Soil DNA Isolation Kit Research Articles

Polyaspartic Acid Urea Increased Maize Yield by Enhancing Leaf N Turnover Efficiency and Soil Microbial Diversity

The release rates of different nitrogen (N) fertilizers and their matching with plant demand determine crop yields. A field experiment was conducted to investigate the effect of using no fertilizer (N0), regular urea applied at rates of 180 kg ha−1 (N180) and 240 kg ha−1 (N240), controlled-release urea applied at a rate of 180 kg ha−1 (H180), and polyaspartic acid urea (PASP) on maize seed yield, soil microbial community diversity, and leaf N-converting enzymes. XianYu 688 was selected as the test maize variety. All cobs in the sample plots were collected per unit area to estimate maize yield. The enzyme-linked immunosorbent assay (ELISA) was used to determine leaf N-converting enzyme activities. Soil DNA was extracted using the Power Max Soil DNA Isolation Kit and subsequently sequenced using the Illumina HiSeq platform (PE 2500) to determine the microbial diversity and communities. The results showed that the highest seed yields were obtained under N240 and PASP180 treatments. The N-partial factor productivity of the PASP180 fertilizer was significantly higher than that of the other treatments. PASP treatment significantly increased maize seed yield due to the potential of storing more N in the ear leaves. Additionally, partial N productivity showed a significant positive correlation with the soil microbial Shannon, Chao1, and ACE indices, indicating that increased soil microbial diversity promoted N efficiency in maize. Further analysis revealed that PASP treatment increased seed yield by promoting leaf N-converting enzyme activity and soil microbial diversity. The results revealed that nitrate reductase (NR), glutamate synthase (GOGAT), and glutaminase (GLNS) enzyme activities in maize leaves were higher under the PASP treatment than under other fertilizer treatments. The PASP treatment significantly enhanced soil microbial diversity at different maize stages. Our study revealed the effects of using different N fertilizers on seed yield by examining their impact on soil microbial diversity and leaf N-converting enzyme activity. This study provides essential insights into maize production and soil fertility maintenance in the North China Plain.

PD31-04 LOWER URINARY TRACT MICROBIOTA DYSBIOSIS IS ASSOCIATED WITH URETHRAL FIBROSIS

You have accessJournal of UrologyCME1 May 2022PD31-04 LOWER URINARY TRACT MICROBIOTA DYSBIOSIS IS ASSOCIATED WITH URETHRAL FIBROSIS Michael Witthaus, Valmik Bhargava, Jill Buckley, and Mahadevan Rajasekaran Michael WitthausMichael Witthaus More articles by this author , Valmik BhargavaValmik Bhargava More articles by this author , Jill BuckleyJill Buckley More articles by this author , and Mahadevan RajasekaranMahadevan Rajasekaran More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002582.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Cellular mechanisms of stricture progression after repeated transurethral interventions (TUI) are unclear. We hypothesize urethral injury and mechanical stretch can cause tears on the urethral epithelial cell lining leading to leaking epithelium and urine extravasation (Fig A). Urinary microbiota traverses the epithelial lining and fibrogenesis can be mediated by inflammation due to microbial metabolites. Our objective is to characterize the urinary microbiome of patients with urethral stricture disease (USD) using 16S rRNA taxonomic profiling to elucidate the clinical relevance of the urinary microbiome in USD progression. METHODS: First catch urine samples from USD patients and healthy volunteers were collected and frozen to −80°C. Prior to analysis, samples were thawed at 2-8°C and centrifuged at 4°C for 15 mins to form a pellet. The pellet was then used for bacterial genomic DNA extraction using the PowerMag Soil DNA Isolation Kit. PCR amplification of the V3 and V4 regions of the 16S rRNA gene was performed. Illumina sequencing adapters and unique dual-index barcodes were added by PCR. Samples were then pooled and sequenced with paired-end 150 base pair reads (PE150) on an Illumina MiSeq Instrument. Data was analyzed using Qiime, which was used to determine alpha and beta diversity metrics per sample and to determine which microbes were presented differentially between the two cohorts. RESULTS: Our results are summarized in Fig B-D. Visualizing the specific microbes using a relative abundance bar plot (Fig B), the microbiome of healthy urine was more diverse than patients with USD. A divergence between the two groups supported distinct microbiomes, and a significant difference between patients with USD and controls was observed with beta diversity analysis (Fig C, D). The findings were indicative of the overall composition of microbial communities in each patient population. CONCLUSIONS: Our findings suggest a potential role for urinary microbiota alterations in developing an inflammatory environment and consequent fibrosis progression in USD. Future studies will determine specific urinary microbes between the two cohorts and their relevance to USD. Further potential for targeting specific microbes using next-generation probiotics to correct the urinary microbiota dysbiosis may be beneficial in preventing stricture progression. Source of Funding: UCSD Academic Senate © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e542 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Michael Witthaus More articles by this author Valmik Bhargava More articles by this author Jill Buckley More articles by this author Mahadevan Rajasekaran More articles by this author Expand All Advertisement PDF DownloadLoading ...

MP03-09 DESCRIBING THE URINARY MICROBIOME IN PRE-OPERATIVE URINE SPECIMENS OF LICHEN SCLEROSUS INDUCED AND NON-LICHEN SCLEROSUS INDUCED URETHRAL STRICTURE DISEASE

You have accessJournal of UrologyTrauma/Reconstruction/Diversion: Urethral Reconstruction (including Stricture, Diverticulum) I (MP03)1 Sep 2021MP03-09 DESCRIBING THE URINARY MICROBIOME IN PRE-OPERATIVE URINE SPECIMENS OF LICHEN SCLEROSUS INDUCED AND NON-LICHEN SCLEROSUS INDUCED URETHRAL STRICTURE DISEASE Amanda Sherman, Travis Sullivan, Harjivan Kohli, Eric Burks, Kimberly Rieger-Christ, and Alex J Vanni Amanda ShermanAmanda Sherman More articles by this author , Travis SullivanTravis Sullivan More articles by this author , Harjivan KohliHarjivan Kohli More articles by this author , Eric BurksEric Burks More articles by this author , Kimberly Rieger-ChristKimberly Rieger-Christ More articles by this author , and Alex J VanniAlex J Vanni More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000001964.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Lichen sclerosus (LS) is a chronic inflammatory condition that can affect both the penile skin and urethral epithelium in men. Over time, severe impairment of sexual and urinary function can result, from preputial adhesions and phimosis, skin tearing with erections, acquired buried penis, and urethral stricture disease (USD). The pathophysiology of LS USD is poorly understood, with a heterogeneous presentation and disease severity. This study seeks to describe differences in the urinary microbiome of patients with pathologically confirmed LS USD vs non-LS USD. METHODS: An IRB approved protocol of men with USD was performed. Pre-operative clean catch voided urine was collected in 34 men with pathologically confirmed LS USD and non-LS USD. Bacterial genomic DNA was extracted using the PowerMag Soil DNA Isolation Kit. 16S rRNA gene sequencing was performed using the MiSeq platform for paired-end sequencing. Gene sequences were clustered into Operational Taxonomic Units (OTUs) at a similarity cutoff value of 97%. Taxonomic identities were assigned to each using the scikit-learn classifier and an optimized, variable region-specific version of the SILVA Database. RESULTS: Sufficient bacterial DNA for analysis was obtained from 19 patients (10 non-LS USD and 9 LS USD). Significant differences in alpha diversity (within sample variance) and beta diversity (between sample variance) were observed (p <0.05) between the two cohorts. Likewise, differences in taxonomic profiles and differential abundance of taxa (Phylum, Class, Order, Family, Genus) were noted between the cohorts. CONCLUSIONS: LS USD have significant alterations in diversity and differential abundance of urine microbiota compared to non-LS USD controls. While further validation in a larger sample is necessary, this finding could be used to guide further investigation into the role of bacteria and the urinary microbiome in LS USD pathogenesis, severity of presentation, and stricture recurrence. Source of Funding: Support for this project generously provided by The Ellison Foundation © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e24-e24 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Amanda Sherman More articles by this author Travis Sullivan More articles by this author Harjivan Kohli More articles by this author Eric Burks More articles by this author Kimberly Rieger-Christ More articles by this author Alex J Vanni More articles by this author Expand All Advertisement Loading ...

Comparative study of benthic diatom community using 18S rDNA metabarcoding and light microscopy in the Bosphorus, Turkey

Diatoms are one of the major algal groups having an important role in the aquatic systems in which they constitute the main primary production with dinoflagellates in the coastal regions, and can contribute to photosynthesis at great rates. There had been intense effort to reveal diatom community with light microscopy (LM) and scanning electron microscopy (SEM), on the other hand, DNA barcoding techniques had been an interest to understand the microbiome in the last decades. Barcoding provided rapid response on the targeted organisms and introduced many new species. Recently, monocultural molecular studies showed that species could be described with accurate and reliable results. DNA metabarcoding approaches yielded to determine the whole organisms with available DNA data in the sample and major advances on next-generation sequencing platforms enable to widen the application of metabarcoding approach to various environmental samples. The aim of this study was to perform a comparative assessment of the diatom community structures in Bosphorus of Istanbul, Turkey by LM and 18S rDNA metabarcoding. Diatom samples were collected by brushing from the submerged stones of 10 cm2 of area taken from the coast of Beykoz on the Asian part of the Bosphorus, Istanbul and processed for LM and metabarcoding in June 2020. To concentrate the sample, the scrapes of the biofilm filtered from a 0.22-micron filter, and eDNA was extracted from that filter paper using MN NucleoSpin Soil DNA isolation kit (Macherey-Nagel, Germany) following the manufacturer’s protocol and diatom community profile was analyzed by targeting the V4 region of the 18S rRNA gene using Illumina® MiSeq™. DNA metabarcoding results revealed two classes Bacillariophyceae (91%) and Mediophyceae (9%) (Fig. 1). While small-celled Hyalosira delicatula Kützing was the most abundant taxa with 40% abundance and it was followed by Licmophora spp. (33%). 28% of Licmophora sequences could not be assigned to any species and remained as unidentified, Licmophora gracilis (Ehrenberg) Grunow (4%), and L. flabellata (0.9%) detected at lower abundances. Besides, one of the common genera of diatoms, Navicula spp., showed a relative abundance of 4% and another common genus Nitzschia spp. were represented only by N. commutata Grunow and composed 0.5% of the community. While 18S rDNA metabarcoding revealed 11 genera, LM investigation identified 17 genera belonging to 21 species. 4 genera were common in both techniques and Licmophora flabellata was detected in both LM and metabarcoding methods. Interestingly, small celled taxa which could be easily overlooked in LM was detected with 18S metabarcoding. The results presented a promising number of genera which could be detected by both methods (Fig. 2). DNA metabarcoding of diatoms is a new area of research in the coastal waters and there are few studies performed so far and this is the first study relying on the rDNA metabarcoding of diatoms in the aquatic systems in Turkey. The comparison of the taxa using microscopy methods and metabarcoding techniques indicated some significant differences in the diatom composition. However, the results here with 18 species with metabarcoding vice-versa 21 taxa with microscopy methods confirms poor biodiversity in the Bosphorus. However, this study based on one sampling effort in one station for the comparison of the two methods, we had LM results from more stations in different seasons supporting the lower biodiversity. eDNA data is scarce from the coastal areas and our results comprise a promising number of genera. Results of this study could provide data for further research, which high number of diatoms could be determined with eDNA metabarcoding.

The effects of humic substances on DNA isolation from soils.

BackgroundHumic substances (HS) are compounds with a complicated structure, present in the humus soil layer, water, lake sediments, peat, brown coal and shales. Due to their similar physicochemical properties to DNA, they may have an adverse effect on the subsequent use of the isolated material. The main aim of this research was to examine the effect of HS on DNA isolation depending on the soil type and land use, taking into account the spectroscopic full characteristics of HS fractions.MethodsThe research was conducted on eight types of soil sample. Soils represented the most important Soil Reference Groups for temperate climates: Fluvisols, Regosols, Cambisols, Arenosols, Histosols and Luvisols. Soil samples were also collected from areas diversified in terms of use: arable land, grassland and forest. The extraction of HS fractions was performed using the procedure recommended by the International HS Society. The fractional composition of HS was characterized by UV–Vis and fluorescence methods. Soil DNA is extracted by direct cell lysis in the using a CTAB-based method with a commonly-used commercial soil DNA isolation kit. The basis for assessing the quantity and quality of extracted DNA was the Polymerase chain reaction (PCR) reaction since the analysis of soil DNA often relies on the use of PCR to study soil microorganisms.ResultsBased on the results, it can be concluded that in the presence of a high concentration of HS, the isolated DNA was low quality and the additional purification procedure was necessary. Despite the differentiation of the internal structure of HS fractions, the decisive factor in the efficiency of DNA isolation from soil samples was the total carbon content in HS. Reduced DNA yields can significantly constrain PCR detection limits to levels inadequate for metagenomic analysis, especially from humus-rich soils.

LAMP Detection of Four Plant-Pathogenic Oomycetes and Its Application in Lettuce Fields.

In Kagawa Prefecture, Japan, the pathogens Phytophthora pseudolactucae, Pythium irregulare, Pythium uncinulatum, and Pythium spinosum have caused huge losses in lettuce production. We used loop-mediated isothermal amplification (LAMP) to analyze soil and plants in lettuce fields for the presence of these four pathogens. To develop an effective on-site detection method, we contrasted the Plant-LAMP and Plant Culture-LAMP procedures for plant samples, and five soil DNA extraction methods for soil samples. Plant-LAMP and a Soil DNA Isolation kit were selected to analyze three fields for the pathogen species present, infected sites, and level of soil contamination. We found that the same wilting symptoms could be caused by Phytophthora or Pythium, or a mixture of species from both genera. Ph. pseudolactucae infects the pith of the lettuce in aboveground parts, whereas Pythium spp. mainly infect roots. Ph. pseudolactucae and Py. uncinulatum caused disease more frequently than the other two pathogens. Furthermore, not all of the pathogens existed in the soil near infected lettuce plants. Therefore, the LAMP method can be used to diagnose pathogenic oomycetes in the field, and will be useful in the development of control strategies in lettuce production.

Assessing impacts of DNA extraction methods on next generation sequencing of water and wastewater samples

Next Generation Sequencing (NGS) is increasingly affordable and easier to perform. However, standard protocols prior to the sequencing step are only available for few selected sample types. Here we investigated the impact of DNA extraction methods on the consistency of NGS results. Four commercial DNA extraction kits (QIAamp DNA Mini Kit, QIAamp DNA Stool Mini Kit, MO BIO Power Water Kit, and MO BIO Power Soil DNA Isolation Kit) were used on sample sources including lake water and wastewater, and sample types including planktonic and biofilm bacteria communities. Sampling locations included a lake water reservoir, a trickling filter, and a moving bed biofilm reactor (MBBR). Unique genera such as Gemmatimonadetes, Elusimicrobia, and Latescibacteria were found in multiple samples. The Stool Mini Kit was least efficient in terms of diversity in sampling results with freshwater lake samples, and surprisingly the Power Water Kit was the least efficient across all sample types examined. Detailed NGS beta diversity comparisons indicated that the Mini Kit and PowerSoil Kit are best suited for studies that extract DNA from a variety of water and wastewater samples. We ultimately recommend application of Mini Kit or PowerSoil Kit as an improvement to NGS protocols for these sampling environments. These results are a step toward achieving accurate comparability of complex samples from water and wastewater environments by applying a single DNA extraction method, further streamlining future investigations.

Gene Encoding Nitrilase from Soil Sample of Lombok Gold Mine Industry using Metagenomics Approach

&lt;p class="Els-body-text"&gt;This paper describes a efficient screening gene nitrilase from contaminated soil from Lombok gold mine industry. DNA was extracted directly from soil using the soil DNA isolation kit based on enzymatic, chemical and mechanical lysis. The existence of nitrilase gene in soil sample can be identified by nitrilase gene amplification using H1F˗H1R primer. BLASTN analysis result revealed that the nitrilase gene fragment which was amplified by H1F˗H1R primer has a high homology with &lt;em&gt;Rhodococcus rhodochrous &lt;/em&gt;strain&lt;em&gt; tg1˗A6 nitrilase gene&lt;/em&gt;. These amplification and DNA fragment sequencing results indicated that nitrilase gene existence on soil sample can be identified by metagenomic approach&lt;/p&gt;&lt;p class="Els-Abstract-text"&gt; &lt;/p&gt;&lt;div&gt;&lt;p class="Els-keywords"&gt;&lt;strong&gt;Keywords:&lt;/strong&gt; Metagenomics; nitrilase gene; &lt;em&gt;Rhodococcus rhodochrous&lt;/em&gt;; soil.&lt;/p&gt;&lt;/div&gt;

Comparison of Total DNA Extraction Methods for Microbial Community Form Polluted Soil

Abstract DNA isolation represents the basic and probably the most important step in molecular biology for microbial strains, and even more, for microbial community analyses. Despite the development of molecular protocols for DNA microbial community isolation, there are still many drawbacks dependent of samples composition, and even the commercially available genomic isolation kits have significant limitations in recovering high genomic DNA amounts, especially from soil samples. Our study is aiming to compare and optimize a total microbial community DNA isolation protocol from polluted soil samples, estimating the amount and the purity of genomic DNA per g of soil, versus time requirements for each protocol, taking under consideration that our soil samples have a high content of humic acids. We checked several protocols for total DNA extraction, CTAB based, including a specific Kit for Soil DNA Isolation Kit NorgenBiotek. We estimated the time needed for each protocol, the amount of the DNA per gram of polluted soil, proteins and RNA contamination grade, by spectrophotometric analysis, but also the grade of PCR amplification inhibition. The most efficient method for our soil samples with high content of humic acid, suitable for further molecular analyses was the total DNA microbial community sample retrieved from Sagova et al. (2008) based protocol, with several adjustments. This protocol will be valuable for molecular analysis on microbial community profiling from environmental samples, especially from polluted soils.

Correction for Morono et al., Hot-Alkaline DNA Extraction Method for Deep-Subseafloor Archaeal Communities

Volume 80, no. 6, p. 1985–1994, 2014. Page 1986, column 1, paragraph 5, lines 7 to 10: “For the PowerMax soil DNA isolation kit, the sediment was vigorously shaken with autoclaved metal beads (5 mm in diameter) using a Shake Master auto (Bio Medical Science, Tokyo, Japan) operated at 1,500 rpm

A method suitable for DNA extraction from humus-rich soil.

A rapid and convenient method for extracting DNA from soil is presented. Soil DNA is extracted by direct cell lysis in the presence of EDTA, SDS, phenol, chloroform and isoamyl alcohol (3-methyl-1-butanol) followed by precipitation with 2-propanol. The extracted DNA is purified by modified DNA purification kit and DNA gel extraction kit. With this method, DNA extracted from humus-rich dark brown forest soil was free from humic substances and, therefore, could be used for efficient PCR amplification and restriction digestion. In contrast, DNA sample extracted with the traditional CTAB-based method had lower yield and purity, and no DNA could be extracted from the same soil sample with a commonly-used commercial soil DNA isolation kit. In addition, this method is time-saving and convenient, providing an efficient choice especially for DNA extraction from humus-rich soils.

An efficient and economical method for extraction of DNA amenable to biotechnological manipulations, from diverse soils and sediments

An attempt was made to optimize a new protocol for isolation of pure metagenomic DNA from soil samples. Various chemicals (FeCl3 , MgCl2 , CaCl2 and activated charcoal) were tested for their efficacy in isolation of metagenomic DNA from different soil and compost samples. Among these trials, charcoal and MgCl2 when used in combination yielded highly pure DNA free from humic acids and other contaminants. The DNA extracted with the optimized protocol was readily digested, amplified and cloned. Moreover, compared with a well-established commercial DNA isolation kit (UltraClean™ Soil DNA Isolation Kit), our method for DNA isolation was found to be economical. This demonstrated that the method developed can be applied to a wide variety of soil samples and allows handling of multiple samples at a given time. The optimized protocol developed has successfully yielded pure metagenomic DNA amenable to biotechnological manipulations. A user-friendly and economical protocol for isolation of DNA from soil and compost samples has been developed.

First Report of Fusarium solani on Utah Sweetvetch in the United States.

Utah sweetvetch (Hedysarum boreale Nutt.) is a native American perennial nitrogen fixing legume used mainly in rangeland reclamation, soil rejuvenation, and erosion control. In June 2011, a field of Utah sweetvetch grown for seeds in central Oregon had approximately 15% of the plants exhibiting chlorosis, defoliation, stunting, wilting, and/or death. Dissection of the crown of symptomatic plants revealed discolored pinkish brown vascular tissue. Symptomatic tissues from six random plants were surface sterilized, placed on acidified potato dextrose agar (PDA) medium, and cultured for 7 days at room temperature, which allowed six fungal isolates (SS1 through SS6) to be collected. On PDA, all six isolates had rapid, creamy white colored growth. Based on observations of 1-week-old isolates, microconidia were oval to kidney shaped, single celled, 8 to 10 × 2.5 to 4 μm, and formed at the tips of long unbranched monophialides. Macroconidia were three to four septate, cylindrical to slightly curved, with characteristic foot shaped basal cell and blunt apical cell, 37 to 49 × 4.4 to 5.3 μm. Chlaymydospores observed were 8.5 to 11 × 7.6 to 9 μm. Based on fungal references (1,2,3), the isolates were identified as Fusarium solani (Mart.) Sacc. Identification of the isolates at the molecular level was determined by amplification of the internal transcribed spacer (ITS) region using PCR and amplicon sequencing. Botrytis cinerea and F. graminearum cultures were used as controls for the extraction, amplification, and sequencing steps. Genomic DNA was extracted from mycelia using protocols of the MOBIO Ultraclean Soil DNA Isolation Kit (MO-BIO Laboratories Inc, Carlsbad, CA, USA). PCR was performed using ITS1/ITS4 primers and resulted in 563- to 573-bp amplicons, which were sequenced. Analysis of the ITS sequences (GenBank Accession Nos. JX524018 to JX524023) for the six fungal isolates using BLASTn revealed a 99% sequence identity with F. solani strains (AB470903, AB513851, AJ608989, EF152426, EU029589, and HM214456). Pathogenicity was confirmed on Utah sweetvetch plants in the greenhouse. Seeds of Utah sweetvetch were first plated on acidified PDA for germination; healthy seedlings were then selected and transplanted into pots with sterilized soil after 2 weeks of growth. The plants were kept in a greenhouse at Central Oregon Agricultural Research Center, Madras, Oregon. Ten 40-day-old healthy vetch plants were inoculated by drenching with a mixed conidial suspension (107 conidia/ml) of the six F. solani isolates. Ten plants drenched with sterile distilled water were included as controls. Symptoms of chlorosis and stunting similar to those in the commercial field were observed within 30 days of inoculation on 8 of 10 inoculated plants, while control plants were symptomless. Fungal isolates identical to F. solani were reisolated from the symptomatic plants. To our knowledge, this is the first report of F. solani on Utah sweetvetch plants.

Assessment of three commercial DNA extraction kits and a laboratory-developed method for detecting Cryptosporidium and Cyclospora in raspberry wash, basil wash and pesto

Polymerase chain reaction (PCR) methods are often used to identify the parasitic protozoa Cryptosporidium parvum and Cyclospora cayetanensis in foods although little has been published regarding the efficacy of available DNA extraction methods. This study reviewed three commonly used commercial DNA extraction kits: FastDNA SPIN Kit for soil, QBiogene (FastDNA), UltraClean™ Soil DNA Isolation Kit, MO BIO Laboratories (MoBio), and QIAamp DNA Mini Stool Kit, Qiagen (QIAamp), as well as a ‘homebrew’ Universal Nucleic Acid Extraction (UNEX) method.Washes from raspberry and basil as well as commercial pesto samples were seeded with 5000, 500, or 50 C. parvum and C. cayetanensis oocysts. The protocols were assessed for: quantity and quality of the extracted DNA, time to completion, presence of PCR inhibitors and the percentage of samples correctly identified as positive for the two parasites.Real-time and conventional nested PCR assays were used to detect the seeded pathogens. Of the commercial kits, PCR results of samples extracted using FastDNA were statistically similar to QIAamp and both were superior to MoBio. Differences in PCR results among FastDNA, QIAamp and UNEX for detection of Cyclospora were not statistically significant although the UNEX method proved best with Cryptosporidium. Real-time PCR assays targeted the 18S rRNA and the hsp70 genes of C. cayetanensis; overall results were similar to those found using conventional nested PCR targeting the 18S rRNA gene.

Comparison of Methodologies for the Extraction of Bacterial DNA from Mussels—Relevance for Food Safety

The control of the microbiological quality of bivalve molluscs assumes particular importance because they are among the most produced seafood products and mostly consumed as a whole, raw, or lightly cooked. The composition of the bacterial community associated with bivalves depends mostly on the microbiology of the surrounding environment at growing sites. Once the relationship between microbiology of bivalves and environment is established, a better classification and monitoring of the shellfish beds and evaluation of depuration strategies can be achieved. In this work, we tested if the methods of DNA extraction commonly used for the culture-independent microbiological analysis of sediment and water could be used directly, or with modifications, for the analysis of bacteria in mussels. The commercial kits Genomic DNA Purification Kit (MBI Fermentas, Vilnius, Lithuania), UltraCleanTM Soil DNA Isolation Kit (MOBIO Laboratories, Inc., Carlsbad, CA) and the method described by Griffiths and collaborators for DNA/RNA co-extraction were compared. The efficiency of extraction was assessed by DNA fluorescence and the denaturing gradient gel electrophoresis gel patterns of 16S ribosomal RNA gene fragments were used to compare the reproducibility and representativeness of the extraction methods. Results showed that the DNA/RNA co-extraction method with modifications was the most suitable. However, the results must be interpreted in the light of the purpose of the study and the relevance of maximizing extraction yield or diversity estimate, without compromising reproducibility. To our knowledge, this was the first attempt to transpose the procedure currently used for DNA extraction from sediments or waters, to the analysis of whole mussels.

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

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

A rapid DNA extraction method for PCR amplification from wetland soils

We tested a method of rapid DNA extraction from wetland soil samples for use in the polymerase chain reaction. The glass bead/calcium chloride/SDS method obtained in the present study was compared with the calcium chloride/SDS/enzymatic extraction method and the UltraClean™ Soil DNA Isolation Kit. Rapid DNA extraction could be completed within about two hours without purification steps. This study succeeded in establishing a fast soil DNA extraction protocol that can be applied to various environmental sources that are rich in humic acid content. The method provides a technology with high-quality DNA extraction from soils for testing the diversity of AOB and AOA.

Development and evaluation of a Taqman duplex real-time PCR quantification method for reliable enumeration of Legionella pneumophila in water samples

This study describes the development and evaluation of a specific Legionella pneumophila Taqman duplex real-time PCR (qPCR) for fast and reliable quantification of this human pathogen in suspected man-made water systems. The qPCR assay was 100% specific for all L. pneumophila serogroups 1–15 with a sensitivity of 60 genome units/l and an amplification efficiency of 98%. Amplification inhibitors were detected via an exogenous internal positive control, which was amplified simultaneously with L. pneumophila DNA using its own primer and probe set. Mean recovery rates of the qPCR assay for tap water and cooling circuit water, spiked with a known number L. pneumophila bacteria, were 93.0% and 56.3%, respectively. Additionally, by using the Ultraclean™ Soil DNA isolation kit, we were able to remove amplification inhibitors ubiquitously present in cooling water. The practical value of our qPCR assay was evaluated through analysis of 30 water samples from showers, taps, eyewash stations, fire sprinklers and recirculation loops with qPCR and traditional culture. In conclusion, the described L. pneumophila Taqman duplex real-time assay proved to be specific, sensitive and reproducible. This makes it a promising method complementing the current time-consuming culture standard method.

Isolation of polymerase chain reaction-ready bacterial DNA from Lake Baikal sediments by carboxyl-functionalised magnetic polymer microspheres

Carboxyl group-containing magnetic nonporous poly(2-hydroxyethyl methacrylate- co-ethylene dimethacrylate) (P(HEMA- co-EDMA)) microspheres were used for the isolation of polymerase chain reaction (PCR)-ready DNA from samples of Baikal sediments. DNA was isolated using the phenol extraction method or the Soil DNA Isolation Kit. The occurrence of false-negative results in PCR caused by the presence of extracellular inhibitors in DNA samples was solved using solid phase reversible DNA immobilisation. PCR-ready DNA was reversibly adsorbed to the microspheres in the presence of 8.0% (w/v) poly(ethylene glycol) (PEG 6000) and 2.0 M sodium chloride concentrations. The adsorbed DNA was released from the microspheres in a low ionic strength TE buffer. The quality of isolated DNA was checked by PCR amplification.

Extraction of inhibitor-free metagenomic DNA from polluted sediments, compatible with molecular diversity analysis using adsorption and ion-exchange treatments

PCR inhibitor-free metagenomic DNA of high quality and high yield was extracted from highly polluted sediments using a simple remediation strategy of adsorption and ion-exchange chromatography. Extraction procedure was optimized with series of steps, which involved gentle mechanical lysis, treatment with powdered activated charcoal (PAC) and ion-exchange chromatography with amberlite resin. Quality of the extracted DNA for molecular diversity analysis was tested by amplifying bacterial 16S rDNA (16S rRNA gene) with eubacterial specific universal primers (8f and 1492r), cloning of the amplified 16S rDNA and ARDRA (amplified rDNA restriction analysis) of the 16S rDNA clones. The presence of discrete differences in ARDRA banding profiles provided evidence for expediency of the DNA extraction protocol in molecular diversity studies. A comparison of the optimized protocol with commercial Ultraclean Soil DNA isolation kit suggested that method described in this report would be more efficient in removing metallic and organic inhibitors, from polluted sediment samples.