Improving PCR and qPCR detection of hydrogenase A (<Emphasis Type="Italic">hyd</Emphasis>A) associated with Clostridia in pure cultures and environmental sludges using bovine serum albumin

Detection of hydA genes of Clostridia spp. using degenerative and species specific primers for C. butyricum were optimized by the addition of bovine serum albumin (BSA) to polymerase chain reaction (PCR) and quantitative PCR (qPCR) reactions. BSA concentrations ranging from 100 to 400 ng/μl were examined using pure cultures and a variety of environmental samples as test targets. A BSA concentration of 100 ng/μl, which is lower than previously reported in the literature, was found to be most effective in improving the detection limit. The brightness of amplicons with 100 ng/μl BSA increased in ethidium bromide-treated gels, the minimum detection limit with BSA was at least one log greater, and cycle threshold (C _T) values were lower than without BSA in qPCR indicating improved detection of target deoxyribonucleic acid for most samples tested. Although amplicon visualization was improved at BSA concentrations greater than or equal to 100 ng/μl, gene copy numbers detected by qPCR were less, C_T values were increased, and T _m values were altered. SYBR Green dissociation curves of qPCR products of DNA from pure culture or sludge samples showed that BSA at 100 ng/μl reduced the variability of peak areas and T _m values.     

人尿嘧啶糖基化酶cDNA的克隆及其在食管鳞癌组织中的检测

尿嘧啶糖基化酶是碱基切除修复过程的起始酶,对于维护基因稳定具有重要意义。在不同组织及不同细胞周期中,该酶的表达水平存在差异。通过反转录PCR克隆了人尿嘧啶糖基化酶的cDNA编码序列,进一步以克隆所得的已知UNG基因拷贝数的重组质粒作为定量标准,通过实时荧光定量RT-PCR测定了食管癌病人手术切除组织中尿嘧啶糖基化酶的mRNA水平,探讨了尿嘧啶糖基化酶表达水平与食管癌之间的联系。     

qPCR-based mitochondrial DNA quantification: influence of template DNA fragmentation on accuracy

Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serial qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze-thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA (λnDNA) and mtDNA (λmtDNA) we present an approach to possibly correct measurements in degraded samples in the future. To our knowledge this is the first time different degradation impact of the two genomes is demonstrated and which evaluates systematically the impact of DNA degradation on quantification of mtDNA copy number.     

Influencing factors of dsDNA dye (high-resolution) melting curves and improved genotype call based on thermodynamic considerations

High-resolution melting of dsDNA using suitable dyes is a simple and cost-effective alternative for mutation scanning. Analytical variation can result from salt and template concentration (C_T). To overcome this problem the van’t Hoff transition enthalpy ΔH_vH from dsDNA melting curves was estimated and used for robust genotype calling or mutation scanning. Model calculations show the effect of salt, C_T, and temperature resolution on (1) T_m, (2) difference plots, (3) melting peaks, and (4) calculated ΔH_vH. Using the LightCycler480, the influence of dye (ResoLight) and scanning speed was assessed. The model calculations show that only ΔH_vH is not influenced by salt and C_T. Higher amplicon enthalpy ameliorates the ability to discriminate mutations. Temperature resolution is important for peak- but not for curve-based genotyping. ResoLight increases T_m by 3.4 °C, while lowering ΔH_vH. Using a 4-bp deletion in a 200-bp amplicon as a model, the miscalling rate improved substantially, when using ΔH_vH instead of difference plots. Melting curves of duplex DNA are influenced by dye and salt and less so by duplex concentrations. As predicted from theory, ΔH_vH is a robust measure for mutation detection in two-state melting. The influence of dyes on enthalpy is of general impact for PCR assays.     

The HIV1 protein Vpr acts to enhance constitutive DCAF1-dependent UNG2 turnover

Background

The HIV1 protein Vpr assembles with and acts through an ubiquitin ligase complex that includes DDB1 and cullin 4 (CRL4) to cause G2 cell cycle arrest and to promote degradation of both uracil DNA glycosylase 2 (UNG2) and single-strand selective mono-functional uracil DNA glycosylase 1 (SMUG1). DCAF1, an adaptor protein, is required for Vpr-mediated G2 arrest through the ubiquitin ligase complex. In work described here, we used UNG2 as a model substrate to study how Vpr acts through the ubiquitin ligase complex. We examined whether DCAF1 is essential for Vpr-mediated degradation of UNG2 and SMUG1. We further investigated whether Vpr is required for recruiting substrates to the ubiquitin ligase or acts to enhance its function and whether this parallels Vpr-mediated G2 arrest.

Methodology/Principal Findings

We found that DCAF1 plays an important role in Vpr-independent UNG2 and SMUG1 depletion. UNG2 assembled with the ubiquitin ligase complex in the absence of Vpr, but Vpr enhanced this interaction. Further, Vpr-mediated enhancement of UNG2 degradation correlated with low Vpr expression levels. Vpr concentrations exceeding a threshold blocked UNG2 depletion and enhanced its accumulation in the cell nucleus. A similar dose-dependent trend was seen for Vpr-mediated cell cycle arrest.

Conclusions/Significance

This work identifies UNG2 and SMUG1 as novel targets for CRL4^DCAF1-mediated degradation. It further shows that Vpr enhances rather than enables the interaction between UNG2 and the ubiquitin ligase. Vpr augments CRL4^DCAF1-mediated UNG2 degradation at low concentrations but antagonizes it at high concentrations, allowing nuclear accumulation of UNG2. Further, the protein that is targeted to cause G2 arrest behaves much like UNG2. Our findings provide the basis for determining whether the CRL4^DCAF1 complex is alone responsible for cell cycle-dependent UNG2 turnover and will also aid in establishing conditions necessary for the identification of additional targets of Vpr-enhanced degradation.     

Demonstration of preferential binding of SYBR Green I to specific DNA fragments in real-time multiplex PCR

SYBR Green I (SG) is widely used in real-time PCR applications as an intercalating dye and is included in many commercially available kits at undisclosed concentrations. Binding of SG to double-stranded DNA is non-specific and additional testing, such as DNA melting curve analysis, is required to confirm the generation of a specific amplicon. The use of melt curve analysis eliminates the necessity for agarose gel electrophoresis because the melting temperature (T_m) of the specific amplicon is analogous to the detection of an electrophoretic band. When using SG for real-time PCR multiplex reactions, discrimination of amplicons should be possible, provided the T_m values are sufficiently different. Real-time multiplex assays for Vibrio cholerae and Legionella pneumophila using commercially available kits and in-house SG mastermixes have highlighted variability in performance characteristics, in particular the detection of only a single product as assessed by T_m analysis but multiple products as assessed by agarose gel electrophoresis. The detected T_m corresponds to the amplicon with the higher G+C% and larger size, suggesting preferential binding of SG during PCR and resulting in the failure to detect multiple amplicons in multiplex reactions when the amount of SG present is limiting. This has implications for the design and routine application of diagnostic real-time PCR assays employing SG.     

DNA isolation by a rapid method from human blood samples: Effects of MgCl2, EDTA,storage time,and temperature on DNA yield and quality

The isolation of DNA from whole blood by a modified rapid method (RM) was tested using various detergents and buffer conditions. Extraction of DNA with either NP-40 or Triton X-100 gave a high yield of undegraded DNA in less than an hour. The concentration of magnesium ion in the buffers was critical to obtaining intact, high molecular weight (HMW) DNA. Greater than 10 mM MgCl₂ led to degradation. Addition of EDTA to the buffer inhibits this degradation. Preparation of DNA from blood stored at room temperature or incubated at 37°C for 24 hr resulted in the same amount and quality of DNA as from samples frozen at −70°C. DNA from blood samples that had undergone more than four freeze-thaw cycles was found to be partially degraded. The modified RM can be applied to extract DNA from as little as 10 μl of blood (340 ng of DNA) and from dried blood samples. DNA samples remained intact and undegraded for longer times when DNA was dissolved in higher concentrations of EDTA. This work was supported by grants from the Indiana Department of Mental Health and PHS RO1 AG10297.     

A stable and differentiable RNA positive control for reverse transcription-polymerase chain reaction

Most RNA positive controls currently used for monitoring the quality of RT-PCR assays have some disadvantages, such as instability, inability to monitor the quality of the relevant primers and/or causing indifferentiable false positives. To avoid these disadvantages, a simple method to prepare stable and differentiable RNA positive controls is now demonstrated with a real-time RT-PCR assay for the detection of Nipah virus (NiV). A DNA sequence which was shorter than its counterpart in the NiV genome and contained the binding sites of the primers of the RT-PCR assay was designed, synthesized and inserted into a vector, and then amplified by PCR with two vector-specific primers both of which contained a T7 promoter at the 5' terminal. The RNA positive control was the dsRNA in vitro transcribed from the PCR amplicons flanked by two T7 promoters. The RNA positive control was stable and able to monitor the quality of the whole concerned RT-PCR assay. False positives caused by contaminations of the RNA positive control or its amplicons could be easily identified because the amplicons of the RNA positive control were obviously shorter than those of real positive samples. Thus, the RNA positive control reported in this study avoided some common disadvantages of current RNA positive controls.     

Replication levels,false presences and the estimation of the presence/absence from eDNA metabarcoding data

Environmental DNA (eDNA) metabarcoding is increasingly used to study the present and past biodiversity. eDNA analyses often rely on amplification of very small quantities or degraded DNA. To avoid missing detection of taxa that are actually present (false negatives), multiple extractions and amplifications of the same samples are often performed. However, the level of replication needed for reliable estimates of the presence/absence patterns remains an unaddressed topic. Furthermore, degraded DNA and PCR/sequencing errors might produce false positives. We used simulations and empirical data to evaluate the level of replication required for accurate detection of targeted taxa in different contexts and to assess the performance of methods used to reduce the risk of false detections. Furthermore, we evaluated whether statistical approaches developed to estimate occupancy in the presence of observational errors can successfully estimate true prevalence, detection probability and false‐positive rates. Replications reduced the rate of false negatives; the optimal level of replication was strongly dependent on the detection probability of taxa. Occupancy models successfully estimated true prevalence, detection probability and false‐positive rates, but their performance increased with the number of replicates. At least eight PCR replicates should be performed if detection probability is not high, such as in ancient DNA studies. Multiple DNA extractions from the same sample yielded consistent results; in some cases, collecting multiple samples from the same locality allowed detecting more species. The optimal level of replication for accurate species detection strongly varies among studies and could be explicitly estimated to improve the reliability of results.     

Microaerobic enrichment of benzene-degrading bacteria and description of Ideonella benzenivorans sp. nov., capable of degrading benzene,toluene and ethylbenzene under microaerobic conditions

In the present study, the bacterial community structure of enrichment cultures degrading benzene under microaerobic conditions was investigated through culturing and 16S rRNA gene Illumina amplicon sequencing. Enrichments were dominated by members of the genus Rhodoferax followed by Pseudomonas and Acidovorax. Additionally, a pale amber-coloured, motile, Gram-stain-negative bacterium, designated B7^T was isolated from the microaerobic benzene-degrading enrichment cultures and characterized using a polyphasic approach to determine its taxonomic position. The 16S rRNA gene and whole genome-based phylogenetic analyses revealed that strain B7^T formed a lineage within the family Comamonadaceae, clustered as a member of the genus Ideonella and most closely related to Ideonella dechloratans CCUG 30977^T. The sole respiratory quinone is ubiquinone-8. The major fatty acids are C_16:0 and summed feature 3 (C_16:1 ω7c/iso-C_15:0 2-OH). The DNA G?+?C content of the type strain is 68.8?mol%. The orthologous average nucleotide identity (OrthoANI) and in silico DNA–DNA hybridization (dDDH) relatedness values between strain B7^T and closest relatives were below the threshold values for species demarcation. The genome of strain B7^T, which is approximately 4.5?Mb, contains a phenol degradation gene cluster, encoding a multicomponent phenol hydroxylase (mPH) together with a complete meta-cleavage pathway including a I.2.C-type catechol 2,3-dioxygenase (C23O) gene. As predicted by the genome, the type strain is involved in aromatic hydrocarbon-degradation: benzene, toluene and ethylbenzene are degraded aerobically and also microaerobically as sole source of carbon and energy. Based on phenotypic characteristics and phylogenetic analysis, strain B7^T is a member of the genus Ideonella and represents a novel species for which the name Ideonella benzenivorans sp. nov. is proposed. The type strain of the species is strain B7^T (=?LMG 32,345^T?=?NCAIM B.02664^T).

     

Development of real-time RT-PCR for detecting viable Cochlodinium polykrikoides (Dinophyceae) cysts in sediment

Morphological observations have confirmed that cysts are produced by dinoflagellates. However, finding a seed bed or unknown cysts in field samples by microscopy is extremely time consuming. Real-time PCR has been used to facilitate the detection of dinoflagellate cysts in sediment. However, DNA from dead vegetative cells remaining on the surface sediment may persist for a long period of time, which can cause false positive DNA detection. In this study, a non-quantitative RNA targeted probe using real-time RT-PCR was developed for detection of viable cysts in sediment. Large-subunit rRNA was used to develop a species-specific RNA targeted probe for the ichthyotoxic dinoflagellate Cochlodinium polykrikoides. The sediment samples were sieved and incubated at 30 °C for 3 h prior to RNA extraction to remove RNA from dead cells remaining in the sediment. Nested-PCR was conducted to maximize assay sensitivity. A field survey to determine the distribution of cysts at 155 sampling stations in the western and southern part of the Korean peninsula showed that C. polykrikoides cysts were detected at five sampling stations.     

Evaluation of DNA Extraction Methods for Use in Combination with SYBR Green I Real-Time PCR To Detect Salmonella enterica Serotype Enteritidis in Poultry

The objective of this study was to develop a rapid, reproducible, and robust method for detecting Salmonella enterica serotype Enteritidis in poultry samples. First, for the extraction and purification of DNA from the preenrichment culture, four methods (boiling, alkaline lysis, Nucleospin, and Dynabeads DNA Direct System I) were compared. The most effective method was then combined with a real-time PCR method based on the double-stranded DNA binding dye SYBR Green I used with the ABI Prism 7700 system. The specificity of the reaction was determined by the melting temperature (T_m) of the amplicon obtained. The experiments were conducted both on samples of chicken experimentally contaminated with serotype Enteritidis and on commercially available poultry samples, which were also used for comparisons with the standard cultural method (i.e., ISO 6579/2001). The results of comparisons among the four DNA extraction methods showed significant differences except for the results from the boiling and Nucleospin methods (the two methods that produced the lowest threshold cycles). Boiling was selected as the preferred extraction method because it is the simplest and most rapid. This method was then combined with SYBR Green I real-time PCR, using primers SEFA-1 and SEFA-2. The specificity of the reaction was confirmed by the T_m, which was consistently specific for the amplicon obtained; the mean peak T_m obtained with curves specific for serotype Enteritidis was 82.56 ± 0.22°C. The standard curve constructed using the mean threshold cycle and various concentrations of serotype Enteritidis (ranging from 10³ to 10⁸ CFU/ml) showed good linearity (R² = 0.9767) and a sensitivity limit of less than 10³ CFU/ml. The results of this study demonstrate that the SYBR Green I real-time PCR constitutes an effective and easy-to-perform method for detecting serotype Enteritidis in poultry samples.     

A rapid and sensitive one step-SYBR green based semi quantitative real time RT-PCR for the detection of <Emphasis Type="Italic">peste des petits ruminants</Emphasis> virus in the clinical samples

A sensitive and rapid single step real time (rt) RT-PCR was standardized using one-step Brilliant SYBR Green kit® for detection and semi-quantitation of peste des petitis ruminants virus (PPRV) using the virus RNA and matrix (M) protein gene-specific primers and compared with established conventional RT-PCR and TaqMan RT-PCR. The assay amplifies a 124 bp fragment of the PPRV M gene with T_m of 78.28 to 78.50. The assay was linear within a range of 50 ng to 0.5 fg total virus RNA with a detection limit (sensitivity) of 0.5 fg. Based on the serial dilution of the live-attenuated PPR vaccine virus, the detection limit was ～0.0001 cell culture infectious dose 50% units (TCID₅₀). Additionally, swab materials spiked with known titre of vaccine virus were equally well detected in the assay. The standardized rt RT-PCR was easily employed for the detection of PPRV nucleic acid directly in the field and experimental clinical samples. The assay detected the PPRV nucleic acid as early as 3 day post infection (dpi) and up to 20 dpi in swab materials from the experimental samples. The assay was rapid and more sensitive than TaqMan and conventional RT-PCR in the detection of PPRV nucleic acid from the PPR suspected clinical samples of sheep and goats. Therefore, the established, simplified SYBR green rt RT-PCR is an alternative test to the already existing various diagnostic assays and could be useful for rapid clinical diagnosis with advantage in reducing risk of contamination.