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.     

More than skin and bones: Comparing extraction methods and alternative sources of DNA from avian museum specimens

Next‐generation sequencing has greatly expanded the utility and value of museum collections by revealing specimens as genomic resources. As the field of museum genomics grows, so does the need for extraction methods that maximize DNA yields. For avian museum specimens, the established method of extracting DNA from toe pads works well for most specimens. However, for some specimens, especially those of birds that are very small or very large, toe pads can be a poor source of DNA. In this study, we apply two DNA extraction methods (phenol–chloroform and silica column) to three different sources of DNA (toe pad, skin punch and bone) from 10 historical avian museum specimens. We show that a modified phenol–chloroform protocol yielded significantly more DNA than a silica column protocol (e.g., Qiagen DNeasy Blood & Tissue Kit) across all tissue types. However, extractions using the silica column protocol contained longer fragments on average than those using the phenol–chloroform protocol, probably as a result of loss of small fragments through the silica column. While toe pads yielded more DNA than skin punches and bone fragments, skin punches proved to be a reliable alternative source of DNA and might be especially appealing when toe pad extractions are impractical. Overall, we found that historical bird museum specimens contain substantial amounts of DNA for genomic studies under most extraction scenarios, but that a phenol–chloroform protocol consistently provides the high quantities of DNA required for most current genomic protocols.     

Evaluation of six methods for extraction and purification of viral DNA from urine and serum samples

The sensitivity and reliability of PCR for diagnostic and research purposes require efficient unbiased procedures of extraction and purification of nucleic acids. One of the major limitations of PCR-based tests is the inhibition of the amplification process by substances present in clinical samples. This study used specimens spiked with a known amount of plasmid pBKV (ATCC 33-1) to compare six methods for extraction and purification of viral DNA from urine and serum samples based on recovery efficiency in terms of yield of DNA and percentage of plasmid pBKV recovered, purity of extracted DNA, and percentage of inhibition. The most effective extraction methods were the phenol/chloroform technique and the silica gel extraction procedure for urine and serum samples, respectively. Considering DNA purity, the silica gel extraction procedure and the phenol/chloroform method produced the most satisfactory results in urine and serum samples, respectively. The presence of inhibitors was overcome by all DNA extraction techniques in urine samples, as evidenced by semiquantitative PCR amplification. In serum samples, the lysis method and the proteinase K procedure did not completely overcome the presence of inhibitors.     

A comparison of DNA extraction and purification methods to detect Escherichia coli O157:H7 in cattle manure

The extraction of DNA from manure and the subsequent polymerase chain reaction (PCR) amplification of virulence genes to detect pathogens require an effective method of purification. Four different methods were assessed for their effectiveness in extracting and purifying Escherichia coli O157:H7 DNA from cattle manure: phenol/chloroform purification, phenol/chloroform/Sepharose B4 spin columns, phenol/chloroform/polyvinylpolypyrrolidone (PVPP) spun columns, and Mo Bio UltraClean kit. A PCR assay targeting the shiga-like toxin I gene (sltI) was carried out to determine the effectiveness of the four methods in removing PCR inhibitors from the manure samples. All methods were used to extract a manure slurry and the cleanliness of the samples was tested by the PCR with varying concentrations of spiked E. coli O157:H7 target DNA. The PVPP spun columns and the UltraClean kit had the best detection limit, detecting 20 pg of E. coli DNA (about 2x10(3) cells) per 100 mg of manure. The UltraClean kit and the PVPP spun columns also had the best and similar detection limits of 3x10(4) CFU/100 mg manure when E. coli O157:H7 cells were spiked into the manure sample and purified by all four methods. The enrichment of cells after inoculation into manure was performed using tryptic soy broth at 37 degrees C for 5 h. Both the PVPP spun columns and the UltraClean kit methods were used to purify the enriched samples and were able to detect initial inocula of 6 CFU/100 mg manure, indicating that the two methods were highly efficient in purifying DNA from manure samples.     

Developmentally regulated synthesis of an unusually small, basic peptide by Coxiella burnetii

Coxiella burnetii undergoes a poorly defined developmental cycle within phagolysosomes of eukaryotic host cells. Two distinct developmental forms are part of this cycle: a small-cell variant (SCV) and large-cell variant (LCV). Ultrastructurally, the SCV is distinguished from the LCV by its smaller size and condensed chromatin. At a molecular level, little is known about morphogenesis in C. burnetii, and no proteins specific to the SCV have been identified. Preparative isoelectric focusing was conducted to purify basic proteins possibly involved in SCV chromatin structure. A predominant protein of low M_r was present in the most basic fraction, eluting with a pH of approx. 11. Degenerate deoxyoligonucleotides corresponding to the N-terminal sequence of this protein were used to recover a cosmid clone from a C. burnetii genomic library. Nucleotide sequencing of insert DNA revealed an open reading frame designated scvA (small-cell-variant protein A) with coding potential for a 30 amino acid protein (ScvA) with a predicted M_r of 3610. ScvA is 46% arginine plus 46% glutamine with a predicted pi of 12.6. SDS-PAGE and silver staining of lysates of SCV and LCV purified by caesium chloride-equilibrium density centrifugation revealed a number of proteins unique to each cell type. Immunoblot analysis with ScvA antiserum demonstrated the presence of ScvA only in the SCV. By immunoelectron microscopy, ScvA antiserum labelled only the SCV, with the label concentrated on the condensed nucleoid. In addition, ScvA bound double-stranded DNA in gel mobility-shift assays. A 66% reduction in the mean number of gold particles per Coxiella cell was observed at 12 h post-infection when compared with the starting inoculum. Collectively, these data suggest that synthesis of ScvA is developmentally regulated, and that the protein may serve a structural or functional role as an integral component of the SCV chromatin. Moreover, degradation of this protein may be a necessary prerequisite for morphogenesis from SCV to LCV.     

PCR Detection of Coxiella burnetii from Different Clinical Specimens, Especially Bovine Milk, on the Basis of DNA Preparation with a Silica Matrix

For PCR detection of Coxiella burnetii in various clinical specimens we developed a sample preparation method in which silica binding of DNA was used. This method was found to be fast, easily performed with large numbers of samples, and equally sensitive for all of the specimens tested (livers, spleens, placentas, heart valves, milk, blood). The DNA preparation method described here can also be used as an initial step in any PCR-based examination of specimens. The procedure was tested with more than 600 milk samples, which were taken from 21 cows that were seropositive for C. burnetii and reportedly had fertility problems (and therefore were suspected of shedding the agent through milk intermittently or continuously). Of the 21 cows tested, 6 were shedding C. burnetii through milk. Altogether, C. burnetii DNA was detected in 6% of the samples. There was no correlation between the shedding pattern and the serological results.     

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.      

Statistical assessment of DNA extraction reagent lot variability in real‐time quantitative PCR

Aims: The aim of this study was to evaluate the variability in lots of a DNA extraction kit using real‐time PCR assays for Bacillus anthracis, Francisella tularensis and Vibrio cholerae. Methods and Results: Replicate aliquots of three bacteria were processed in duplicate with three different lots of a commercial DNA extraction kit. This experiment was repeated in triplicate. Results showed that cycle threshold values were statistically different among the different lots. Conclusions: Differences in DNA extraction reagent lots were found to be a significant source of variability for qPCR results. Steps should be taken to ensure the quality and consistency of reagents. Minimally, we propose that standard curves should be constructed for each new lot of extraction reagents, so that lot‐to‐lot variation is accounted for in data interpretation. Significance and Impact of the Study: This study highlights the importance of evaluating variability in DNA extraction procedures, especially when different reagent lots are used. Consideration of this variability in data interpretation should be an integral part of studies investigating environmental samples with unknown concentrations of organisms.     

Evaluating genomic DNA extraction methods from human whole blood using endpoint and real-time PCR assays

The extraction of genomic DNA is the crucial first step in large-scale epidemiological studies. Though there are many popular DNA isolation methods from human whole blood, only a few reports have compared their efficiencies using both end-point and real-time PCR assays. Genomic DNA was extracted from coronary artery disease patients using solution-based conventional protocols such as the phenol–chloroform/proteinase-K method and a non-phenolic non-enzymatic Rapid-Method, which were evaluated and compared vis-a-vis a commercially available silica column-based Blood DNA isolation kit. The appropriate method for efficiently extracting relatively pure DNA was assessed based on the total DNA yield, concentration, purity ratios (A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀), spectral profile and agarose gel electrophoresis analysis. The quality of the isolated DNA was further analysed for PCR inhibition using a murine specific ATP1A3 qPCR assay and mtDNA/Y-chromosome ratio determination assay. The suitability of the extracted DNA for downstream applications such as end-point SNP genotyping, was tested using PCR-RFLP analysis of the AGTR1-1166A>C variant, a mirSNP having pharmacogenetic relevance in cardiovascular diseases. Compared to the traditional phenol–chloroform/proteinase-K method, our results indicated the Rapid-Method to be a more suitable protocol for genomic DNA extraction from human whole blood in terms of DNA quantity, quality, safety, processing time and cost. The Rapid-Method, which is based on a simple salting-out procedure, is not only safe and cost-effective, but also has the added advantage of being scaled up to process variable sample volumes, thus enabling it to be applied in large-scale epidemiological studies.     

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.     

Rapid and simple DNA extraction method for the detection of enterotoxigenic Staphylococcus aureus directly from food samples: comparison of PCR and LAMP methods

Aims: The study describes the development of simple and rapid DNA extraction method in combination with loop‐mediated isothermal amplification (LAMP) to detect enterotoxigenic Staphylococcus aureus in food samples. Methods and Results: In this study, isolation of genomic DNA of enterotoxigenic Staph. aureus from spiked milk, milk burfi, khoa, sugarcane juice and boiled rice was carried out by boiling the isolated sample pellets for 10 min with 1% Triton X‐100. The isolated DNA was evaluated by polymerase chain reaction (PCR) and LAMP method. The LAMP was found to be 100 times more sensitive than PCR. The LAMP assay was very specific for Staph. aureus, and the presence of other contaminating bacterial DNAs and food matrix did not interfere or inhibit the LAMP assay. Conclusions: The template DNA extraction method developed in this study for food samples is simple, rapid and cost‐effective. LAMP was found to be less sensitive to matrix effect of food, compared to PCR. Significance and Impact of the Study: The method is suitable for direct detection of Staph. aureus without any enrichment in contaminated food samples and hence finds its application in food safety analysis, in permutation with LAMP.     

A novel DNA extraction and duplex polymerase chain reaction assay for the rapid detection of Prototheca zopfii genotype 2 in milk

Aims: To develop a PCR‐based assay to detect Prototheca zopfii (P. zopfii) and its mastitis‐related subtype (genotype 2) directly from milk samples. Methods and Results: The DNA extraction method herein is based on the lysing properties of chemical agents, mechanical grinding and DNA‐binding properties of silica particles; this method was developed to rapidly extract DNA directly from P. zopfii in bovine milk. Two pairs of primers specific for P. zopfii and genotype 2 were used in the duplex PCR, and a sensitivity test showed that the detection level was 5 × 10² colony‐forming units (CFU) ml^?1 for P. zopfii and 5 × 10³ CFU ml^?1 for genotype 2. Furthermore, a practical survey of 23 milk samples showed that the assay produced results that were in accordance with those obtained by the conventional microbiology method. Conclusions: The DNA extraction method is effective in isolating sufficient quantities of DNA from P. zopfii in milk for PCR analysis. The PCR assay is economical, sensitive and more rapid than the conventional culture method. Significance and Impact of the Study: The assay could be used as an alternative method for the rapid the detection of bovine mastitis resulting from P. zopfii genotype 2.     

Electron-microscopic study of the effect of various extractants on the morphology ofCoxiella burnetii

This paper presents the results of the effect of trichloroacetic acid (TCA) at two different temperatures and the effect of a mixture of detergents (D) onCoxiella burnetii. Both TCA and D caused a large destruction ofC. burnetii cells. In both cases complexes of high-molar-mass components from the outer membrane were extracted. In the D case not only proteins but a mixture with other high-molar-mass structures were released from the destroyed cells. By extraction of TCA, the antigenic complex composed of lipopolysaccharides (LPS), proteins and phospholipids was released. The effect of the D mixture onC. burnetii causes their complete destruction. The TCA causes a surface destruction of cells but it does not cause complete disintegration. The small-cell variants (SCV) in both cases were shown to be more stable compared to the large-cell variants (LCV).     

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.     

An alternative, sensitive method to detect Helicobacter pylori DNA in feces

Background: Despite the high sensitivity and specificity of PCR, detection of Helicobacter pylori DNA in feces is still challenging. Fecal samples contain inhibitory molecules that can prevent amplification of the target DNA. Even by using specific DNA extraction kits for stools, monitoring of infection by analyzing stool samples remains problematic and endorses the need for improved diagnostic methods. Materials and Methods: The newly proposed method uses selective hybridization of target DNA with biotin‐labeled probes, followed by DNA isolation with streptavidin‐coated magnetic beads. After three washing steps, the purified DNA can be amplified immediately using conventional or quantitative PCR. In order to test this technique on biological samples, Mongolian gerbils were infected with H. pylori ATCC 43504 and fecal samples were analyzed on days 1, 4, and 10 post infection. Results: A detection limit of one bacterial cell per 100 mg stool sample was established, but only after removal of the magnetic beads from the target DNA by heating. This resulted in a 10‐fold increase of sensitivity compared to a commercially available stool DNA extraction kit. Analysis of fecal samples from infected gerbils demonstrated the presence of H. pylori DNA on each time point, while the uninfected animal remained negative. Conclusions: The proposed technique allows detection of very low quantities of H. pylori DNA in biological samples. In laboratory animal models, detailed monitoring of infection and complete clearance of infection can be demonstrated thanks to the low detection limit.     

Back to basics: an evaluation of NaOH and alternative rapid DNA extraction protocols for DNA barcoding,genotyping, and disease diagnostics from fungal and oomycete samples

The ubiquity, high diversity and often‐cryptic manifestations of fungi and oomycetes frequently necessitate molecular tools for detecting and identifying them in the environment. In applications including DNA barcoding, pathogen detection from plant samples, and genotyping for population genetics and epidemiology, rapid and dependable DNA extraction methods scalable from one to hundreds of samples are desirable. We evaluated several rapid extraction methods (NaOH, Rapid one‐step extraction (ROSE), Chelex 100, proteinase K) for their ability to obtain DNA of quantity and quality suitable for the following applications: PCR amplification of the multicopy barcoding locus ITS1/5.8S/ITS2 from various fungal cultures and sporocarps; single‐copy microsatellite amplification from cultures of the phytopathogenic oomycete Phytophthora ramorum; probe‐based P. ramorum detection from leaves. Several methods were effective for most of the applications, with NaOH extraction favored in terms of success rate, cost, speed and simplicity. Frozen dilutions of ROSE and NaOH extracts maintained PCR viability for over 32 months. DNA from rapid extractions performed poorly compared to CTAB/phenol‐chloroform extracts for TaqMan diagnostics from tanoak leaves, suggesting that incomplete removal of PCR inhibitors is an issue for sensitive diagnostic procedures, especially from plants with recalcitrant leaf chemistry. NaOH extracts exhibited lower yield and size than CTAB/phenol‐chloroform extracts; however, NaOH extraction facilitated obtaining clean sequence data from sporocarps contaminated by other fungi, perhaps due to dilution resulting from low DNA yield. We conclude that conventional extractions are often unnecessary for routine DNA sequencing or genotyping of fungi and oomycetes, and recommend simpler strategies where source materials and intended applications warrant such use.     

Detection of Mycobacterium avium ssp. paratuberculosis in cheese, milk powder and milk using IS900 and f57-based qPCR assays

Aims: To develop a quantitative PCR assay for sensitive and specific detection of Mycobacterium avium ssp. paratuberculosis (Map) in a range of dairy products. Methods and Results: TaqMan^® assays were designed to target the IS900 and f57 genetic elements of Map. Both real‐time PCR assays were integrated with the Adiapure^® Map DNA extraction kit and assessed separately for the detection/quantification of Map in spiked milk, Cheddar cheese and milk powder. Assays were validated against Cheddar cheese samples containing known concentrations of Map. The IS900 qPCR assay was significantly more sensitive than the assay based on the f57 primer/probe. At a threshold cycle value of 38, limits of detection (LOD) for the IS900 qPCR assay were 0·6 CFU ml^?1, 2·8 CFU g^?1 and 30 CFU g^?1 for artificially contaminated pasteurized milk, whole milk powder and Cheddar cheese, respectively. The respective LOD’s for the f57 assay were 6·2 CFU ml^?1, 26·7 CFU g^?1 and 316 CFU g^?1. Conclusion: The integrated Adiapure^® extraction – IS900 real time assay described is a sensitive, quantitative method for the detection of Map in dairy products. This is the first study to consider qPCR as a quantitative estimation of Map‐DNA in cheese and whole milk powder. Significance and Impact of the Study: The assay developed allows sensitive detection and quantification of Map DNA in a range of dairy products which is valuable for the screening and surveillance of this potential zoonotic organism.     

Detection of Coxiella burnetii in Ambient Air after a Large Q Fever Outbreak

One of the largest Q fever outbreaks ever occurred in the Netherlands from 2007–2010, with 25 fatalities among 4,026 notified cases. Airborne dispersion of Coxiella burnetii was suspected but not studied extensively at the time. We investigated temporal and spatial variation of Coxiella burnetii in ambient air at residential locations in the most affected area in the Netherlands (the South-East), in the year immediately following the outbreak. One-week average ambient particulate matter < 10 μm samples were collected at eight locations from March till September 2011. Presence of Coxiella burnetii DNA was determined by quantitative polymerase chain reaction. Associations with various spatial and temporal characteristics were analyzed by mixed logistic regression. Coxiella burnetii DNA was detected in 56 out of 202 samples (28%). Airborne Coxiella burnetii presence showed a clear seasonal pattern coinciding with goat kidding. The spatial variation was significantly associated with number of goats on the nearest goat farm weighted by the distance to the farm (OR per IQR: 1.89, CI: 1.31–2.76). We conclude that in the year after a large Q fever outbreak, temporal variation of airborne Coxiella burnetii is suggestive to be associated with goat kidding, and spatial variation with distance to and size of goat farms. Aerosol measurements show to have potential for source identification and attribution of an airborne pathogen, which may also be applicable in early stages of an outbreak.     

Detection of Coxiella burnetii from Dust in a Barn Housing Dairy Cattle

We attempted to detect Coxiella burnetii in dust samples collected from a barn housing dairy cattle by the polymerase chain reaction (PCR) method. Ten dust samples (five from ventilation fans and five from crossbeams) were collected from two areas in a barn on a farm near Sapporo, Hokkaido. C. burnetii was detected in 5 of the 10 dust samples. It was believed that aerial contamination by C. burnetii occurred in the barn.     

Simple and efficient genomic DNA extraction protocol for molecular characterisation of Phytophthora colocasiae causing taro leaf blight

Genomic DNA extraction protocol with relatively high quantity and purity is prerequisite for the successful molecular identification and characterisation of plant pathogens. Conventional DNA extraction methods are often time-consuming and yield only very poor quantity of genomic DNA for samples with higher mycelial age. In our laboratory, we have aimed at establishing an efficient DNA isolation procedure, exclusively for the oomycete pathogen Phytophthora colocasiae causing serious leaf blight disease in taro. For this a phenol free protocol was adopted, which involves SDS/Proteinase K-based inactivation of protein contaminants, extraction of nucleic acids using chloroform: isoamyl alcohol and later precipitation of genomic DNA using isopropanol and sodium acetate. The purity of the isolated DNA was analysed by A_260/280 and A_260/230 spectrophotometric readings and confirmed by restriction digestion with restriction enzyme Eco RI. In this study, a comparative assessment was done with CTAB method and the commercial genomic DNA purification kit (Thermo Fisher Scientific, Fermentas, EU). The extracted DNA was found to be suitable for further downstream applications like ITS amplification of the rDNA ITS region and PCR amplification with species-specific primers.