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This study described a novel multiplex qualitative detection method using pyrosequencing. Based on the principle of the universal primer-multiplex-PCR, only one sequencing primer was employed to realize the detection of the multiple targets. Samples containing three genetically modified (GM) crops in different proportions were used to validate the method. The dNTP dispensing order was designed based on the product sequences. Only 12 rounds (ATCTGATCGACT) of dNTPs addition and, often, as few as three rounds (CAT) under ideal conditions, were required to detect the GM events qualitatively, and sensitivity was as low as 1% of a mixture. However, when considering a mixture, calculating signal values allowed the proportion of each GM to be estimated. Based on these results, we concluded that our novel method not only realized detection but also allowed semi-quantitative detection of individual events. [ABSTRACT FROM AUTHOR]

Many types of diagnostic technologies have been reported for DNA methylation, but they require a standard curve for quantification or only show moderate accuracy. Moreover, most technologies have difficulty providing information on the level of methylation at specific contiguous multi-sites, not to mention easy-to-use detection to eliminate labor-intensive procedures. We have addressed these limitations and report here a cascade strategy that combines proportion competitive quantitative PCR (PCQ-PCR) and lateral flow nucleic acid biosensor (LFNAB), resulting in accurate and easy-to-use assessment. The P16 gene with specific multi-methylated sites, a well-studied tumor suppressor gene, was used as the target DNA sequence model. First, PCQ-PCR provided amplification products with an accurate proportion of multi-methylated sites following the principle of proportionality, and double-labeled duplex DNA was synthesized. Then, a LFNAB strategy was further employed for amplified signal detection via immune affinity recognition, and the exact level of site-specific methylation could be determined by the relative intensity of the test line and internal reference line. This combination resulted in all recoveries being greater than 94%, which are pretty satisfactory recoveries in DNA methylation assessment. Moreover, the developed cascades show significantly high usability as a simple, sensitive, and low-cost tool. Therefore, as a universal platform for sensing systems for the detection of contiguous multi-sites of DNA methylation without external standards and expensive instrumentation, this PCQ-PCR-LFNAB cascade method shows great promise for the point-of-care diagnosis of cancer risk and therapeutics. [ABSTRACT FROM AUTHOR]

Abstract: The authenticity of edible oils has recently attracted increased attention, particularly due to the emergence of swill-cooked dirty oil. There has not been an effective method to detect the authenticity of edible oils due to the diversity of edible oils, the trace DNA in oils and serious DNA degradation. In this study, an efficient method was developed and optimized to detect the authenticity of edible oils. The stability and efficiency of detecting the authenticity of edible oils were dramatically increased by the DNA extraction method, amplification fragment selection and nano-real time PCR. The DNA extraction method was optimized with regard to the type and the amount of organic reagent, the types and the amounts of extraction buffer, and the DNA carriers. The types of organic reagents and DNA carriers determined whether DNA could be successfully extracted from different edible oils. The amount of DNA extracted from edible oils and adulterated edible oils with the optimized extraction method was evaluated by real-time PCR with different amplification fragments and nano-real time PCR. The results showed that 10 mL soybean oil can be detected against a background of 40 mL sesame oil. Additionally, the new nano real-time PCR technology was applied to amplify the DNA in edible oils at the first time. The efficiency and the precision of the PCR were dramatically increased by gold colloid. The results from this research are beneficial for detecting the authenticity of edible oils. [Copyright &y& Elsevier]

Abstract: Fermentability and contamination level of wine can be assessed through the detection of viable fermentation-related and spoilage-related microorganisms. Ethidium monoazide in combination with quantitative PCR (EMA-qPCR) has been considered as a promising method to enumerate viable cells. Milling for 80 s by Ø 500-μm glass beads is demonstrated to be optimal for DNA extraction from yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) in wine to be used as a template for PCR. EMA-qPCR results from experiments using DNA extracted by this method correlate well with the results of a plating assay (R 2 > 0.99), and a PCR efficiency between 96% and 105% was obtained. Moreover, for all of these microorganisms, EMA treatment of pure cultures at a low concentration (10 μg/mL) for 20 min photoactivation resulted in effective differentiation between viable and non-viable cells and had no effect on viable cells. Due to sublethal injury to some cells, underestimation of cell counts was found in most of the wine samples tested using the EMA-qPCR method, and a 40-min incubation in recovery medium could completely offset this error. Our results suggest an optimal glass-bead DNA extraction method and EMA treatment suitable for all of the main microorganisms in wine. The EMA-qPCR method was successfully applied to quantify yeasts, Saccharomyces cerevisiae (S. cerevisiae), LAB, non-Oenococcus oeni LAB (non-O. oeni LAB) and AAB in wine samples. [Copyright &y& Elsevier]

Abstract: A-T linker adapter polymerase chain reaction (PCR) was modified and employed for the isolation of genomic fragments adjacent to a known DNA sequence. The improvements in the method focus on two points. The first is the modification of the PO4 and NH2 groups in the adapter to inhibit the self-ligation of the adapter or the generation of nonspecific products. The second improvement is the use of the capacity of rTaq DNA polymerase to add an adenosine overhang at the 3′ ends of digested DNA to suppress self-ligation in the digested DNA and simultaneously resolve restriction site clone bias. The combination of modifications in the adapter and in the digested DNA leads to T/A-specific ligation, which enhances the flexibility of this method and makes it feasible to use many different restriction enzymes with a single adapter. This novel A-T linker adapter PCR overcomes the inherent limitations of the original ligation-mediated PCR method such as low specificity and a lack of restriction enzyme choice. Moreover, this method also offers higher amplification efficiency, greater flexibility, and easier manipulation compared with other PCR methods for chromosome walking. Experimental results from 143 Arabidopsis mutants illustrate that this method is reliable and efficient in high-throughput experiments. [Copyright &y& Elsevier]

Abstract: With the development of genetically modified organisms (GMOs), labeling regulations have been introduced, which require appropriate detection methods. Event-specific qualitative and quantitative PCR detection methods have become the internationally agreed state-of-art. This paper describes the characterization and event-specific quantitative detection of LY038 maize insert with the application of reference molecule. The flanking regions were characterized by Inverse-PCR (I-PCR). Furthermore, the event-specific PCR primers and TaqMan probe were designed based on the discovered right flanking sequence. In the qualitative PCR assay, two PCR systems were established with the event-specific and specie-specific primers respectively, and the limit of detection (LOD) was 0.1% (approximates to 37 haploid genome copies). In the quantitative TaqMan real-time PCR assay, a reference molecule was constructed by recombinant PCR and standard curves were set up. By using the reference molecule, we obtained standard curves with good linearity and relatively high efficiency of PCR reaction. The results indicated the usability of the plasmid as standard material. From above results, we believed that the established event-specific qualitative and quantitative PCR systems for LY038 maize in this study were acceptable and suitable for LY038 maize detection in mixed samples. [Copyright &y& Elsevier]

Abstract: A novel common primer multiplex PCR (CP-M-PCR) was applied to detect four kinds of meats (chicken, cattle, pig and horse) as raw materials. A common adapter was designed in the 5′-end of species-specific reverse primers which matched with the species-specific DNA sequences for each species and also used as the common primer (CP). CP-M-PCR primers were designed to uncover different length fragments of 239, 292, 412, and 451bp from chicken, cattle, pig and horse meats, respectively. The bands of specific DNA fragments amplified by CP-M-PCR method still appeared until the concentration of species-specific primers diluted to 0.015pmol and primer sensitivity was increased by 100 times compared with conventional multiplex PCR without CP. CP-M-PCR detection limit of the DNA samples was 0.1ng (36.4 copies) for single kind of meat as well as four kinds of meats. CP-M-PCR method simplified the PCR reaction system and conquered the disparate amplified efficiency from different primers. The CP-M-PCR method could be widely applied in practical detection for simultaneous identification of other meat species and their products. [Copyright &y& Elsevier]

Here, based on the design of rational primers and copper nanoclusters (CuNCs), we present a method for the accurate detection of the SARS-CoV-2 Delta variant, which is capable of distinguishing the Delta variant with its single nucleotide polymorphism from the 'wild type' coronavirus (NC_045512.2), and realizing visualization signal out. Specifically, we show that dual priming oligonucleotide (DPO) primers and AT primers can be used to distinguish between wild types and mutations of this virus by polymerase chain reaction (PCR) analysis and that visualization can be achieved via the red fluorescence of CuNCs in ultraviolet radiation. Among the results, it was found that the R-1-down (DPO)-6I and F-1-30 AT, with the single nucleotide deletion site designed at the 3′ end of the downstream primer, showed the best specificity towards the Delta variant. Moreover, the use of AT primers increased the AT contents of the PCR products, thus meeting the template requirements generated by the CuNCs. It was also found that the AT primers could assist with improving detection specificity. Finally, we demonstrate that the visualization of the CuNCs-based detection assay exhibited a linear relationship in 0.5 pg μL−1–50 ng μL−1, with a limit of quantitation (LOQ) of 0.5 pg μL−1. [Display omitted] • Developed a novel detection method for SARS-CoV-2 Delta variant with single nucleotide deletion. • Rational primers design includes DPO primers and AT primers. • Achieved single nucleotide specificity based on DPO primers. • Improved the fluorescence of copper nanoclusters via AT primers, thereby increasing the AT contents of PCR products. • Realized visualized signal output PCR products via copper nanoclusters. [ABSTRACT FROM AUTHOR]

Abstract: In this study, a novel single universal primer multiplex ligation-dependent probe amplification (SUP-MLPA) technique that uses only one universal primer to perform multiplex polymerase chain reaction (PCR) was developed. Two reversely complementary common sequences were designed on the 5′ or 3′ end of the ligation probes (LPs), which allowed the ligation products to be amplified through only a single universal primer (SUP). SUP-MLPA products were analyzed on sequencing gel electrophoresis with extraordinary resolution. This method avoided the high expenses associated with capillary electrophoresis, which was the commonly used detection instrument. In comparison with conventional multiplex PCR, which suffers from low sensitivity, nonspecificity, and amplification disparity, SUP-MLPA had higher specificity and sensitivity and a low detection limit of 0.1ng for detecting single crop species when screening the presence of genetically modified crops. We also studied the effect of different lengths of stuffer sequences on the probes for the first time. Through comparing the results of quantitative PCR, the LPs with different stuffer sequences did not affect the ligation efficiency, which further increased the multiplicity of this assay. The improved SUP–MLPA and sequencing gel electrophoresis method will be useful for food and animal feed identification, bacterial detection, and verification of genetic modification status of crops. [Copyright &y& Elsevier]

In this article, we developed a novel PCR method, termed loop-linker PCR, to isolate flanking sequences in transgenic crops. The novelty of this approach is its use of a stem-loop structure to design a loop-linker adapter. The adapter is designed to form a nick site when ligated with restricted DNA. This modification not only can prevent the self-ligation of adapters but also promotes the elongation of the 3′ end of the loop-linker adapter to generate a stem-loop structure in the ligation products. Moreover, the suppressive effect of the stem-loop structure decreases nonspecific amplification and increases the success rate of the approach; all extension products will suppress exponential amplification except from the ligation product that contains the specific primer binding site. Using this method, 442, 1830, 107, and 512 bp left border flanking sequences were obtained from the transgenic maizes LY038, DAS-59122-7, Event 3272, and the transgenic soybean MON89788, respectively. The experimental results demonstrated that loop-linker PCR is an efficient, reliable, and cost-effective method for identifying flanking sequences in transgenic crops and could be applied for other genome walking applications. © 2012 IUBMB IUBMB Life, 64(10): 841-845, 2012 [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is a well-known opportunistic pathogen that can cause acute nosocomial necrotizing pneumonia and genetic disorder cystic fibrosis of lung patients. Pathogenic interactions between P. aeruginosa and hosts are often guided by the secreted virulence determinants that interact with specific host targets. Exotoxin A, pyocyanin, elastase, and type III secretion system are the most significant virulence determinants and cause great concern. However, P. aeruginosa in various environments has high genotypic diversity, leading to deficiency of exotoxin genes for some P. aeruginosa strains. In current study, a universal primer-multiplex PCR method (UP-MPCR) was employed for the detection of five significant enterotoxin genes ( toxA, phzM, lasB, ExoU, and ExoS) and one internal control gene ecfX in P. aeruginosa. Owing to the application of universal primer (UP), different targeted products have identical amplified efficiency and the sensitivity of multiplex PCR is improved. In addition, the complexity of multiplex PCR system is reduced and the compatibility of primers in a reaction is greatly increased. This UP-MPCR method can detect the presence of five P. aeruginosa enterotoxin genes in a single assay more rapidly and sensitively than conventional methods. In 214 drinking water and environmental isolates, the ExoU, ExoS, phzM, toxA, and lasB genes were detected in 20 (9 %), 180 (84 %), 179 (84 %), 196 (92 %), and 171 (80 %) isolates, respectively. [ABSTRACT FROM AUTHOR]

Cadmium is a heavy metal that is exceedingly hazardous to humans and can enter the body through tainted food or drink, causing severe harm. It is critical to develop a technology for detecting cadmium in food and water that is sensitive and accurate. One such approach, which employs nucleases, is uncommon. A cadmium(II) turn-on biosensor was successfully created in this work using repetitive cleavage of certain specific nucleases for signal conversion and sophisticated stem-loop qPCR (quantitative polymerase chain reaction) for quick signal amplification and output. The method has strong selectivity and sensitivity for precise quantification, with a detection limit of 6 nmol L−1, i.e. 0.948 g L−1, which is far lower than the 5.0 g L−1 set by the United States Environmental Protection Agency, and it also operates well in retail rice samples. [Display omitted] • The first speedy and accurate cadmium biosensor based on DNAzyme and stem-loop primers. • Excellent Cd2+ selectivity, target (Cd2+) cycling features, and increased signal intensity at low target concentrations. • Constructed stem-loop primer enhances the signal of the tiny single-stranded nucleic acid fragments generated by cleavage. • Resolving the issue of difficulties amplification of DNAzyme single-stranded cleavage products. • The label-free qPCR quantification approach, with a detection limit of 6 nmol L−1. [ABSTRACT FROM AUTHOR]

Highlights • An ultrafast, universal and visual screening method has been developed. • GM elements can be detected based on super PCR and lateral flow biosensor in 10-min. • Dual GM events can be amplified via Dual Super PCR in 2.5-min. • Visual results can be interpreted by the universal lateral flow biosensor. • The LOD of the biosensor achieves nine copies in maize. Abstract In this study, a cascade screening system has been developed combining D ual S uper P olymerase C hain R eaction (DSPCR) with the universal L ateral F low B iosensor (LFB) for the ultrafast, universal and visual screening of dual GM elements, taking P- 35s × T- nos for example. In the design of DSPCR for universal screening, gene-specific forward primers were labelled with biotin and gene-specific reverse primers were tagged with Cy5 and digoxin, respectively. In 2.5-min, DSPCR effectively amplified the dual target fragments through our prototype facility. Then, through specific antigen-antibody binding, a universal lateral flow biosensor exported visually dual-amplified results simultaneously without cross contamination. After optimization, the detection limit allowed 0.05% GM maize, corresponding to nine copies in maize. The entire detection process could be achieved in 10 min without any large-scale instrumentation. This method may be useful for the ultrafast, universal and visual screening of dual GM elements (P- 35s × T- nos) in GM crop lines and is expected to be of great promise for rapid GMO screening and point-of-care tests. [ABSTRACT FROM AUTHOR]

Highlights • The detection system was driven by Zn2+-dependent Cleavage DNAzyme (17E). • A "turn-off"fluorescent quantitative biosensor based on the cleaved substrate chains unsuitable for RT-qPCR. • This detection system is ultra-sensitive with the minimum limit of 58.61 pM. • In this study, the most active cDNAzyme was selected. • No largE–Scale instruments were needed during the entire detection process. Abstract Although it is normal for trace elements of zinc (Zn2+) to be present in the human body, the lack or excess thereof will affect general human health. Due to the low content of Zn2+ found in the environment, detection technology possessing enhanced sensitivity capabilities is necessary for its identification. For the purpose of this study, a "turn-off" ultra-sensitive fluorescent quantitative biosensor was established, based on Zn2+-dependent Cleavage DNAzyme for the sensitive and specific recognition of Zn2+. Furthermore, the original sequence of the substrate chain, as well as the added primer-binding sequences on either side of the substrate chain were examined without destroying the catalytic and cleavage sites of the enzyme. In the presence of Zn2+, the biosensor circulates continuously to promote catalyzation of the substrate chains cleavage by 17E at the proper temperature. The cleaved substrate chains cannot be used as templates for amplification reactions, while the uncleaved substrate chains can be expanded by PCR amplification and synthesized into large amounts of double-stranded DNA to produce a strong fluorescent signal. Consequently, the cutting effect is demonstrated by the change in the Cq value (ΔCq) both before and after the separation process. The larger the ΔCq value, the smaller the remaining substrate chain, and the better the cutting effect. Sensitivity, detection limit, and specificity analysis, indicates that the Zn2+ quantitative detection system established in this paper displays high sensitivity and specificity. The minimum limit of detection (LOD) can reach at 58.61 pM, which dramatically improves the sensitivity of Zn2+ detection. In conclusion, a simple, rapid, accurate quantitative detection system for Zn2+ was established with both high sensitivity and specificity. [ABSTRACT FROM AUTHOR]

To prevent the propagation of pathogens and minimize the damage to humans, an ultrasensitive and colorimetric dual detection method for pathogens was developed and validated based on the Multiplex Super Polymerase Chain Reaction (MS-PCR) and Asymmetric Tailing Hybridization Chain Reaction (AT-HCR). In the 5-min MS-PCR, dual pathogens were recognized and amplified simultaneously, generating plenty of dsDNA products with protruding ssDNA toeholds. Since a blocker of oxyethyleneglycol bridge was exquisitely inserted into two-set forward primers, which hindered the polymerase extension. Then, two-set ssDNA toeholds initiated two-set AT-HCR in 20-min and 60-min visual analysis depending on G-quadruplex DNAzyme was induced by Terminal deoxynucleotidyl Transferase (TdT) at the tails of AT-HCR at 3′-OH. During the process, the first magnetic separation was conducted after MS-PCR based on our home-made XP beads, to remove primer-dimers and other impurities in 30-min. The second magnetic separation was performed before TdT catalysis based on dynabeads, to eliminate unreacted AT-HCR hairpins in 15-min. To challenge the practical application capability of this strategy, the detection of analyte in fat-free milk was also tested and demonstrated similar detection capability to visualize single cell. Therefore, the proposed sensor exhibited a high selectivity and sensitivity and is thus applicable for visual single cell detection of dual-pathogens of Salmonella spp. and Staphylococcus aureus simultaneously in 130-min. Generally, it provides a visual, ultrasensitive, rapid and universal detective method for dual-target analysis. [ABSTRACT FROM AUTHOR]

The genes commonly used to determine meat species are mainly mitochondrial, but the copy numbers of such genes are high, meaning they cannot be accurately quantified. In this paper, for the first time, the chromosomal gene Actb was selected as an endogenous reference gene for chicken species. It was assayed in four different chicken varieties and 16 other species using both qualitative and quantitative PCR. No amplification of the Actb gene was found in species other than chicken and no allelic variations were detected in chicken. Southern blot and digital-PCR confirmed the Actb gene was present as a single copy in the chicken genome. The quantitative detection limit was 10 pg of DNA, which is equivalent to eight copies. All experiments indicated that the Actb gene is a useful endogenous reference gene for chicken, and provides a convenient and accurate approach for detection of chicken in feed and food. [ABSTRACT FROM AUTHOR]

Here, we developed an ultra-sensitive and absolute quantitative detection method of Cu 2+ based on DNAzyme and digital PCR. The binding model between DNAzyme and Cu 2+ and the influence caused by the additional primer sequence were revealed to ensure quantitation independent of standard curves. The binding model of DNAzyme and Cu 2+ showed that one molecular DNAzyme could bind one Cu 2+ in the biosensor step. Thus, the final quantitative results, evaluated by three parallels, showed that the limit of quantitation (LOQ) was as low as 0.5 pmol, while the sensitivity was evaluated as 50 fmol. The specificity evaluation of our methodologies shows that extremely low crossing signal is existed within the non-specific ions. Moreover, the results of practical detection have shown that the quantitative results were stable and accurate among different food substrates. In conclusion, a flexible quantitative detection method with ultra-sensitivity was developed to detect trace amounts Cu 2+ within different substrates. [ABSTRACT FROM AUTHOR]

The first example of droplet digital PCR logic gates (“YES”, “OR” and “AND”) for Hg (II) and Ag (I) ion detection has been constructed based on two amplification events triggered by a metal-ion-mediated base mispairing (T-Hg(II)-T and C-Ag(I)-C). In this work, Hg(II) and Ag(I) were used as the input, and the “true” hierarchical colors or “false” green were the output. Through accurate molecular recognition and high sensitivity amplification, positive droplets were generated by droplet digital PCR and viewed as the basis of hierarchical digital signals. Based on this principle, YES gate for Hg(II) (or Ag(I)) detection, OR gate for Hg(II) or Ag(I) detection and AND gate for Hg(II) and Ag(I) detection were developed, and their sensitively and selectivity were reported. The results indicate that the ddPCR logic system developed based on the different indicators for Hg(II) and Ag(I) ions provides a useful strategy for developing advanced detection methods, which are promising for multiplex metal ion analysis and intelligent DNA calculator design applications. [ABSTRACT FROM AUTHOR]

Sensor-based detection methods have inspired the idea that chemical or physical signals could be converted to nucleic acid signals to be quantitatively detected using a combination of appropriate detection tools. To achieve ultra-sensitive and absolute quantitative detection of mercury ion (Hg 2+ ), we have combined a mispairing biosensor for Hg 2+ and emulsion PCR. The parameters that might influence the biosensor step, such as the duration of isothermal amplification and the concentration of the sensor oligonucleotide, have been firstly optimized in our study to achieve the most efficient biosensor detection. The evaluation results of secondary structures between the biosensors with different number of T-Hg-T structures achieved by Circular Dichroism have indicated that the secondary hairpin structure would be varied according to the change of number of T-Hg-T structures, which could influence the quantitative detection results. Further optimization of number of T-Hg-T within the biosensor sequences showed that 5 T-Hg-T structures could generate the most efficient amplification. After the above optimizations, the emulsion PCR has been employed to achieve the absolute quantitation of nucleic acid signals. The final results have shown that the limit of quantitation (LOQ) in our study was as low as 40 fmol, and the limit of detection (LOD) was 10 fmol. The practical detection tests showed that the quantitative results were stable and accurate for all substrates. In conclusion, by combining a mispairing biosensor with emulsion PCR, we developed a flexible and stable quantitative “turn-on” detection method with ultra-sensitivity that can detect trace amounts Hg 2+ within different substrates. [ABSTRACT FROM AUTHOR]

The possibility of the absolute quantitation of GMO events by digital PCR was recently reported. However, most absolute quantitation methods based on the digital PCR required pretreatment steps. Meanwhile, singleplex detection could not meet the demand of the absolute quantitation of GMO events that is based on the ratio of foreign fragments and reference genes. Thus, to promote the absolute quantitative detection of different GMO events by digital PCR, we developed a quantitative detection method based on duplex digital PCR without pretreatment. Moreover, we tested 7 GMO events in our study to evaluate the fitness of our method. The optimized combination of foreign and reference primers, limit of quantitation (LOQ), limit of detection (LOD) and specificity were validated. The results showed that the LOQ of our method for different GMO events was 0.5%, while the LOD is 0.1%. Additionally, we found that duplex digital PCR could achieve the detection results with lower RSD compared with singleplex digital PCR. In summary, the duplex digital PCR detection system is a simple and stable way to achieve the absolute quantitation of different GMO events. Moreover, the LOQ and LOD indicated that this method is suitable for the daily detection and quantitation of GMO events. [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance Many Chinese herbs are traditionally used as medicine to improve the functions of gastrointestinal tract. Some of these herbs are also promising agents for the improvement of the gut microbiota and the treatment of ulcerative colitis. Materials and methods By screening seven traditional Chinese herbs, we found that Red Ginseng and Semen Coicis were the most effective in promoting the growth of probiotics including Lactobacillus and Bifidobacterium in vitro . We then evaluated the effects of Red Ginseng and Semen Coicis on the growth of the bacterial pathogens (Escherichia coli , Staphylococcus aureus , and Salmonella spp.) in vitro . In in vivo experiment, we gavage administrated trinitro–benzene–sulfonic acid induced ulcerative colitis (UC) rats with Red Ginseng and Semen Coicis extracts. After two weeks treatment, we analyzed the structure of the gut microbiota and examined the UC symptoms by employing qPCR and animal pathology detection techniques. Results Both Red Ginseng and Semen Coicis promoted the growth of probiotics – Bifidobacterium and Lactobacillus in vitro . Red Ginseng also inhibited the growth of some pathogen strains. In vivo, Red Ginseng and Semen Coicis improved the structure of gut microbiota and relieved the symptoms of ulcerative colitis in vivo . Compared with Semen Coicis , Red Ginseng was more effective in relieving the symptoms of ulcerative colitis. Conclusions Red Ginseng could promote the growth of probiotic bacteria in vitro. Red Ginseng and, to a lesser extent Semen Coicis , gave positive results in an experimental in vivo model for ulcerative colitis. [ABSTRACT FROM AUTHOR]

To develop an on-site, thermostatic and rapid sensor for the detection of Vibrio parahaemolyticus (V. parahaemolyticus) , A single cross-priming fluorescence (SCPF) sensor was designed using a 3D nano-nucleic acid hybrid material that termed mesoporous silica nanoparticle/nucleic acid-doped nanoflower (MSN/NA-doped nanoflower). In addition, a portable polymerase chain reaction (PCR) tube fluorescence reader was built. Further analysis of the MSN/NA-doped nanoflower morphology and the enhancement mechanism indicated that the MSNs aggregated into larger nanoclusters by adsorbing single cross-priming amplification (sCPA) components, forming MSNs/NAs-doped nanoflowers, and increasing the local concentrations to enhance sCPA efficiency. Cyclic amplification relied mainly on the self-folding hairpin-like structure of the amplified product that continuously formed, opened, re-formed, and opened again. The target DNA was detected with a detection limit of 2.4 copies/μL. [Display omitted] • MSNs/NAs-doped nano-flower enhanced SCPF sensor was firstly developed. • The formation mechanism and composition of MSNs/NAs-doped nano-flower were firstly discovered and analyzed. • MSNs preferentially adsorb single-stranded primers and dNTPs rather than ds templates, and the adsorption of buffers is weak. • MSNs/NAs-doped nano-flower increased the amplification efficiency to improve the detection sensitivity. • Design of an on-site PCR tube fluorescence reader. [ABSTRACT FROM AUTHOR]

Abstract: The restriction enzyme cutting site distribution regularity and looping conditions were studied systematically. We obtained the restriction enzyme cutting site distributions of 13 commonly used restriction enzymes in 5 model organism genomes through two novel self-compiled software programs. All of the average distances between two adjacent restriction sites fell sharply with increasing statistic intervals, and most fragments were 0–499bp. A shorter DNA fragment resulted in a lower looping rate, which was also directly proportional to the DNA concentration. When the length was more than 500bp, the concentration did not affect the looping rate. Therefore, the best known fragment length was longer than 500bp, and did not contain the restriction enzyme cutting sites which would be used for digestion. In order to make the looping efficiencies reach nearly 100%, 4–5 single cohesive end systems were recommended to digest the genome separately. [Copyright &y& Elsevier]

Abstract: Among the methods used to detect food adulteration, the amplification of endogenous reference genes is particularly important. Endogenous reference genes for many different species, such as cotton, papaya, maize and others, have been reported, yet an endogenous reference gene for the peach is still lacking. In this paper, the chlorophyll a/b-binding protein (Lhcb2) gene was identified as a species-specific gene for the peach. Lhcb2 was assayed in 4 species of peaches and 8 non-peach species by both qualitative and quantitative PCR. No amplification was observed in other species. The detection limit of quantitative PCR was as low as 5 pg of DNA, equal to 9 copies, and Southern blot analysis confirmed that the Lhcb2 gene was present in a single copy in the peach genome. All of these experiments indicated that the Lhcb2 gene is a useful endogenous reference gene for the detection of peach material via both qualitative and quantitative PCR assays, even in the processed food samples such as juices containing peach. [Copyright &y& Elsevier]

Abstract: The genetically-modified (GM) soybean 3Ø5423×40-3-2 expresses siRNA for the fatty acid desaturase-2 enzyme which results in higher concentrations of oleic acid (18:1) relative to linoleic acid (18:2) compared with non-GM soybeans. It also expresses the CP4 EPSPS protein for tolerance to glyphosate. In this study, three different dietary concentrations (7.5%, 15% and 30% wt/wt) of 3Ø5423×40-3-2 or non-GM soybeans were fed to Sprague–Dawley rats for 90days during which in-life nutritional and growth performance variables were evaluated followed by analysis of standard clinical chemistry, hematology and organ variables. Compared with rats fed the non-GM control diet, some statistically significant differences were observed in rats fed the 3Ø5423×40-3-2 diet. However the differences were not considered treatment-related and commonly fell within the normal ranges of the control group consuming the commercial diet. These results demonstrated that the GM soybean 3Ø5423×40-3-2 is as safe as non-GM soybeans. [Copyright &y& Elsevier]

The polymerase chain reaction (PCR)-based genome walking method has been extensively used to isolate unknown flanking sequences, whereas nonspecific products are always inevitable. To resolve these problems, we developed a new strategy to isolate the unknown flanking sequences by combining A-T linker adapter PCR with inverse PCR (I–PCR) or thermal asymmetric interlaced PCR (TAIL–PCR). The result showed that this method can be efficiently achieved with the flanking sequence from the Arabidopsis mutant and papain gene. Our study provides researchers with an additional method for determining genomic DNA flanking sequences to identify the target band from bulk of bands and to eliminate the cloning step for sequencing. [ABSTRACT FROM AUTHOR]

• A novel multiplex quantitative detection method uses pyrosequencing is proposed. • Pyrosequencing quantitative result analysis software was first self-compiled. • One unique sequencing peak represents for the existence of the specific target. • The detection sensitivity is as low as 1.47% of single GM maize in a mixture. • UP-M-PCR combined with pyrosequencing can realize multiplex quantitative detection. A multiplex pyrosequencing quantitative detection technique combined with universal primer-multiplex-PCR (UP-M-PCR) was established. In this study, a pyrosequencing results analysis software was first self-compiled, which realized the DNA sequences degeneration, and converted the pyrosequencing results and base composition of the target sequences into mathematic relations. Five calculation models were put forward based on the actual situation, which adjusted the values smaller than zero or the detection limit. By applying this method, samples containing five genetically modified (GM) lines mixed in random ratio were quantified, it showed that the quantification was very close to the actual value, and the detection sensitivity was as low as 1.47% of a single component, which satisfied most labeling policies. This novel method is realized without fluorescent group labeling, hence the number of targets is not limited by factors inherent in method or equipment, and is proven to be a reliable tool for the quantitative detection. [ABSTRACT FROM AUTHOR]

Tricholoma matsutake is a rare, precious, and wild edible fungus that could not be cultivated artificially until now. This situation has given way to the introduction of fake T. matsutake commodities to the mushroom market. Among the methods used to detect food adulteration, amplification of species-specific diagnostic marker is particularly important and accurate. In this study, the Pol gene is reported as a species-specific diagnostic marker to identify three T. matsutake varieties and 10 other types of edible mushrooms through qualitative and quantitative PCR. The PCR results did not reveal variations in the amplified region, and the detection limits of qualitative and quantitative PCR were found to be 8 ng and 32 pg, respectively. Southern blot showed that the Pol gene exists as a single copy in the T. matsutake genome. The method that produced the purest DNA of T. matsutake in this study was also determined, and the high-concentration salt precipitation method was confirmed to be the most suitable among the methods tested. The assay proposed in this work is applicable not only to the detection of raw materials but also to the examination of processed products containing T. matsutake. [ABSTRACT FROM AUTHOR]

Digital PCR has developed rapidly since it was first reported in the 1990s. It was recently reported that an improved method facilitated the detection of genetically modified organisms (GMOs). However, to use this improved method, the samples must be pretreated, which could introduce inaccuracy into the results. In our study, we explored a pretreatment-free digital PCR detection method for the screening for GMOs. We chose the CaMV35s promoter and the NOS terminator as the templates in our assay. To determine the specificity of our method, 9 events of GMOs were collected, including MON810, MON863, TC1507, MIR604, MIR162, GA21, T25, NK603 and Bt176. Moreover, the sensitivity, intra-laboratory and inter-laboratory reproducibility of our detection method were assessed. The results showed that the limit of detection of our method was 0.1%, which was lower than the labeling threshold level of the EU. The specificity and stability among the 9 events were consistent, respectively. The intra-laboratory and inter-laboratory reproducibility were both good. Finally, the perfect fitness for the detection of eight double-blind samples indicated the good practicability of our method. In conclusion, the method in our study would allow more sensitive, specific and stable screening detection of the GMO content of international trading products. [ABSTRACT FROM AUTHOR]