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 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.     

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.     

Comparison of DNA extraction methods for human gut microbial community profiling

The human gut harbors a vast range of microbes that have significant impact on health and disease. Therefore, gut microbiome profiling holds promise for use in early diagnosis and precision medicine development. Accurate profiling of the highly complex gut microbiome requires DNA extraction methods that provide sufficient coverage of the original community as well as adequate quality and quantity. We tested nine different DNA extraction methods using three commercial kits (TianLong Stool DNA/RNA Extraction Kit (TS), QIAamp DNA Stool Mini Kit (QS), and QIAamp PowerFecal DNA Kit (QP)) with or without additional bead-beating step using manual or automated methods and compared them in terms of DNA extraction ability from human fecal sample. All methods produced DNA in sufficient concentration and quality for use in sequencing, and the samples were clustered according to the DNA extraction method. Inclusion of bead-beating step especially resulted in higher degrees of microbial diversity and had the greatest effect on gut microbiome composition. Among the samples subjected to bead-beating method, TS kit samples were more similar to QP kit samples than QS kit samples. Our results emphasize the importance of mechanical disruption step for a more comprehensive profiling of the human gut microbiome.     

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.     

PCR and real time PCR for the detection of Cryptosporidium parvum oocyst DNA

Three DNA extraction kits were used, all without preliminary procedures, then DNA extraction was preceded with freeze/thaw cycles in three versions. A lack of desired effect resulted in the application of liquid nitrogen/water bath cycles before the use of the extractions in further experiments. The effectiveness of DNA extraction was measured by PCR signal and C(T) values of real time PCR. A comparison of the efficiency of various Cryptosporidium parvum undiluted oocyst treatments prior to DNA extraction with the use of three kits has shown that the best results were obtained after extraction of DNA with the QIAamp DNA Tissue Mini Kit (T kit), preceded by triple liquid nitrogen/water bath in 100 degrees C for 2 minutes and with overnight proteinase K digestion. After extraction with the T kit, the detection limit was 50 oocysts per 200 microl when effectiveness was evaluated with PCR and 10 oocysts in the case of real time PCR.     

Evaluation of QIAamp DNA Stool Mini Kit for ecological studies of gut microbiota

Cell lysis efficiency and the quality of DNA extracts from complex bacterial ecosystems are two major concerns in molecular ecological studies of gut microbiota. In this study, we use PCR-denaturing gradient gel electrophoresis (DGGE) DNA profiling, random cloning and sequence analysis of 16S rRNA genes to compare the QIAamp DNA Stool Mini Kit with the bead beating technique in the preparation of DNA extracts from gut microbiota of pigs. We also developed a washing procedure that can release more than 93% of bacterial cells attached to the gut mucosa. Both the QIAamp kit and bead beating method lysed approximately 95% of bacterial cells. PCR-DGGE DNA profiles of ileal and cecal microbiota from both digesta and mucosa that were generated from the DNA extracts using the two methods were nearly identical. Random cloning and sequence analysis also demonstrated the high quality of DNA extracts using the two methods. Two random clone sets of 16S rRNA genes generated from the DNA extracts had a similar degree of bacterial diversity. Different preparations of DNA extract from a single sample using the QIAamp kit consistently produced similar PCR-DGGE DNA profiles with similarity indexes higher than 99%. Our data suggest the appropriateness of the QIAamp DNA Stool Mini Kit for the studies of gut microbial ecology and the effectiveness of the QIAamp kit in processing multiple samples for cell lysis and DNA extraction.     

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.     

Comparison of nucleic acid extraction platforms for detection of select biothreat agents for use in clinical resource limited settings

High-quality nucleic acids are critical for optimal PCR-based diagnostics and pathogen detection. Rapid sample processing time is important for the earliest administration of therapeutic and containment measures, especially in the case of biothreat agents. In this context, we compared the Fujifilm QuickGene-Mini80 to Qiagen's QIAamp Mini Purification kits for extraction of DNA and RNA for potential use in austere settings. Qiagen (QIAamp) column-based extraction is the currently recommended purification platform by United States Army Medical Research Institute for Infectious Diseases for both DNA and RNA extraction. However, this sample processing system requires dedicated laboratory equipment including a centrifuge. In this study, we investigated the QuickGene-Mini80, which does not require centrifugation, as a suitable platform for nucleic acid extraction for use in resource-limited locations. Quality of the sample extraction was evaluated using pathogen-specific, real-time PCR assays for nucleic acids extracted from viable and γ-irradiated Bacillus anthracis, Yersinia pestis, vaccinia virus, Venezuelan equine encephalitis virus, or B. anthracis spores in buffer or human whole blood. QuickGene-Mini80 and QIAamp performed similarly for DNA extraction regardless of organism viability. It was noteworthy that γ-irradiation did not have a significant impact on real-time PCR for organism detection. Comparison with QIAamp showed a less than adequate performance of the Fujifilm instrument for RNA extraction. However, QuickGene-Mini80 remains a viable alternative to QIAamp for DNA extraction for use in remote settings due to extraction quality, time efficiency, reduced instrument requirements, and ease of use.     

体重差异双胎新生儿 粪便样本DNA提取的优化及分析

摘要：目的 优化新生儿粪便样本DNA提取方法,提取及分析体重差异双胎新生儿粪便样本DNA。方法 从7种DNA提取试剂盒方法中选择对成人粪便样本DNA提取效果最佳的QIAamp DNA Stool Mini Kit法为基准方法,通过钢珠打断前处理、DNA吸附柱收集全部裂解液上清和洗脱液重复洗脱的优化,建立了用于新生儿粪便样本DNA的提取方法。结果 该优化方法用于婴儿（出生1个月）粪便样本,结果显示DNA提取浓度平均提高了2.4倍。用于48对双胞胎新生儿出生第1天和第3天粪便样本DNA的提取,经酶标仪及PCR扩增检测,结果显示出生第1天粪便样本DNA提取率为32%,出生3天提取率达83%。RT-PCR显示新生儿第1天到第3天肠道微生物量呈现增长趋势。结论 优化的QIAamp DNA Stool Mini Kit法适用于新生儿粪便样本DNA的快速提取,为后续扩增子高通量测序和研究体重差异双胎新生儿肠道菌群构成规律奠定基础。     

Pathogen quantitation in complex matrices: a multi-operator comparison of DNA extraction methods with a novel assessment of PCR inhibition

Background

Mycobacterium bovis is the aetiological agent of bovine tuberculosis (bTB), an important recrudescent zoonosis, significantly increasing in British herds in recent years. Wildlife reservoirs have been identified for this disease but the mode of transmission to cattle remains unclear. There is evidence that viable M. bovis cells can survive in soil and faeces for over a year.

Methodology/Principal Findings

We report a multi-operator blinded trial for a rigorous comparison of five DNA extraction methods from a variety of soil and faecal samples to assess recovery of M. bovis via real-time PCR detection. The methods included four commercial kits: the QIAamp Stool Mini kit with a pre-treatment step, the FastDNA® Spin kit, the UltraClean™ and PowerSoil™ soil kits and a published manual method based on phenol:chloroform purification, termed Griffiths. M. bovis BCG Pasteur spiked samples were extracted by four operators and evaluated using a specific real-time PCR assay. A novel inhibition control assay was used alongside spectrophotometric ratios to monitor the level of inhibitory compounds affecting PCR, DNA yield, and purity. There were statistically significant differences in M. bovis detection between methods of extraction and types of environmental samples; no significant differences were observed between operators. Processing times and costs were also evaluated. To improve M. bovis detection further, the two best performing methods, FastDNA® Spin kit and Griffiths, were optimised and the ABI TaqMan environmental PCR Master mix was adopted, leading to improved sensitivities.

Conclusions

M. bovis was successfully detected in all environmental samples; DNA extraction using FastDNA® Spin kit was the most sensitive method with highest recoveries from all soil types tested. For troublesome faecal samples, we have used and recommend an improved assay based on a reduced volume, resulting in detection limits of 4.25×10⁵ cells g⁻¹ using Griffiths and 4.25×10⁶ cells g⁻¹ using FastDNA® Spin kit.     

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.     

Practical Method for Extraction of PCR-Quality DNA from Environmental Soil Samples

Methods for the extraction of PCR-quality DNA from environmental soil samples by using pairs of commercially available kits were evaluated. Coxiella burnetii DNA was detected in spiked soil samples at <1,000 genome equivalents per gram of soil and in 12 (16.4%) of 73 environmental soil samples.The detection of pathogenic organisms in the environment often relies on PCR analysis of DNA purified from environmental soil (6). For effective detection, a reliable method to obtain PCR-quality DNA from soil is necessary. Although a variety of complex techniques have been effective for specific soil samples (1-3, 7, 8), it is not clear which methods would be the best for the wide variety of samples encountered in a large-scale environmental sampling study. In addition, many published techniques would be difficult to use on a large number of samples (1-3, 7, 8).This study evaluates the abilities of commercially available DNA extraction kits to provide DNA from environmental soil samples that are suitable for PCR detection of Coxiella burnetii. C. burnetii is an obligate intracellular, Gram-negative, zoonotic pathogen and the causative agent of Q fever (5). It is classified as a category B agent of bioterrorism by the CDC.Three commercially available DNA purification kits were evaluated. Twenty different soil samples obtained from diverse locations in the southeastern United States were used for testing. These samples consisted of light sandy soil and were all initially processed through one of three DNA purification kits, the UltraClean soil DNA isolation kit (MoBio Laboratories, Carlsbad CA), the QIAamp DNA minikit (Qiagen, Valencia, CA), or the QIAamp DNA stool minikit (Qiagen), or through a combination of two of the kits used sequentially. Thus, all 20 samples were each processed through nine extraction protocols. To process soil samples, five grams of soil was mixed with 10 to 30 ml of phosphate-buffered saline (PBS) to create a homogenized slurry. Samples were mixed for 1 h at room temperature and then centrifuged for 5 min at 123 × g. The supernatant was removed and centrifuged at 20,000 × g for 15 min. The supernatant was then carefully discarded and the pellet resuspended in 1 ml of PBS.For the UltraClean soil kit, 700 μl of the resuspended soil extraction pellet was processed by the manufacturer''s alternative protocol (for maximum yields). For preps done using the QIAamp DNA minikit (tissue protocol) and the QIAamp stool kit (stool protocol), 700 μl (high volume) of the soil extract was processed according to the instructions for the particular kit. For 17 of the samples the tissue protocol and stool protocol were applied using only 200 μl of the soil extract (low volume). For all of the kits, the final elutions were performed with 55 μl of water.To further purify the products of the commercial DNA isolation kits, eluates were passed through a second round of extraction. When the MoBio UltraClean kit was used for the second round of extraction, eluates were added to the bead-containing tubes and mixed with 60 μl of solution 1 and 200 μl of the MoBio inhibitor removal solution (IRS). The manufacturer''s protocol was then followed. When the QIAamp tissue protocol was utilized for the second round of extraction, eluates were diluted to 200 μl with water and then mixed with 200 μl of buffer ATL plus 200 μl of buffer AL and then incubated at 70°C for 10 min. Following this step, the manufacturer''s protocol was followed. When the QIAamp stool protocol was used for the second round of extraction, eluates were mixed with 1.2 ml of the ASL buffer, followed by addition of the InhibitEX tablet. The manufacturer''s protocol was then followed.PCR inhibition in all of the DNA samples was then evaluated by running a quantitative PCR that detects the IS1111 gene from C. burnetii (4). PCRs were run on 200 genome equivalents of C. burnetii (strain Nine Mile Phase 1) DNA. Reaction mixtures spiked with 1-μl aliquots of the environmental DNA samples were compared to reaction mixtures spiked with 1 μl of water. Inhibition was considered present if the DNA sample caused an increase of 1 in the threshold cycle value.Use of the MoBio UltraClean procedure by itself resulted in removal of inhibitors from 35% of the samples, whereas after use of the Qiagen tissue protocol (high volume) only 4% of the samples were free of inhibition (Fig. ​(Fig.1).1). The Qiagen stool kit (high volume) resulted in 96% of the samples showing lack of inhibition with a low volume of soil eluate and 62.5% of the samples when the high volume was used. The DNA extracted from these three kits was then used as starting material for a subsequent DNA extraction step using the same set of three commercial kits. The MoBio UltraClean kit followed by the Qiagen stool kit eliminated inhibition in all samples, as did these two kits when used in the reverse order, even if the Qiagen stool kit was loaded with 700 μl of material (high volume). When a low volume of starting material was used, combinations of the two Qiagen kits also removed inhibitors from 100% of the samples when either the Qiagen tissue protocol was used first or the Qiagen stool protocol was used first (Fig. ​(Fig.1).1). The raw data for all of the inhibition assays are included as supplemental data (see Table S1 in the supplemental material).Open in a separate windowFIG. 1.Twenty environmental soil samples were used for the isolation of DNA with the indicated protocols. The samples were then tested for the ability to inhibit an IS1111 PCR with C. burnetii Nine Mile DNA as template. The percentages of samples that did not show any inhibition are indicated.To determine the yield of DNA obtained by the various protocols, nine aliquots (5 g each) of a single rich organic soil sample were each mixed with 5 ml PBS, spiked with 1 × 10⁶ Nine Mile Phase 2 C. burnetii organisms, and then processed by the nine (high-volume) extraction protocols described above. An additional 1 × 10⁶ Nine Mile Phase 2 C. burnetii organisms were used directly in the Qiagen tissue protocol to prepare DNA for the purpose of determining the exact amount of C. burnetii input into the assays. The quantitative IS1111 PCR assay (4) was used to determine the yield of C. burnetii DNA by using the various methods for processing soil. The yield was calculated by dividing the number of genome equivalents of C. burnetii DNA obtained from the spiked soil samples by the number of genome equivalents obtained when C. burnetii was included directly in the Qiagen tissue protocol. A common feature of all of the protocols was that they all produced a low yield of C. burnetii DNA when purified from a complex soil mixture (Fig. ​(Fig.2).2). The yields ranged from 0.02% to 4.3% and were variable. Although the 4.3% yield obtained when the stool kit was used alone was the highest on average, the high variability observed with these extractions suggests that most of these protocols provide similar yields. The stool kit followed by the MoBio kit clearly resulted in the lowest yield.Open in a separate windowFIG. 2.Five-gram aliquots of a single soil sample were all spiked with approximately 1 × 10⁶ C. burnetii Phase 2 Nine Mile strain cells. The samples were then subjected to the indicated extraction protocol(s). The resulting DNA was tested for inhibition, and then the genome equivalents of C. burnetii DNA were determined by quantitative IS1111 PCR. The exact input amount of C. burnetii was determined by running an aliquot directly through the QIAamp tissue protocol followed by IS1111 PCR. Yield was calculated as genome equivalents obtained from the spiked soil samples divided by the genome equivalents obtained from the direct extraction through the QIAamp tissue protocol. Values represent the mean ± standard deviation of five experiments. Statistically significant differences (Student''s t test) were found between stool versus MoBio plus stool kits (P = 0.05), stool plus tissue versus MoBio plus stool kits (P = 0.01), and stool plus tissue versus tissue plus MoBio kits (P = 0.03). For the protocol using the stool kit followed by the MoBio kit the yield was significantly different from stool, stool plus tissue, MoBio plus tissue, and MoBio protocols (P < 0.05).Although these yields are low, the IS1111 PCR assay used to detect C. burnetii DNA amplifies a multicopy gene, and the assay can detect a single genome equivalent (4). This suggests that these protocols are adequate for the detection of C. burnetii in soil samples with 500 to 2,000 organisms per gram of soil. To test this, a 5-g sample of organic soil was spiked with 800 C. burnetii organisms per gram, and the DNA was extracted using the MoBio UltraClean kit followed by the QIAamp stool protocol. C. burnetii DNA was detected after 38 cycles using the IS1111 PCR assay.While these results are focused on soil samples, the procedures described also work well on vacuum samples and sponge wipe samples (data not shown). Based on removal of inhibitors and yield, our data suggest that the QIAamp tissue protocol (high volume) followed by the QIAamp stool protocol and the MoBio UltraClean kit followed by the QIAamp stool protocol are both suitable for extraction of DNA from environmental soil samples. To test the application of the latter method to a larger number of samples, 73 bulk soil samples from the southeastern United States were processed according to this method. Inhibition was removed from all 73 samples, and 12 of the samples were positive in the C. burnetii IS1111 PCR assay. This suggests that this practical method for extraction of PCR-quality DNA can be successfully used to detect DNA from C. burnetii and other pathogens in large numbers of environmental samples.      

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.     

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.     

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.     

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.     

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.     

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.     

On-line cell lysis of bacteria and its spores using a microfluidic biochip

Optimal detection of pathogens by molecular methods in water samples depends on the ability to extract DNA rapidly and efficiently. In this study, an innovative method was developed using a microfluidic biochip, produced by microelectrochemical system technology, and capable of performing online cell lysis and DNA extraction during a continuous flow process. On-chip cell lysis based on chemical/physical methods was performed by employing a sufficient blend of water with the lysing buffer. The efficiency of lysis with microfluidic biochip was compared with thermal lysis in Eppendorf tubes and with two commercial DNA extraction kits: Power Water DNA isolation kit and ForensicGEM Saliva isolation kit in parallel tests. Two lysing buffers containing 1% Triton X-100 or 5% Chelex were assessed for their lysis effectiveness on a microfluidic biochip. SYBR Green real-time PCR analysis revealed that cell lysis on a microfluidic biochip using 5% Chelex buffer provided better or comparable recovery of DNA than commercial isolation kits. The system yielded better results for Gram-positive bacteria than for Gram-negative bacteria and spores of Gram-positive bacteria, within the limits of detection at 10³ CFU/ml. During the continuous flow process in the system, rapid cells lysis with PCR-amplifiable genomic DNA were achieved within 20 minutes.