) and Thymallus thymallus (Linnaeus, 1758) as an alternative method to conventional fin‐clipping

The first objective of the actual project was to investigate if mucus from Salmo trutta (Linnaeus, 1758) and Thymallus thymallus (Linnaeus, 1758) is suitable for DNA extraction. In the second step, it was analyzed if the avoidance of environmentally hazardous chemicals could lead to sufficiently pure DNA isolation. Finally, it was examined if samples taken from different fish body parts yielded different grades of purity of the extracted DNA. Mucus was collected from 552 individuals of S. truttaand T. thymallusfrom 2012 to 2015. DNA was extracted with two different kits (E.Z.N.A.^® Insect DNA Kit and my‐Budget DNA Mini Kit) and two different swabs (my‐Budget roughened laminated sterile cotton swabs and common autoclaved cotton swabs). The results showed that the my‐Budget DNA Mini Kit in combination with the my‐Budget roughened laminated sterile cotton swabs was the most suitable method. It is a non‐destructive and swift technique that results in the concentration of DNA being high and pure.     

Optimized microRNA purification from TRIzol-treated plasma

Background

MicroRNAs (miRNAs) represent new and potentially informative diagnostic targets for disease detection and prognosis. However, little work exists documenting the effect of TRIzol, a common viral inactivation and nucleic acid extraction reagent, on miRNA purification. Here, we developed an optimized protocol for miRNA extraction from plasma samples by evaluating five different RNA extraction kits, TRIzol phase separation, purification additives, and initial plasma sample volume. This method was then used for downstream profiling of plasma miRNAs found in archived samples from one nonhuman primate (NHP) experimentally challenged with Ebola virus by the aerosol route.

Results

Comparison of real-time RT-PCR results for spiked-in and endogenous miRNA sequences determined extraction efficiencies from five different RNA purification kits. These experiments showed that 50 μL plasma processed using the QIAGEN miRNeasy Mini Kit with 5 μg of glycogen as a co-precipitant yielded the highest recovery of endogenous miRNAs. Using this optimized protocol, miRNAs from archived plasma samples of one rhesus macaque challenged with aerosolized Ebola virus was profiled using a targeted real-time PCR array. A total of 519 of the 752 unique miRNAs assayed were present in the plasma samples at day 0 and day 7 (time of death) post-exposure. Statistical analyses revealed 25 sequences significantly up- or down-regulated between day 0 and day 7 post infection, validating the utility of the extraction method for plasma miRNA profiling.

Conclusions

This study contributes to the knowledgebase of circulating miRNA extraction methods and expands on the potential applications of cell-free miRNA profiling for diagnostics and pathogenesis studies. Specifically, we optimized an extraction protocol for miRNAs from TRIzol-inactivated plasma samples that can be used for highly pathogenic viruses.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1299-5) contains supplementary material, which is available to authorized users.     

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.