Comparison of rapid methods for the extraction of bacterial DNA from colonic and caecal lumen contents of the pig

AIMS: The increasing uses of DNA methodologies to study the micro flora of the pig gastrointestinal tract requires an efficient recovery of bacterial DNA from the intestinal sample. Thus, the objective of this study was to determine which DNA extraction methods are most effective for luminal samples from pigs. Several routinely used nucleic acid extraction procedures were compared based upon quantity and purity of extracted DNA. METHODS AND RESULTS: DNA was extracted from pig colonic and caecal lumen samples using 19 methods for bacterial DNA extraction. The quantity of total DNA recovered by each extraction method was determined and compared. Two methods using extraction with polyvinylpolypyrrolidone (PVPP) or phenol and two methods involving bead mill homogenization were found to provide the greatest quantity of extracted DNA for both colonic and caecal lumen. Extracted DNA from these four methods was further analysed for purity based upon the presence of PCR inhibitors, which was ascertained by determining the efficiency of amplification of a segment of the 16S rDNA. PCR amplification could be readily achieved with DNA extracted by each of these four methods, but efficiency of amplification tended to be higher with DNA from two of the methods (one extracted with PVPP and one with bead mill homogenization). CONCLUSIONS: Four extraction methods proved to be significantly superior in quantity of DNA extracted from luminal samples. Of these four, no strong inhibitors of PCR amplification were detected in any of the extracted DNA. However, the efficiency of amplification tended to be lower in DNA samples from two of the methods, suggesting the presence of low levels of PCR inhibitors. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this study provide a basis for choosing which DNA extraction procedures are most effective for use with samples of pig lumen.     

Ion pair-reversed phase HPLC approach facilitates subcloning of PCR products and screening of recombinant colonies

The isolation of a single DNA molecule for cloning is technically difficult, and the subsequent screening of colonies for recombinant DNA clones is time consuming. Ion pair-reversed phase HPLC (IP-RP-HPLC) analysis of nucleic acids improves the resolution and isolation of PCR products to be cloned. We demonstrate that PCR products analyzed and collected using the IP-RP-HPLC approach (WAVE DNA Fragment Analysis System) can be cloned directly into a plasmid vector. In addition, we demonstrate that when IP-RP-HPLC analysis is extended to the colony screening process, the time required for these procedures is reduced.     

Assessment of the specificity of Burkholderia and Pseudomonas qPCR assays for detection of these genera in soil using 454 pyrosequencing

In this study, two highly specific quantitative PCR assays targeting the bacterial genera Burkholderia and Pseudomonas were developed and evaluated on soil samples. The primers were targeting different multivariate regions of the 16S rRNA gene and designed to be compatible with quantitative PCR and the high throughput 454 pyrosequencing technique. The developed assays were validated using the standard methods. All tests with the new developed assays showed very high specificity. Pyrosequencing was used for direct analysis of the PCR product and applied as a specificity measurement of the primers. The Pseudomonas primers showed a 99% primer specificity, which covered 200 different Pseudomonas sequence clusters in 0.5 g of soil. In contrast to that the same approach using the genus-specific Burkholderia primers showed only 8% primer specificity. This discrepancy in primer specificity between the normal procedures compared with pyrosequencing illustrates that the common validation procedures for quantitative PCR primers may be misleading. Our results exemplify the fact that current 16S RNA gene sequence databases might lack resolution within many taxonomic groups and emphasize the necessity for a standardized and functional primer validation protocol. A possible solution to this could be to supplement the normal verification of quantitative PCR assays with a pyrosequencing approach.     

A new technique for genome-wide mapping of nucleotide excision repair without immunopurification of damaged DNA

Nucleotide excision repair functions to protect genome integrity, and ongoing studies using excision repair sequencing (XR-seq) have contributed to our understanding of how cells prioritize repair across the genome. In this method, the products of excision repair bearing damaged DNA are captured, sequenced, and then mapped genome-wide at single-nucleotide resolution. However, reagent requirements and complex procedures have limited widespread usage of this technique. In addition to the expense of these reagents, it has been hypothesized that the immunoprecipitation step using antibodies directed against damaged DNA may introduce bias in different sequence contexts. Here, we describe a newly developed adaptation called dA-tailing and adaptor ligation (ATL)–XR-seq, a relatively simple XR-seq method that avoids the use of immunoprecipitation targeting damaged DNA. ATL-XR-seq captures repair products by 3′-dA-tailing and 5′-adapter ligation instead of the original 5′- and 3′-dual adapter ligation. This new approach avoids adapter dimer formation during subsequent PCR, omits inefficient and time-consuming purification steps, and is very sensitive. In addition, poly(dA) tail length heterogeneity can serve as a molecular identifier, allowing more repair hotspots to be mapped. Importantly, a comparison of both repair mapping methods showed that no major bias is introduced by the anti-UV damage antibodies used in the original XR-seq procedure. Finally, we also coupled the described dA-tailing approach with quantitative PCR in a new method to quantify repair products. These new methods provide powerful and user-friendly tools to qualitatively and quantitatively measure excision repair.     

Construction and use of spotted large-insert clone DNA microarrays for the detection of genomic copy number changes

Microarray-based comparative genomic hybridization has become a widespread method for the analysis of DNA copy number changes across the human genome. Initial methods for microarray construction using large-insert clones required the preparation of DNA from large-scale cultures. This rapidly became an expensive and time-consuming process when expanded to the number of clones needed for higher resolution arrays. To overcome this problem, several PCR-based strategies have been developed to enable array construction from small amounts of cloned DNA. Here, we describe the construction of microarrays composed of human-specific large-insert clones (40-200 kb) using a specific degenerate oligonucleotide PCR strategy. In addition, we also describe array hybridization using manual and automated procedures and methods for array analysis. The technology and protocols described in this article can easily be adapted for other species dependent on the availability of clone libraries. According to our protocols, the procedure will take approximately 3 days from labeling the DNA to scanning the hybridized slides.     

Profiling DNA methylation by melting analysis

The idea of modifying DNA with bisulfite has paved the way for a variety of polymerase chain reaction (PCR) methods for accurately mapping 5-methylcytosine at specific genes. Bisulfite selectively deaminates cytosine to uracil under conditions where 5-methylcytosine remains unreacted. Following conventional PCR amplification of bisulfite-treated DNA, original cytosines appear as thymine while 5-methylcytosines appear as cytosine. Because the relative thermostability of a DNA duplex increases with increasing content of G:C base pairs, PCR products originating from DNA templates with different contents of 5-methylcytosine differ in melting temperature, i.e., the temperature required to convert the double helix into random coils. We describe two methods that resolve differentially methylated DNA sequences on the basis of differences in melting temperature. The first method integrates PCR amplification of bisulfite-treated DNA and subsequent melting analysis by using a thermal cycler coupled with a fluorometer. By including in the reaction a PCR-compatible, fluorescent dye that specifically binds to double-stranded DNA, the melting properties of the PCR product can be examined directly in the PCR tube by continuous fluorescence monitoring during a temperature transition. The second method relies on resolution of alleles with different 5-methylcytosine contents by analysis of PCR products in a polyacrylamide gel containing a gradient of chemical denaturants. Optimal resolution of differences in melting temperature is achieved by a special design of PCR primers. Both methods allow resolution of "heterogeneous" methylation, i.e., the situation where the content and distribution of 5-methylcytosine in a target gene differ between different molecules in the same sample.     

AmpliconDuo: A Split-Sample Filtering Protocol for High-Throughput Amplicon Sequencing of Microbial Communities

High throughput sequencing (HTSeq) of small ribosomal subunit amplicons has the potential for a comprehensive characterization of microbial community compositions, down to rare species. However, the error-prone nature of the multi-step experimental process requires that the resulting raw sequences are subjected to quality control procedures. These procedures often involve an abundance cutoff for rare sequences or clustering of sequences, both of which limit genetic resolution. Here we propose a simple experimental protocol that retains the high genetic resolution granted by HTSeq methods while effectively removing many low abundance sequences that are likely due to PCR and sequencing errors. According to this protocol, we split samples and submit both halves to independent PCR and sequencing runs. The resulting sequence data is graphically and quantitatively characterized by the discordance between the two experimental branches, allowing for a quick identification of problematic samples. Further, we discard sequences that are not found in both branches (“AmpliconDuo filter”). We show that the majority of sequences removed in this way, mostly low abundance but also some higher abundance sequences, show features expected from random modifications of true sequences as introduced by PCR and sequencing errors. On the other hand, the filter retains many low abundance sequences observed in both branches and thus provides a more reliable census of the rare biosphere. We find that the AmpliconDuo filter increases biological resolution as it increases apparent community similarity between biologically similar communities, while it does not affect apparent community similarities between biologically dissimilar communities. The filter does not distort overall apparent community compositions. Finally, we quantitatively explain the effect of the AmpliconDuo filter by a simple mathematical model.     

猪单个精子单倍型分析方法研究

应用引物延伸预扩增,内嵌引物设计策略进行了ＰＣＲ扩增并结合聚丙烯酰胺凝胶电泳及银染技术,对猪单个精子１２号染色体上４个微卫星位点进行了ＰＣＲ扩增。结果表明,上述技术的应用可以清晰地鉴定出每个精子的单倍型,单精子分型技术的应用为猪高精度遗传作图及需要样本量足够的大遗传现象研究提供了独特的工具。     

Comparison of real-time PCR and microscopy to evaluate sclerotial colonisation by a biocontrol fungus

The biocontrol agent Trichoderma harzianum colonises sclerotia of the plant pathogenic fungus Sclerotinia sclerotiorum. Plating of sclerotia typically has been used to determine the incidence of mycoparasitism, but does not quantify the extent to which individual sclerotia are colonised. We developed a specific PCR primer/probe set for the green fluorescent protein (GFP)-transformant T. harzianum ThzID1-M3, which exhibited high precision and reproducibility. Quantitative real-time PCR was evaluated along with epifluorescence microscopy and image analysis to investigate dynamics of colonisation of sclerotia in non-sterile soil. Amounts of ThzID1-M3 DNA and S. sclerotiorum DNA from entire individual sclerotia were quantified using real-time PCR. Epifluorescence micrographs were captured from sclerotial thin-section samples, and GFP fluorescence from these was quantified using computer image analysis in order to estimate colonisation on a per-sclerotium basis. As determined by either method, ThzID1-M3 colonised sclerotia in soil, and both methods quantified colonisation dynamics over time. In a separate experiment, colonisation of sclerotia on agar plates was observed using confocal laser scanning microscopy to view the GFP-fluorescing hyphae of ThzID1-M3. This method, while highly labour-intensive, provided high spatial resolution of colonisation dynamics. Thus, each method has advantages: microscopy combined with image analysis can provide useful information on the spatial and temporal dynamics of colonisation, while real-time PCR can provide a more precise assessment of the extent of sclerotial colonisation over time and can more easily be used to sample entire sclerotia.     

Cellular context in epigenetics: quantitative multicolor imaging and automated per-cell analysis of miRNAs and their putative targets

This paper assesses the quantitative resolution of qPCR using copy number variation (CNV) as a paradigm. An error model is developed for real-time qPCR data showing how the precision of CNV determination varies with the number of replicates. Using samples with varying numbers of X chromosomes, experimental data demonstrates that real-time qPCR can readily distinguish four copes from five copies, which corresponds to a 1.25-fold difference in relative quantity. Digital PCR is considered as an alternative form of qPCR. For digital PCR, an error model is shown that relates the precision of CNV determination to the number of reaction chambers. The quantitative capability of digital PCR is illustrated with an experiment distinguishing four and five copies of the human gene MRGPRX1. For either real-time qPCR or digital PCR, practical application of these models to achieve enhanced quantitative resolution requires use of a high throughput PCR platform that can simultaneously perform thousands of reactions. Comparing the two methods, real-time qPCR has the advantage of throughput and digital PCR has the advantage of simplicity in terms of the assumptions made for data analysis.     

A hybrid LC-Gel-MS method for proteomics research and its application to protease functional pathway mapping

Two-dimensional (2D) gel electrophoresis is the most common protein separation method in proteomics research. It can provide high resolution and high sensitivity. However, 2D gel methods have several limitations, such as labor-intensive procedures, poor reproducibility, and limited dynamic range of detection. In fact, many investigators have returned to couple the one-dimensional (1D) SDS-PAGE with mass spectrometry for protein identification. The limitation of this approach is the increased protein complexity in each one-dimensional gel band. To overcome this problem and provide reproducible quantitative information, we describe here a 2D method for protein mixture separation using a combination of high performance liquid chromatography (HPLC) and 1D SDS-PAGE. The study shows that the step-gradient fractionation method we have applied provides excellent reproducibility. In addition, high mass accuracy of LC-FTICR-MS can allow more confident protein identifications by high resolution and ultra-high mass measurement accuracy. This approach was applied to comparative proteomics since protein abundance level changes can be easily visualized with side-by-side vertical comparison in one gel. Furthermore, separation of multi-samples in the same gel significantly reduces run-to-run variation, as is shown with differential image gel electrophoresis (DIGE). Finally, this approach readily incorporates immunological methods to normalize relative abundances of multiple samples within a single gel. This paper presents the results of our developments and our initial application of this strategy for mapping protease function of beta amyloid cleaving enzyme (BACE) in biological systems.     

Evaluation of procedures for reliable PCR detection of Ralstonia solanacearum in common natural substrates

Several procedures were compared for reliable PCR detection of Ralstonia solanacearum in common substrates (plant, seed, water and soil). In order to prevent the inhibition of PCR by substances contained in crude extracts, numerous DNA extraction procedures as well as additives to buffers or PCR mixtures were checked. Our results showed that the efficiency of these methods or compounds depended greatly upon the nature of the sample. Consequently, preparation of samples prior to PCR depended upon sample origin. Simple methods such as a combined PVPP/BSA treatment or the association of filtration and centrifugation for detecting the bacterium in plant or water samples were very powerful. DNA capture also efficiently overcame PCR inhibition problems and ensured the detection of R. solanacearum in environmental samples. However, the commercial DNA extraction QIAamp kit appeared to be the most effective tool to guarantee the accurate PCR detection of the pathogen whatever the origin of the sample; this was particularly true for soil samples where the commonly used methods for the detection of R. solanacearum were inefficient. This study demonstrates that using an appropriate procedure, PCR is a useful and powerful tool for detecting low levels of R. solanacearum populations in their natural habitats.     

Immunocytochemistry of the amphibian embryo--from overview to ultrastructure

Amphibian embryos are standard research objects to study pattern formation and morphogenesis. Due to their external development and robust nature, experimental manipulations such as microinjections or transplantations can be easily performed. However, most immunocytochemical approaches addressing the specific localization of proteins are hampered by the fragility of the large and yolky embryonic cells which render high resolution staining difficult. Immunocytochemical data are therefore often restricted to either overall patterns in whole embryo preparations or to immunofluorescent localization with limited resolution on sections. High resolution or ultrastructural protein localization data are rare and can be achieved only with time consuming procedures. Here, a comparative study of immunocytochemical methods suitable for light and electron microscopy using different kinds of plastic resins is presented. Three main approaches are described: preembedding staining of whole embryos, postembedding staining of ultrathin sections and preembedding staining of vibratome sections. All the procedures are designed to study protein expression in early amphibian embryos en gros as well as en detail and the described techniques are suitable to combine two or three levels of resolution on the very same biological specimen. Examples are presented and advantages and disadvantages of the different protocols are discussed.     

Application of real-time PCR for quantitative detection of Vibrio parahaemolyticus from seafood in eastern China

Vibrio parahaemolyticus is recognized as a leading human food-borne pathogen. A TaqMan PCR assay based on the gyrase B gene (gyrB) sequence of V. parahaemolyticus was developed for quantitative detection of V. parahaemolyticus in seafood. The study involving 27 V. parahaemolyticus and 10 strains of other species indicated that the real-time PCR test was highly specific. The sensitivity of the assay was approximately a single CFU per PCR in pure culture and six to eight CFU per PCR in spiked raw oyster, respectively. Real-time PCR values of artificially inoculated oyster homogenates correlated well with plate counts determined using culture methods. A total of 300 seafood samples were analyzed and 78 (26%) of these samples were positive for V. parahaemolyticus using a conventional culture method and 97 (32.3%) using the real-time PCR assay. All culture-positive samples were PCR-positive. However, 19 samples positive by PCR were culture-negative. The results show that retail seafood is commonly contaminated with V. parahaemolyticus in harvest season in eastern China. These data also indicate that real-time PCR can provide sensitive species-specific detection and quantification of V. parahaemolyticus in seafood without prior isolation and characterization of the bacteria by traditional microbiological methods.     

Challenges in Laboratory Detection of Fungal Pathogens in the Airways of Cystic Fibrosis Patients

Study of the clinical significance of fungal colonization/infection in the airways of cystic fibrosis (CF) patients, especially by filamentous fungi, is challenged by the absence of standardized methodology for the detection and identification of an ever-broadening range of fungal pathogens. Culture-based methods remain the cornerstone diagnostic approaches, but current methods used in many clinical laboratories are insensitive and unstandardized, rendering comparative studies unfeasible. Guidelines for standardized processing of respiratory specimens and for their culture are urgently needed and should include recommendations for specific processing procedures, inoculum density, culture media, incubation temperature and duration of culture. Molecular techniques to detect fungi directly from clinical specimens include panfungal PCR assays, multiplex or pathogen-directed assays, real-time PCR, isothermal methods and probe-based assays. In general, these are used to complement culture. Fungal identification by DNA sequencing methods is often required to identify cultured isolates, but matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is increasingly used as an alternative to DNA sequencing. Genotyping of isolates is undertaken to investigate relatedness between isolates, to pinpoint the infection source and to study the population structure. Methods range from PCR fingerprinting and amplified fragment length polymorphism analysis, to short tandem repeat typing, multilocus sequencing typing (MLST) and whole genome sequencing (WGS). MLST is the current preferred method, whilst WGS offers best case resolution but currently is understudied.     

丙型肝炎病毒RNA打点杂交检测方法同RT－PCR方法的比较

采用ＨＣＶ基因组结构区Ｃ区ｃＤＮＡ探针和非结构区ＮＳ３－４区ｃＤＮＡ探针,建立了用打点杂交（ｄｏｔｂｌｏｔｈｙｂｒｉｄｉｚａｔｉｏｎ）检测血清中ＨＣＶＲＮＡ的方法,同采用ＨＣＶ基因组５’端非编码区的一对寡核苷酸引物通过逆转录－聚合酶链式反应（ＲＴ－ＰＣＲ）检测血清中ＨＣＶＲＮＡ的方法相比较,发现两种方法都能快速早期和特异地检出血清中ＨＣＶＲＮＡ,但ＲＴ－ＰＣＲ法敏感性优于ＲＮＡ打点杂交法。对于无血清学指标的慢性ＮＡＮＢ肝炎病人的诊断,可采用这两种方法。这两种方法的敏感性在很大程度上依赖于引物和探针的敏感性,以及ＲＮＡ提取方法。ＲＴ－ＰＣＲ法适用于诊断病毒血症和复制,打点杂交法适用于研究ＨＣＶＲＮＡ量的变化,对治疗的评价,以及为实验筛选较高滴度的ＨＣＶＲＮＡ阳性样本。     

基于PCR的染色体步移技术研究进展

基于PCR的染色体步移技术主要用于分离已知序列侧翼的未知序列,为分离基因、步移调控区域及填补基因组测序的空隙提供极大便利。基于PCR的染色体步移技术依照原理可分成依赖连接介导PCR法和不需要酶切连接PCR法。综述了近年来以PCR为基础的染色体步移技术,比较了这些方法的原理及操作步骤,同时总结了依赖连接介导PCR法和不需要酶切连接PCR法的优点与缺点,以期对研究起到借鉴作用。     

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.