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Genetic engineering (GM) has great potential to improve maize productivity, but rises some concerns on unintended effects, and equivalent as their comparators. There are some limitations through targeted analysis to detect the UE in genetically modified organisms in many previous studies. We here reported a case-study on the effects of introducing herbicides and insect resistance (HIR) gene cassette on molecular profiling (transcripts and metabolites) in a popular maize variety Zhengdan958 (ZD958) in China. We found that introducing HIR gene cassette bring a limited numbers of differential abundant genes (DAGs) or differential abundant metabolites (DAMs) between transgenic events and non-transgenic control. In contrast, averaged 10 times more DAGs and DAMs were observed when performed comparison under different growing environments in three different ecological regions of China than the numbers induced by gene effects. Major biological pathways relating to stress response or signaling transduction could explain somehow the effects of growing environments. We further compared two transgenic events mediated ZD958 (GM-ZD958) with either transgenic parent GM-Z58, and other genetic background nonGM-Z58, nonGM-ZD958, and Chang7-2. We found that the numbers of DAGs and DAMs between GM-ZD958 and its one parent maize variety, Z58 or GM-Z58 is equivalent, but not Chang7-2. These findings suggest that greater effects due to different genetic background on altered molecular profiling than gene modification itself. This study provides a case evidence indicating marginal effects of gene pleiotropic effects, and environmental effects should be emphasized.

Seed vigour is an important trait and is often used to evaluate seed quality. A rapid and accurate evaluation of seed vigour is very important for agricultural production. Ca2+ is an important secondary messenger in plants, responding to various biotic and abiotic stimuli by Ca2+ flux into cytoplasm. To the best of our knowledge, how-ever, no report has been published about seed vigour and Ca2+ influx. In this study, we used two hybrid maize (Zea mays L.) lines and their corresponding female parent lines as materials, and performed ageing and ‘ageing + priming’ treatments to obtain seeds with different vigour levels. After seeds were imbibed for 24 h, the intact seeds or embryos were used as materials for determining the Ca2+ influx rate using non-invasive micro-test technique (NMT). Results showed that, with the intact embryos as materials, the Ca2+ influx rate showed higher stability and higher values. Correlation analysis indicated that there was a significant, positive linear correlation between the shoot dry weight vigour index and Ca2+ flux rate into embryo. The results demonstrated that the Ca2+ influx rate can be used to evaluate the vigour levels of maize seeds.

Genetically modified (GM) organisms have been developed for decades. However, unintended effects are the main concerns of safety assessment that needs to be carefully investigated. Here, eight varieties of GM rice that were developed in China were selected to assess the unintended effects through transcriptome and metabolism. There are 2892–8758 differentially expressed genes (DEGs) and 7–50 metabolites at significant level between GM varieties and their isogenic counterparts, which were far fewer than that between traditional rice varieties. The function enrichment analysis showed altered transcription in stress-related pathway and starch and sucrose metabolism. DEGs shared among eight GM samples constitute less than 1% of the genes in the genome, and none of them is reported more than four times. The insertion effect on the nearby gene expression and the associated metabolism is only restricted to 50 genes. All the results provide a comprehensive analysis of unintended effects and indication of difference in Chinese transgenic rice based on their backgrounds, transformation, and insertion elements.

Seed vigor is influenced by seed position in plant. However, current understanding of its underlying mechanism is limited. In this study, we used isobaric tags for relative and absolute quantitation technique to study the comparative proteomes between middle seeds (with higher vigor) and top seeds of maize (Zea mays L.) ears at 0 h, 24 h, and 48 h of imbibition. A total of 159 differentially accumulated proteins were identified. Among these, the largest number of proteins was from the functional categories of Disease/Defense and Metabolism. Compared with top seeds, most of the differentially accumulated proteins of Protein Synthesis and Energy showed higher accumulation in middle seeds at 0 h and 24 h of imbibition, but lower accumulation at 48 h of imbibition. Seed water absorption activates metabolic processes. The water content of middle seeds was significantly lower than that of top seeds at between 12 h and 30 h of imbibition, but energy production would be higher in the middle seeds at 24 h of imbibition. Meanwhile, tonoplast intrinsic proteins 3.1 and 3.2, which mediate water inflow into protein storage vacuoles, then activating enzymes involved in reserve mobilization, showed higher accumulation in middle seeds at 24 h of imbibition. In addition, our data also showed middle seeds may suffer less fungal damages. Our results contribute to understanding the mechanisms underlying the effects of growth position on seed vigor.

Genetically modified organisms (GMO) are increasingly gaining acceptance, which has led to an increasing demand for high-throughput methods for their detection. This study describes development of a high-throughput and low-cost multiplex tandem PCR (MT-PCR) assay for the screening of transgenic maize of 14 targets, containing 7 genetically modified (GM) common screening elements, 6 transgenic maize events and a maize reference gene, using SYBR Green I for quantification. The 14-target high-throughput multiplex tandem PCR assay successfully amplified products from single-event samples with a GM maize DNA amount less to 0.001 ng, and displayed high specificity. This assay can efficiently be used for screening GM maize in foods and feeds.

Among all of the high-throughput detection methods, PCR-based methodologies are regarded as the most cost-efficient and feasible methodologies compared with the next-generation sequencing or ChIP-based methods. However, the PCR-based methods can only achieve multiplex detection up to 15-plex due to limitations imposed by the multiplex primer interactions. The detection throughput cannot meet the demands of high-throughput detection, such as SNP or gene expression analysis. Therefore, in our study, we have developed a new high-throughput PCR-based detection method, multiplex enrichment quantitative PCR (ME-qPCR), which is a combination of qPCR and nested PCR. The GMO content detection results in our study showed that ME-qPCR could achieve high-throughput detection up to 26-plex. Compared to the original qPCR, the Ct values of ME-qPCR were lower for the same group, which showed that ME-qPCR sensitivity is higher than the original qPCR. The absolute limit of detection for ME-qPCR could achieve levels as low as a single copy of the plant genome. Moreover, the specificity results showed that no cross-amplification occurred for irrelevant GMO events. After evaluation of all of the parameters, a practical evaluation was performed with different foods. The more stable amplification results, compared to qPCR, showed that ME-qPCR was suitable for GMO detection in foods. In conclusion, ME-qPCR achieved sensitive, high-throughput GMO detection in complex substrates, such as crops or food samples. In the future, ME-qPCR-based GMO content identification may positively impact SNP analysis or multiplex gene expression of food or agricultural samples. Graphical abstract For the first-step amplification, four primers (A, B, C, and D) have been added into the reaction volume. In this manner, four kinds of amplicons have been generated. All of these four amplicons could be regarded as the target of second-step PCR. For the second-step amplification, three parallels have been taken for the final evaluation. After the second evaluation, the final amplification curves and melting curves have been achieved.

Techniques of isothermal amplification have recently made great strides, and have generated significant interest in the field of point-of-care detection. Nicking endonuclease-mediated isothermal amplification (NEMA) is an example of simple isothermal technology. In this paper, a real-time quantitative nicking endonuclease-mediated isothermal amplification with small molecular beacons (SMB-NEMA) of improved specificity and sensitivity is described. First, we optimized the prohibition of de novo synthesis by choosing Nt·BstNBI endonuclease. Second, the whole genome was successfully amplified with Nt·BstNBI (6 U), betaine (1 M) and trehalose (60 mM) for the first time. Third, we achieved 10 pg sensitivity for the first time after adding a small molecular beacon that spontaneously undergoes a conformational change when hybridizing to target, and the practical test validated the assay's application. The small molecular beacon has a similar melting temperature to the reaction temperature, but is approximately 10 bp shorter than the length of a traditional molecular beacon. A new threshold regulation was also established for isothermal conditions. Finally, we established a thermodynamic model for designing small molecular beacons. This multistate model is more correct than the traditional algorithm. This theoretical and practical basis will help us to monitor SMB-NEMA in a quantitative way. In summary, our SMB-NEMA method allows the simple, specific and sensitive assessment of isothermal DNA quantification.

The purpose of this study is to apply the visual qualitative (LFD-LAMP) and advanced quantitative loop-mediated isothermal amplification (EVA-LAMP) methods to detect Listeria monocytogenes, which is an important pathogenic foodborne bacterium. This research designed a new set of primers to amplify the hlyA gene of L. monocytogenes. The LFD-LAMP products were detected by lateral flow device (LFD) with the naked eyes, and the test results were consistent with agarose gel electrophoresis. Meanwhile, an advanced quantitative LAMP assay was established which depended on the EVA Green. The detection limit of the EVA-LAMP method was 10 pg/μL, and a good linear relationship was obtained. The subsequent analysis of contaminated raw food samples showed that the primers were able to identify L. monocytogenes in food. The LFD-LAMP and EVA-LAMP reactions can be used as sensitive, rapid and simple methods for the detection of L. monocytogenes, especially on field detection. Practical Applications This work suggests a specific, sensitive, cost-effective and nonpollution LAMP assay for the detection of Listeria monocytogenes. A new set of hlyA-based primers was designed and successfully used to detect L. monocytogenes in raw food samples. Amplicon detection by visual LFD method proved to be as useful as the gel, but more convenient and clean. The LFD-LAMP detection method in this study testifies as a significant advancement toward making LAMP a potential on-site detection. Otherwise, in contrast to the traditional real-time LAMP approach, this is a novel study to describe a real-time quantitative LAMP assay based on EVA Green to detect L. monocytogenes in the fields of food safety.

Purpose. This study aimed to investigate the possible effects and underlying mechanisms of Kangzhi syrup on ovalbumin- (OVA-) induced cough variant asthma (CVA) in guinea pigs. Methods. All 48 guinea pigs were randomly assigned to four experimental groups: normal, OVA model with or without Kangzhi syrup (OVA and OVA + KZ), and OVA with Dexamethasone (OVA + DM). After sensitizing the guinea pigs, a cough challenge was performed by the inhalation of capsaicin. The antitussive effect, inflammatory cells, cytokines in bronchoalveolar lavage fluid (BALF) and lung tissue, and morphological changes were examined. Results. Compared with model group, Kangzhi syrup effectively exerted an antitussive effect (p<0.0001) and reduced the pneumonic anaphylacticitis by inhibiting the infiltration of total inflammatory cells (p<0.0001) and reducing the percentage of eosinophil in BALF (p<0.0001). Moreover, evidence from morphological studies also demonstrated that Kangzhi syrup inhibited the infiltration of inflammatory cells and ameliorated the structure changes. NF-κB and TGF-β1 expression were attenuated in the OVA + KZ group versus the OVA group (p<0.0001). Additionally, a semiquantitative analysis of TGF-β1 expression also demonstrated that the Kangzhi syrup attenuated this profibrogenic growth factor (p<0.001). Conclusions. The results demonstrated that Kangzhi syrup exerted a considerable antitussive effect in CVA animal model, which depended on its marked impact on the anti-anaphylactic inflammation. Additionally, it could ameliorate the airway remodeling by inhibiting NF-κB and TGF-β1 signal pathway.

BACKGROUND A SYBR Green I-based quantitative loop-mediated isothermal amplification (LAMP) assay was developed for the rapid detection of genetically modified maize MON863. A set of primers was designed based on the integration region of the Cry3Bb1 and tahsp17 genes. RESULTS The qualitative and quantitative reaction conditions (dNTPs, betaine, primers, Mg2+, Bst polymerase, temperature, reaction time) were optimized. The concentrations of Mg2+ and betaine were found to be important to the LAMP assay. The detection limits of both qualitative and quantitative LAMP for MON863 were as low as 4 haploid genomic DNA, and the LAMP reactions can be completed within 1 h at an isothermal temperature of 65 °C. CONCLUSION The results of this study demonstrate that this new SYBR Green I-based quantitative LAMP assay system is reliable, sensitive and accurate. © 2014 Society of Chemical Industry

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been widely applied in functional genomics research and plant breeding. In contrast to the off-target studies of mammalian cells, there is little evidence for the common occurrence of off-target sites in plants and a great need exists for accurate detection of editing sites. Here, we summarized the precision of CRISPR/Cas9-mediated mutations for 281 targets and found that there is a preference for single nucleotide deletions/insertions and longer deletions starting from 40 nt upstream or ending at 30 nt downstream of the cleavage site, which suggested the candidate sequences for editing sites detection by whole-genome sequencing (WGS). We analyzed the on-/off-target sites of 6 CRISPR/Cas9-mediated Arabidopsis plants by the optimized method. The results showed that the on-target editing frequency ranged from 38.1% to 100%, and one off target at a frequency of 9.8%&ndash, 97.3% cannot be prevented by increasing the specificity or reducing the expression level of the Cas9 enzyme. These results indicated that designing guide RNA with high specificity may be the preferred factor to avoid the off-target events, and it is necessary to predict or detect off-target sites by WGS-based methods for preventing off targets caused by genome differences in different individuals.

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.

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 0-499 bp. 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 500 bp, the concentration did not affect the looping rate. Therefore, the best known fragment length was longer than 500 bp, 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.

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.

High throughput screening systems are the preferred solution to meet the urgent requirement of increasing number of genetically modified organisms (GMOs). In this study, we have successfully developed a multiplex GMO element screening system with dual priming oligonucleotide (DPO) primers. This system can detect the cauliflower mosaic virus 35S (CaMV 35S), terminator of nopaline synthase gene (NOS), figwort mosaic virus 35S (FMV 35S) promoter, neomycin phosphotransferaseII (NPTII), Bt Cry 1Ab, phosphinothricin acetyltransferase genes (bar) and Streptomyces viridochromogenes (pat) simultaneously, which covers more than 90% of all authorized GMO species worldwide. This system exhibits a high tolerance to annealing temperatures, high specificity and a limit of detection equal to conventional PCR. A total of 214 samples from markets, national entry-exit agencies, the Institute for Reference Materials and Measurement (IRMM) and the American Oil Chemists' Society (AOCS) were also tested for applicability. This screening system is therefore suitable for GMO screening.

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.

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 10pg 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.

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.

Many genome walking methods based on polymerase chain reaction (PCR) are available, including those with and without restriction enzyme modification. Nevertheless, these methods suffer from low reproducibility, inefficiency, and non-specificity. Here, we present a traceable and efficient PCR strategy: randomly broken fragment PCR with 5′ end-directed adaptor for genome walking. The genome is first fragmented randomly. After blunting ends, the fragments are ligated to the 5′ end-directed adaptors. Semi-nested PCR is then performed. Thus, we can obtain an unknown sequence by cloning the fragments of interest, followed by sequencing. This method effectively bypasses the above-mentioned obstacles and offers the advances: 1) genome fragmentation without using restriction enzymes; 2) enhancement of primer specificity and the prevention of self-ligation between the adaptors by employing a 5′ end-directed adaptor. All of the steps in this new method are straightforward, and the unknown sequence can be definitively obtained by merely applying the method once.

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 0.1 ng 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.

Digital polymerase chain reaction (PCR) has developed rapidly since it was first reported in the 1990s. However, pretreatments are often required during preparation for digital PCR, which can increase operation error. The single-plex amplification of both the target and reference genes may cause uncertainties due to the different reaction volumes and the matrix effect. In the current study, a quantitative detection system based on the pretreatment-free duplex chamber digital PCR was developed. The dynamic range, limit of quantitation (LOQ), sensitivity and specificity were evaluated taking the GA21 event as the experimental object. Moreover, to determine the factors that may influence the stability of the duplex system, we evaluated whether the pretreatments, the primary and secondary structures of the probes and the SNP effect influence the detection. The results showed that the LOQ was 0.5% and the sensitivity was 0.1%. We also found that genome digestion and single nucleotide polymorphism (SNP) sites affect the detection results, whereas the unspecific hybridization within different probes had little side effect. This indicated that the detection system was suited for both chamber-based and droplet-based digital PCR. In conclusion, we have provided a simple and flexible way of achieving absolute quantitation for genetically modified organism (GMO) genome samples using commercial digital PCR detection systems.