[Show abstract][Hide abstract] ABSTRACT: Protoplasts have been widely used for genetic transformation, cell fusion, and somatic mutation due to the absence of a cell wall. However, without the protection of a cell wall, protoplasts are easy to rupture and aggregate during washing, collecting, and gene transfection. In this work, we propose a simple and effective silica/alginate two-step method to immobilize protoplasts with advantages in experimental manipulation and microscopic imaging, as well as in potentially studying cell biological processes such as secretion and metabolism. The proposed two-step immobilization method adopts Transwell with clear tissue culture-treated membrane to support protoplasts in the form of uniform thin layer, which has three unique properties.
[Show abstract][Hide abstract] ABSTRACT: The number of species and planting areas of genetically modified organisms (GMOs) has been rapidly developed during the past ten years. For the purpose of GMO inspection, quarantine and manipulation, we have now devised a high-throughput Bt-based GMOs screening method based on the liquid bead array. This novel method is based on the direct competitive recognition between biotinylated antibodies and beads-coupled antigens, searching for Bt presence in samples if it contains Bt Cry1 Aa, Bt Cry1 Ab, Bt Cry1 Ac, Bt Cry1 Ah, Bt Cry1 B, Bt Cry1 C, Bt Cry1 F, Bt Cry2 A, Bt Cry3 or Bt Cry9 C. Our method has a wide GMO species coverage so that more than 90% of the whole commercialized GMO species can be identified throughout the world. Under our optimization, specificity, sensitivity, repeatability and availability validation, the method shows a high specificity and 10-50ng/mL sensitivity of quantification. We then assessed more than 1800 samples in the field and food market to prove capacity of our method in performing a high throughput screening work for GMO manipulation. Our method offers an applicant platform for further inspection and research on GMO plants.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Though special nodes play an important role in spreading invasive species, most attempts have focused on modeling the invasive pathway. Few invasion hotspots, such as botanic gardens and greenhouses, as well as their roles in introducing alien species, have been investigated. There are some investigations about the importance of botanic gardens in the biological invasion process, but few centers on the same role that greenhouses play. This study pays more attention on the intermediate role of greenhouses in spreading alien species which can escape into neighboring habitats without appropriate supervision and management. The pest risk analysis and IPM should take greenhouses as a hotspot of invasive network, and more information on invasive species of greenhouse must be recorded and shared.
Biodiversity and Conservation 07/2015; 24(7). DOI:10.1007/s10531-015-0876-x · 2.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The number of species in an area is a fundamental parameter in species-area theory. Besides sampling size and statistical extrapolation, the inaccurate number of species can intensify the existing controversy on the species richness—productivity and extinction rate overestimate/underestimate. This problem can be caused by Matthew effect from sampling process and taxonomic identification: it is much easier to identify and record common species than rare species. Therefore, it is necessary to prevent and control the Matthew effect, which can distort the number of species (especially the rare species) and the individuals of each species.
Biodiversity and Conservation 07/2015; 24(11). DOI:10.1007/s10531-015-0956-y · 2.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: With ongoing development of global economy and increasing trade between countries, China faces increasingly serious invasions by alien species, causing great harm and potential disasters to agriculture, forestry and natural environment. According to a national survey of invasive alien species in China, there are 265 species of invasive alien plants and 171 species of invasive alien animals in China, most of which are widely distributed. In general, there are more invasive species, either plants or animals, in the south than in the north, and more in coastal areas than in interior areas. The distribution of first detection locations of invasive alien plants and animals shows a similar pattern. The cluster analyses showed that the distribution of invasive alien plants and animals was significantly influenced by geographical region, and the alien species of invasive plants and animals were similar in the same geographical region. Thus, the overall distribution of invasive alien plants and animals are spatially similar in China. The results remind us an ongoing invasion pressure from other countries to China and from the provinces with more invasive species to the provinces with less invasive species. Considering different biological and ecological characteristics of plants and animals, common social-economic factors and environmental conditions in each province lead to such similar spatial patterns, supporting the distribution prediction of establishment possibility based on the invasive pest assemblages.
[Show abstract][Hide abstract] ABSTRACT: A fast, accurate, and full indexing of viruses and viroids in a sample for the inspection and quarantine services and disease management is desirable but was unrealistic until recently. This article reviews the rapid and exciting recent progress in the use of next-generation sequencing (NGS) technologies for the identification of viruses and viroids in plants. A total of four viroids/viroid-like RNAs and 49 new plant RNA and DNA viruses from 18 known or unassigned virus families have been identified from plants since 2009. A comparison of enrichment strategies reveals that full indexing of RNA and DNA viruses as well as viroids in a plant sample at single-nucleotide resolution is made possible by one NGS run of total small RNAs, followed by data mining with homology-dependent and homology-independent computational algorithms. Major challenges in the application of NGS technologies to pathogen discovery are discussed. Expected final online publication date for the Annual Review of Phytopathology Volume 53 is August 04, 2015. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
[Show abstract][Hide abstract] ABSTRACT: The gypsy moth, Lymantria dispar, is an important economic pest that causes large-scale damage to forests worldwide. Because of its important role in initiating and controlling insect behavior, olfaction-and olfaction-based pest management-has drawn increasing attention from entomologists. In this study, we identified the gene that encodes the olfactory receptor co-receptor (OrCo). Through amino acid sequence alignment, we found that LdisOrCo shares high identity with other OrCo proteins from different insect orders. Next, we performed RNA-interference (RNAi) to assess the role of OrCo in olfaction. Electroantennographic assays showed that after RNAi, the average value of males' response to sex pheromones was 0.636 mV, significantly lower than that of the positive control (average = 1.472 mV). Females showed no response to sex pheromones before or after RNAi. Finally, quantitative PCR showed a strong decrease in the expression of OrCo after RNAi, by ~74% in males and by 23% in females relative to the positive controls. These results indicate that OrCo is not only critical to odor recognition, but it may also represent a new target for development of semiochemicals that can influence insect behavior.
International journal of biological sciences 05/2015; 11(7):772-80. DOI:10.7150/ijbs.11898 · 4.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transgenic rice 114-7-2 is a newly developed transgenic rice line of producing human serum albumin (HSA). It has attracted much attention because of its economic potential. This paper was designated to discover the integration site of the transgenic HSA rice line 114-7-2 and to establish event-specific methods for qualitative and quantitative detection of the transgenic HSA rice based on the border junction fragment. One gene fragment of 5' flanking region was successfully isolated using the TAIL-PCR methods. The fragment sequence showed that a 454-bp junction fragment contained 75 bp of T-DNA sequence and 379 bp of rice genome DNA, which is located in chromosome 4. Event-specific real-time PCR method for HSA rice line 114-7-2 was established with the primers (HSA-F/HSA-R) and the probe (HSA-P) targeting the 454-bp junction region. The qualitative PCR assay showed the limit of detection was 0.01 %. In the event-specific quantitative detection method, the LOQ for 114-7-2 HSA rice was estimated to be 0.025 ng or 50 copies. The method developed in this study is highly specific, sensitive, and reliable for transgenic HSA rice sample detection.
European Food Research and Technology 09/2014; 239(3):403-408. DOI:10.1007/s00217-014-2234-8 · 1.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Alien species are brought into countries world wide on a massive scale for agricultural production, ex situ conservation, landscape aesthetics, gardens, and ecosystem restoration. Unfortunately, some of these species have escaped and adversely impacted on regional as well as global biodiversity conservation and agricultural production. To reduce such risks, it is necessary to implement specific and effective measures. Since various government departments and institutions are involved in the management of alien species, it is difficult to prevent native and agroecosystems from being invaded by invited species. We propose the establishment of a supervision and inspection continuum over intentional species introduction, similar to that which exists in some countries over unintentional species introductions. Namely, a justification of the necessity to import, a risk assessment, assurances as to provision of an adequate containment facility assessment, and a damage-limitation protocol should that need to be invoked. These requirements should be satisfied before an alien species is knowingly imported, and the necessary follow-up supervision is important post- importation.
Biodiversity and Conservation 09/2014; 23(10). DOI:10.1007/s10531-014-0728-0 · 2.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transgenic maize plant expressing high phytase activity has been reported and approved by Chinese government in 2009. Here, we report a highly specific loop-mediated isothermal amplification (LAMP) method to detect the phytase gene in the GMO maize. The LAMP reaction takes less than 20 min and the amplification is visible without gel electrophoresis. The detection sensitivity of the LAMP method is about 30 copies of phytase genomic DNA, which is 33.3 times greater than the conventional PCR method with gel electrophoresis. The quantitative detection results showed that the LAMP method has a good linear correlation between the DNA copy number and the associated Tt values over a large dynamic range of template concentration from 6 × 101 to 6 × 107 copies, with a quantification limit of 60 copies. Therefore, the LAMP method is visual, faster, and more sensitive, and does not need special equipment compared to traditional PCR technique, which is very useful for field tests and fast screening of GMO feeds.
[Show abstract][Hide abstract] ABSTRACT: Background: Adapter trimming is a prerequisite step for analyzing next-generation sequencing (NGS) data when the reads are longer than the target DNA/RNA fragments. Although typically used in small RNA sequencing, adapter trimming is also used widely in other applications, such as genome DNA sequencing and transcriptome RNA/cDNA sequencing, where fragments shorter than a read are sometimes obtained because of the limitations of NGS protocols. For the newly emerged Nextera long mate-pair (LMP) protocol, junction adapters are located in the middle of all properly constructed fragments; hence, adapter trimming is essential to gain the correct paired reads. However, our investigations have shown that few adapter trimming tools meet both efficiency and accuracy requirements simultaneously. The performances of these tools can be even worse for paired-end and/or mate-pair sequencing.
Results: To improve the efficiency of adapter trimming, we devised a novel algorithm, the bit-masked k-difference matching algorithm, which has O(kn) expected time with O(m) space, where k is the maximum number of differences allowed, n is the read length, and m is the adapter length. This algorithm makes it possible to fully enumerate all candidates that meet a specified threshold, e.g. error ratio, within a short period of time. To improve the accuracy of this algorithm, we designed a simple and easy-to-explain statistical scoring scheme to evaluate candidates in the pattern matching step. We also devised scoring schemes to fully exploit the paired-end/mate-pair information when it is applicable. All these features have been implemented in an industry-standard tool named Skewer (https://sourceforge.net/projects/skewer). Experiments on simulated data, real data of small RNA sequencing, paired-end RNA sequencing, and Nextera LMP sequencing showed that Skewer outperforms all other similar tools that have the same utility. Further, Skewer is considerably faster than other tools that have comparative accuracies; namely, one times faster for single-end sequencing, more than 12 times faster for paired-end sequencing, and 49% faster for LMP sequencing.
Conclusions: Skewer achieved as yet unmatched accuracies for adapter trimming with low time bound.
[Show abstract][Hide abstract] ABSTRACT: The aim was to establish an effective screening microarray at genus level for Pospiviroid. We analyzed nucleotide sequences from Pospiviroid viroid and designed 19 probes with genus identification characteristics. The standards of these probes included the characters of (i) a GC content between 40 and 60%, (ii) less than 50% of single nucleotide, (iii) less than 4 continuous mononucleotides, and (iv) less than 6 nucleotides in the inner hairpin. We synthesized microarrays by using these probes on glass slides. The validation results of microarray probes show effective signals from chrysanthemum stunt viroid and tomato planta macho viroid standard samples hybridization. The sensitivity results show that the microarray detected 200 pg/microL of total RNA. The microarray can be used to screen Pospiviroid viroid.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 03/2014; 30(3):514-23. DOI:10.13345/j.cjb.130329
[Show abstract][Hide abstract] ABSTRACT: A major challenge in the agricultural industry is the development of techniques that can screen plant samples for viroid infection. Microarrays are promising in this regard, as their high throughput nature can potentially allow for the detection of a range of viroids in a single test. In this paper we present a microarray that can detect a wide spectrum of all 8 reported viroid genera including 37 known plant viroid species. The array was constructed using an automated probe design protocol which generated a minimal number of probes to detect viroids at the genus level. The designed microarray showed a high specificity and sensitivity when tested with a set of standard virus samples. Finally, the microarray was applied to screen infected field samples, with Hop stunt viroid infection identified as the major disease causing pathogen for an infected citrus sample.
PLoS ONE 10/2013; 8(5):e64474. DOI:10.1371/journal.pone.0064474 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A simple and ultrasensitive DNA biosensor was developed utilizing the color and size changes originating from Ligation Chain Reaction (LCR)-based gold nanoparticle assembly as an indicator. Using synthetic DNA of 10-fold serial dilutions as a target, the limit of detection (LOD) was 1.5aM with a range of DNA concentrations from 0.01 to 1000fM by UV-vis absorption and 0.1aM in the range of 0.01-10,000fM by dynamic light scattering (DLS). This developed method is low-cost, rapid, and much lower LOD for the detection of specific short DNA sequences, and the results can be directly determined either by the color or by the DLS. The capability of high-throughput detection with the aid of PCR amplification apparatus can also be realized.
[Show abstract][Hide abstract] ABSTRACT: An integration event-specific fluorescent liquid bead array was developed for the simultaneous identification of 10 genetically modified (GM) maize, including Bt176, Bt11, MON810, NK603, GA21, MON88017, MON89034, MIR604, T25 and MIR162, as well as one non-GM maize. The system comprised 11 specific oligonucleotide probes labeled with an amino group and coupled to fluorescence-encoded microspheres. To enable fluorescence detection, 11 pairs of primers labeled with biotin at the 5' ends were used. The hybridization signal of biotinylated PCR product to the probe-coupled microspheres was then detected. The limit of detection of this assay was 0.1% for GM maize, which is lower than the current labeling threshold levels enforced in the EU (0.9%). The results of the positive and negative controls were consistent with their expected situation, which showed that the method was highly specific. We detected GM maize in 20 of the 1370 commercial food samples tested, which were labeled as containing maize. The overall sensitivity, specificity, rapidity and high throughput capacity of this liquid chip system suggest that it could provide a significant improvement over current methods, and potentially offer an improved platform for further research into the detection of other GM plants.
[Show abstract][Hide abstract] ABSTRACT: We report a biosensor based on Surface Plasmon resonance (SPR) for the selective detection of Maize Chlorotic Mottle Virus (MCMV). 11-mercaptoundecanoic acid was applied on a gold surface to form a self-assembled monolayer and a layer of anti-MCMV antibody was crosslinked on the surface for specific recognition of MCMV. The effects of coupling reaction time and antibody concentration on detection sensitivity were studied. The coverage mass change is a function of the concentration of MCMV with a dynamic range from 1 ppb to 1000 ppb. The detection limit is approximately 1 ppb which is about two orders of magnitude higher than that of the exiting ELISA method. The developed SPR sensor showed highly specific recognition for both purified MCMV and crude extracts from real-world samples.
[Show abstract][Hide abstract] ABSTRACT: Comprehensive sampling is crucial to DNA barcoding, but it is rarely performed because materials are usually unavailable. In practice, only a few rather than all species of a genus are required to be identified. Thus identification of a given species using a limited sample is of great importance in current application of DNA barcodes. Here, we selected 70 individuals representing 48 species from each major lineage of Solanum, one of the most species-rich genera of seed plants, to explore whether DNA barcodes can provide reliable specific-species discrimination in the context of incomplete sampling. Chloroplast genes ndhF and trnS-trnG and the nuclear gene waxy, the commonly used markers in Solanum phylogeny, were selected as the supplementary barcodes. The tree-building and modified barcode gap methods were employed to assess species resolution. The results showed that four Solanum species of quarantine concern could be successfully identified through the two-step barcoding sampling strategy. In addition, discrepancies between nuclear and cpDNA barcodes in some samples demonstrated the ability to discriminate hybrid species, and highlights the necessity of using barcode regions with different modes of inheritance. We conclude that efficient phylogenetic markers are good candidates as the supplementary barcodes in a given taxonomic group. Critically, we hypothesized that a specific-species could be identified from a phylogenetic framework using incomplete sampling-through this, DNA barcoding will greatly benefit the current fields of its application.
PLoS ONE 02/2013; 8(2):e55927. DOI:10.1371/journal.pone.0055927 · 3.23 Impact Factor