Development of amperometric magnetogenosensors coupled to asymmetric PCR for the specific detection of <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis>

A disposable magnetogenosensor for the rapid, specific and sensitive detection of Streptococcus pneumoniae is reported. The developed procedure involves the use of streptavidin-modified magnetic beads, a specific biotinylated capture probe that hybridizes with a specific region of lytA, the gene encoding the pneumococcal major autolysin, and appropriate primers for asymmetric polymerase chain reaction (PCR) amplification. Capture probes and amplicons specific for S. pneumoniae were selected by a careful analysis of all lytA alleles available. The selected primers amplify a 235-bp fragment of pneumococcal lytA. A detection limit (LOD) of 5.1 nM was obtained for a 20-mer synthetic target DNA without any amplification protocol, while the LOD for the asymmetric PCR amplicon was 1.1 nM. A RSD value of 6.9% was obtained for measurements carried out with seven different genosensors for 1.1-nM aPCR product. The strict specificity of the designed primers was demonstrated by aPCR amplification of genomic DNA prepared from different bacteria, including some closely related streptococci. Direct asymmetric PCR (daPCR), using cells directly from broth cultures of S. pneumoniae, showed that daPCR products could be prepared with as few as 2 colony-forming units (CFU). Furthermore, this methodology did not show any cross-reaction with closely related streptococci such as Streptococcus mitis (or Streptococcus pseudopneumoniae) even when present in the culture at concentrations up to 10⁵ times higher than that of S. pneumoniae. Preliminary data for rapid detection of pneumococcus directly in clinical samples has shown that it is possible to discriminate between non-inoculated blood and urine samples and samples inoculated with only 10³ CFU mL⁻¹  S. pneumoniae.     

A Parametric Study of Sample Lysis and DNA Purification Techniques for Use in Automated Devices

Abstract

We present a parametric study on the efficiency of several automatable procedures for the extraction and purification of DNA from a variety of pathogens. Based on the results of this work, an optimized protocol has been developed for use with both spiked buffers and nasal wash. All steps of this protocol are suitable for incorporation into a field-portable, automated sample preparation device. From introduction of the sample to elution of DNA, the entire process was completed in less than 60 min, and the time could be reduced further by automation. The recovered DNA is of sufficient quality for real-time or multiplex PCR amplification. The protocol was demonstrated on nasal wash samples spiked with E. coli containing the J7R and crmB genes. Subsequent testing on resequencing microarrays correctly identified the samples as Variola major virus.     

Mitochondrial DNA control region typing from highly degraded skeletal remains by single-multiplex next-generation sequencing

Poor nuclear DNA preservation from highly degraded skeletal remains is the most limiting factor for the genetic identification of individuals. Mitochondrial DNA (mtDNA) typing, and especially of the control region (CR), using next-generation sequencing (NGS), enables retrieval of valuable genetic information in forensic contexts where highly degraded human skeletal remains are the only source of genetic material. Currently, NGS commercial kits can type all mtDNA-CR in fewer steps than the conventional Sanger technique. The PowerSeq CRM Nested System kit (Promega Corporation) employs a nested multiplex-polymerase chain reaction (PCR) strategy to amplify and index all mtDNA-CR in a single reaction. Our study analyzes the success of mtDNA-CR typing of highly degraded human skeletons using the PowerSeq CRM Nested System kit. We used samples from 41 individuals from different time periods to test three protocols (M1, M2, and M3) based on modifications of PCR conditions. To analyze the detected variants, two bioinformatic procedures were compared: an in-house pipeline and the GeneMarker HTS software. The results showed that many samples were not analyzed when the standard protocol (M1) was used. In contrast, the M3 protocol, which includes 35 PCR cycles and longer denaturation and extension steps, successfully recovered the mtDNA-CR from highly degraded skeletal samples. Mixed base profiles and the percentage of damaged reads were both indicators of possible contamination and can provide better results if used together. Furthermore, our freely available in-house pipeline can provide variants concordant with the forensic software.     

Use of DNase to eliminate contamination in ancient DNA analysis

Using polymerase chain reaction (PCR) amplification, it is possible to analyze DNA from limited source template. This method has proved especially valuable in studies of ancient DNA and in forensic investigations. However, PCR reactions containing minimal or damaged source template are prone to contamination by DNA from a number of other sources. While standard protocols to prevent and/or detect contamination do exist, methods of eliminating contamination are needed to ensure the validity of results obtained. We present a method to eliminate sources of contamination in reagents and labware through the use of a DNase prior to PCR amplification without damaging even the minimal amounts of template present in ancient DNA samples. This method, suggested previously for forensics applications, appears to be effective in eliminating contamination without interfering with the amplification of ancient template.     

Detection of Ovine or Bovine Milk Components in Commercial Camel Milk Powder Using a PCR-Based Method

Food ingredient adulteration, especially the adulteration of milk and dairy products, is one of the important issues of food safety. The large price difference between camel milk powder, ovine, and bovine milk powder may be an incentive for the incorporation of ovine and bovine derived foods in camel milk products. This study evaluated the use of ordinary PCR and real-time PCR for the detection of camel milk powder adulteration based on the presence of ovine and bovine milk components. DNA was extracted from camel, ovine, and bovine milk powder using a deep-processed product column DNA extraction kit. The quality of the extracted DNA was detected by amplifying the target sequence from the mitochondrial Cytb gene, and the extracted DNA was used for the identification of milk powder based on PCR analysis. In addition, PCR-based methods (both ordinary PCR and real-time PCR) were used to detect laboratory adulteration models of milk powder using primers targeting mitochondrial genes. The results show that the ordinary PCR method had better sensitivity and could qualitatively detect ovine and bovine milk components in the range of 1% to 100% in camel milk powder. The commercial camel milk powder was used to verify the practicability of this method. The real-time PCR normalization system has a good exponential correlation (R² = 0.9822 and 0.9923) between ovine or bovine content and Ct ratio (specific/internal reference gene) and allows for the quantitative determination of ovine or bovine milk contents in adulterated camel milk powder samples. Accuracy was effectively validated using simulated adulterated samples, with recoveries ranging from 80% to 110% with a coefficient of variation of less than 7%, exhibiting sufficient parameters of trueness. The ordinary PCR qualitative detection and real-time PCR quantitative detection method established in this study proved to be a specific, sensitive, and effective technology, which is expected to be used for market detection.     

Determination of Yersinia enterocolitica in Food by Capillary Electrophoresis with Laser Induced Fluorescence Detection

A duplex polymerase chain reaction (PCR)-capillary electrophoresis-laser induced fluorescence (CE-LIF) method was developed to determine Yersinia enterocolitica in food sensitively, rapidly, and reliably. Two sets of primers were selected to amplify the genus-specific 16 S ribosomal RNA gene and ail gene associated with the pathogenicity of Yersinia enterocolitica. The parameters of duplex PCR and the conditions for CE-LIF were optimized. Under the optimum conditions, the PCR products of Yersinia enterocolitica were determined within twenty minutes. Alignment analysis showed favorable agreement with published sequences from GenBank, indicating that the primers were specific and the PCR results were reliable. The method detected 16 colony forming units per milliliter pathogenic Yersinia enterocolitica. The intraday precision of migration time of the DNA marker and the PCR products were between 1.13 and 1.81 percent. In summary, a new method combining duplex PCR and CE-LIF is reported for specific, sensitive, and reproducible detection of Yersinia enterocolitica in food with low sample consumption and cost.     

Determination of the LOQ in real-time PCR by receiver operating characteristic curve analysis: application to qPCR assays for Fusarium verticillioides and F. proliferatum

Real-time PCR (qPCR) is the principal technique for the quantification of pathogen biomass in host tissue, yet no generic methods exist for the determination of the limit of quantification (LOQ) and the limit of detection (LOD) in qPCR. We suggest using the Youden index in the context of the receiver operating characteristic (ROC) curve analysis for this purpose. The LOQ was defined as the amount of target DNA that maximizes the sum of sensitivity and specificity. The LOD was defined as the lowest amount of target DNA that was amplified with a false-negative rate below a given threshold. We applied this concept to qPCR assays for Fusarium verticillioides and Fusarium proliferatum DNA in maize kernels. Spiked matrix and field samples characterized by melting curve analysis of PCR products were used as the source of true positives and true negatives. On the basis of the analysis of sensitivity and specificity of the assays, we estimated the LOQ values as 0.11 pg of DNA for spiked matrix and 0.62 pg of DNA for field samples for F. verticillioides. The LOQ values for F. proliferatum were 0.03 pg for spiked matrix and 0.24 pg for field samples. The mean LOQ values correspond to approximately eight genomes for F. verticillioides and three genomes for F. proliferatum. We demonstrated that the ROC analysis concept, developed for qualitative diagnostics, can be used for the determination of performance parameters of quantitative PCR.     

Chemiluminescent bead-based hybridization assay for the detection of genomic DNA from <Emphasis Type="Italic">E. coli</Emphasis> in purified plasmid samples

A bead-based hybridization assay was developed for detection of traces of E. coli genomic DNA (gDNA) present in purified plasmid DNA (pDNA) samples. Standards of gDNA and pDNA samples were sheared by sonication and adsorbed onto aminopropyl controlled pore glass (CPG) particles (130 μm). A preliminary study was conducted to optimize the amount of DNA adsorbed on the particles. Results indicated that maximum attachment efficiency was obtained by adsorbing DNA for 2 h in 0.2 × SSC, pH 5.7. The DNA-bound particles were hybridized overnight with a 181-bp digoxigenin-labeled probe, specific for gDNA. Following a chemiluminescent detection protocol, signal intensities of the standards were plotted as a function of initial gDNA concentration. The calculated detection limit (LOD) was 1.4 pM of gDNA. The assay was able to detect gDNA in pure plasmid preparations at the 1% level even in the presence of 1,000-fold excess of noncomplementary target. Hybridization results were compared with a quantitative real-time PCR assay. Both methods afforded similar accurate results at the 95% confidence level.     

Determination of Phytate in Urine by High-Performance Liquid Chromatography–Mass Spectrometry

An indirect method is described for determination of phytate in human urine. The method is based on hydrolysis of the phytate and determination of myo-inositol, one of the hydrolysis products. Chromatographic separations were performed on an Aminex HPX-87C column with Milli-Q water as mobile phase; 5 mM ammonium acetate was added post-column. The detector counted positive ions by monitoring m/z=198, which corresponds to the adduct of myo-inositol with the ammonium cation. The relative standard deviations obtained for standards containing 0.5, 1, and 1.5 mg L^–1 phytate were 4.1, 3.0, and 2.7% respectively (n=5). The limit of detection was 60 g L^–1. Different urine samples were analyzed both by this method and by an alternative analytical method based on GC–MS. The results from both methods were comparable.     

A Graphene Oxide‐DNA Electrochemical Sensor Based on Glassy Carbon Electrode for Sensitive Determination of Methotrexate

Methotrexate (MTX) was used as an anti‐cancer drug, but its excessive use can cause serious side effects, it was necessary to monitor MTX in vivo. In this report, DNA was immobilized on a glassy carbon electrode (GCE) modified with graphene oxide (GO) to develop an electrochemical sensor for sensitive determination of MTX for the first time. The adsorptive voltammetric behaviors of MTX on DNA sensor were investigated using differential pulse voltammetry (DPV). The peak current response of guanine in DNA was used as a determination signal of MTX in acetate buffer solution pH 4.6. Voltammetric investigations revealed that the proposed method could determine MTX in the concentration range from 5.5×10⁻⁸ to 2.2×10⁻⁶ mol L⁻¹ with a lower detection limit of 7.6×10⁻9 mol L⁻¹ (S/N=3). The method was applied to detect MTX in human blood serum and diluted urine samples with excellent recoveries of 97.4–102.5 %. Compared with the previous studies, the DNA/GO/GCE electrode constructed by us based on the change rate of guanine current (R%) in DNA, proportionally reflecting the MTX concentration, is simple and sensitive .     

Evaluation of purification procedures of DNA from maize-meal samples by exploiting different analytical techniques for the assessment of DNA quality

Two different approaches generally applied to achieve purification of DNA extracted from cells were compared: precipitation by organic solvents and enzymatic treatments. We investigated various experimental protocols reported in literature by evaluating DNA purity, integrity and yield. Reliability of analytical techniques normally employed to assess DNA purity and quantity was studied and comments and conclusions were suggested by comparing results obtained by different analytical techniques. Enzymatic treatments prove to be unable of increasing DNA purity while determining a significant degradation. In contrast, optimised conditions for solvent precipitation enabled a sharp increase of DNA purity to be obtained, associated with the maintenance of the initial DNA integrity. The application of the optimised protocol to maize-meal samples allowed us to achieve a good PCR amplification even with those samples which gave poor amplification by following the protocol recommended by the Italian legislation in force for GMO detection in food.     

The Nature of Crosslinking in N‐Vinylformamide Free‐Radical Polymerization

N‐Vinylformamide (NVF) free‐radical polymerization was found to form polymer gels at high conversions both in bulk and in solution. The polymerization was conducted at different temperatures, monomer and initiator concentrations to show the gelation conditions. Gel fractions and gel swelling ratios were also measured after separating the gel from the polymer samples. In order to confirm the crosslinking unit, a series of hydrolysis experiments were conducted on the gel samples. The hydrolysis results showed that the crosslinks in PNVF gels could be destroyed by alkaline hydrolysis. The most appropriate explanation to this fact is that crosslinking takes place via the amide group.     

Real-time polymerase chain reaction assay for endogenous reference gene for specific detection and quantification of common wheat-derived DNA (Triticum aestivum L.)

A species-specific endogenous reference gene system was developed for polymerase chain reaction (PCR)-based analysis in common wheat (Triticum aestivum L.) by targeting the ALMT1 gene, an aluminium-activated malate transporter. The primers and probe were elaborated for real-time PCR-based qualitative and quantitative assay. The size of amplified product is 95 base pairs. The specificity was assessed on 17 monocot and dicot plant species. The established real-time PCR assay amplified only T. aestivum-derived DNA; no amplification occurred on other phylogenetically related species, including durum wheat (T. durum). The robustness of the system was tested on the DNA of 15 common wheat cultivars using 20 000 genomic copies per PCR the mean cycle threshold (Ct) values of 24.02 +/- 0.251 were obtained. The absolute limits of detection and quantification of the real-time PCR assay were estimated to 2 and 20 haploid genome copies of common wheat, respectively. The linearity was experimentally validated on 2-fold serial dilutions of DNA from 650 to 20 000 haploid genome copies. All these results show that the real-time PCR assay developed on the ALMT1 gene is suitable to be used as an endogenous reference gene for PCR-based specific detection and quantification of T. aestivum-derived DNA in various applications, in particular for the detection and quantification of genetically modified materials in common wheat.     

Fluorimetric determination of β-carotene in food samples using a fluorescent dye

Abstract

A simple, sensitive, and selective fluorimetric method is reported for the determination of β-carotene in various food samples. Three fluorescent dyes, including fluorescein (F), eosin B (EB), and Congo red (CR), were characterized in a series of measurements to optimize the analytical response for the reliable, suitable, and sustainable determination of β-carotene. 3′,6′-Dihydroxyspiro[isobenzofuran-1(3H),9′-[9H]xanthen]-3-one, commonly known as fluorescein, was selected for the fluorimetric determination of β-carotene using a phosphate buffer. Under the optimum experimental conditions, β-carotene was determined across a range from 0.54 to 536.87?mg L^?1 with a detection limit equal to 0.47?mg L^?1. The novel method was demonstrated to be easy to use, cost effective, rapid, and less complex compared to a standard protocol that involves a time-consuming extraction followed by an expensive HPLC determination.     

BIOTECON diagnostics foodproof Listeria monocytogenes Detection Kit, 5' nuclease in combination with the foodproof ShortPrep II Kit

A method was developed for the detection of Listeria monocytogenes in food. The method is based on real-time PCR using hydrolysis probes (5' Nuclease). This advanced PCR method was designed to reduce the time necessary to achieve results from PCR reactions and to enable the user to monitor the amplification of the PCR product simultaneously, in real-time. After DNA isolation using the BIOTECON foodproof ShortPrep II Kit designed for the rapid preparation of L. monocytogenes DNA for direct use in PCR, the real-time detection of L. monocytogenes DNA is carried out using the foodproof Listeria monocytogenes Detection Kit. The kit provides primers and hydrolysis probes for sequence-specific detection, convenient premixed reagents, and controls for reliable interpretation of results. For the internal comparison study, three different foods (soft cheese, coalfish, and smoked ham) were analyzed, chosen from the 15 food groups recommended by the AOAC Research Institute for detection of L. monocytogenes. From each food, 20 samples were inoculated with a low level (1-10 CFU/25 g) and 20 samples with a high level (10-50 CFU/25 g) of L. monocytogenes. Additionally, five nonspiked samples were prepared from each food. Depending on the matrix, the food samples were examined with the test kits and compared with the cultural methods according to the U.S. Food and Drug Administration's Bacteriological Analytical Manual or the U.S. Department of Agriculture/Food Safety and Inspection Service Microbiology Laboratory Guidebook.     

Hydrolysis of tRNAPhe on Suspensions of Amino Acids

RNA is adsorbed strongly on suspensions of many moderately soluble organic solids. In some cases, the hydrolysis of tRNA^Phe is greatly accelerated by adsorption, and the major sites of hydrolysis are changed from those that are important in homogeneous solution. Here we show that the hydrolysis is greatly accelerated by suspensions of aspartic acid and β‐glutamic acid but not by suspensions of α‐glutamic acid, asparagine, or glutamine.     

Detection and characterization of recombinant DNA expressing <Emphasis Type="Italic">vip3A</Emphasis>-type insecticidal gene in GMOs—standard single,multiplex and construct-specific PCR assays

Vegetative insecticidal protein (Vip), a unique class of insecticidal protein, is now part of transgenic plants for conferring resistance against lepidopteron pests. In order to address the imminent regulatory need for detection and labeling of vip3A carrying genetically modified (GM) products, we have developed a standard single PCR and a multiplex PCR assay. As far as we are aware, this is the first report on PCR-based detection of a vip3A-type gene (vip-s) in transgenic cotton and tobacco. Our assay involves amplification of a 284-bp region of the vip-s gene. This assay can possibly detect as many as 20 natural wild-type isolates bearing a vip3A-like gene and two synthetic genes of vip3A in transgenic plants. The limit of detection as established by our assay for GM trait (vip-s) is 0.1%. Spiking with nontarget DNA originating from diverse plant sources had no inhibitory effect on vip-s detection. Since autoclaving of vip-s bearing GM leaf samples showed no deterioration/interference in detection efficacy, the assay seems to be suitable for processed food products as well. The vip-s amplicon identity was reconfirmed by restriction endonuclease assay. The primer set for vip-s was equally effective in a multiplex PCR assay format (duplex, triplex and quadruplex), used in conjunction with the primer sets for the npt-II selectable marker gene, Cauliflower mosaic virus 35S promoter and nopaline synthetase terminator, enabling concurrent detection of the transgene, regulatory sequences and marker gene. Further, the entire transgene construct was amplified using the forward primer of the promoter and the reverse primer of the terminator. The resultant amplicon served as a template for nested PCR to confirm the construct integrity. The method is suitable for screening any vip3A-carrying GM plant and food. The availability of a reliable PCR assay method prior to commercial release of vip3A-based transgenic crops and food would facilitate rapid and efficient regulatory compliance. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. An application for an Indian patent (1891/DEL2006/17.08.07) comprising a substantive part of this study has been filed. ITRC communication no. 2516.     

Experimental Detection of Induced Damage in Yeast RNA Due to γ‐Irradiation

Non‐isothermal studies by means of thermogravimetric (TG) measurements were carried out using a derivatograph to obtain the number of water molecules per repeat unit of yeast ribonucleic acid (RNA) before and after exposure to γ‐doses of 3 to 600 Gray. IR spectroscopy was utilized to determine which molecular subgroups of RNA adsorb water molecules and to investigate the conformational changes produced in RNA after γ‐irradiation. γ‐Irradiation was found to lead to ring opening or bond cleavage per unit nucleotide, which results in the formation of crosslinked regions (increase in molecular size). Such cleavage becomes more pronounced at doses >300 Gy leading to an increase in the number of water molecules per repeat unit and a decrease in molecular weight (degradation effect). A γ‐dose of 300 Gy may be considered as a critical damaging dose for yeast RNA molecules.     

多吡啶锌配合物键合与切割DNA研究

The complex of Zn[(phen)(dione)Cl]ClO_4·H_2O(where phen is 1,10-phenanthroline and dione is 1,10-phenan-throline-5,6-dione)has been synthesized and characterized.The interaction of the complex with DNA was investi-gated using UV absorption,fluorescence spectroscopy and electrophoresis measurements.The results show that thecomplex mainly binds to the double helix of DNA with intercalation mode and the binding constant K is 2.4×10~4mol~(-1)·L.Moreover,the complex can efficiently cleave plasmid DNA at physiological pH and temperature.Thecleavage occurs via a hydrolysis mechanism,which is showed by adding radical scavengers,rigorously anaerobicexperiments,analysis for malondialdehyde-like products,and the hydrolysis experiment of BDNPP with a rate con-stant k_(obs)of 5.3×10~(-6)s~(-1).