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High‐throughput detection and screening of plants modified by gene editing using quantitative real‐time polymerase chain reaction 下载免费PDF全文
Cheng Peng Hua Wang Xiaoli Xu Xiaofu Wang Xiaoyun Chen Wei Wei Yongmin Lai Guoquan Liu Ian Douglas Godwin Jieqin Li Ling Zhang Junfeng Xu 《The Plant journal : for cell and molecular biology》2018,95(3):557-567
Gene editing techniques are becoming powerful tools for modifying target genes in organisms. Although several methods have been developed to detect gene‐edited organisms, these techniques are time and labour intensive. Meanwhile, few studies have investigated high‐throughput detection and screening strategies for plants modified by gene editing. In this study, we developed a simple, sensitive and high‐throughput quantitative real‐time (qPCR)‐based method. The qPCR‐based method exploits two differently labelled probes that are placed within one amplicon at the gene editing target site to simultaneously detect the wild‐type and a gene‐edited mutant. We showed that the qPCR‐based method can accurately distinguish CRISPR/Cas9‐induced mutants from the wild‐type in several different plant species, such as Oryza sativa, Arabidopsis thaliana, Sorghum bicolor, and Zea mays. Moreover, the method can subsequently determine the mutation type by direct sequencing of the qPCR products of mutations due to gene editing. The qPCR‐based method is also sufficiently sensitive to distinguish between heterozygous and homozygous mutations in T0 transgenic plants. In a 384‐well plate format, the method enabled the simultaneous analysis of up to 128 samples in three replicates without handling the post‐polymerase chain reaction (PCR) products. Thus, we propose that our method is an ideal choice for screening plants modified by gene editing from many candidates in T0 transgenic plants, which will be widely used in the area of plant gene editing. 相似文献
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Zhi‐Wei Kang Fang‐Hua Liu Hong‐Gang Tian Meng Zhang Shan‐Shan Guo Tong‐Xian Liu 《Insect Science》2017,24(2):222-234
The green peach aphid, Myzus persicae Sulzer (Hemiptera, Aphididae), is an important cosmopolitan pest. Real time qRT‐PCR has been used for target gene expression analysis on M. persicae. Using real time qRT‐PCR, the expression levels are normalized on the basis of the reliable reference genes. However, to date, the stability of available reference genes has been insufficient. In this study, we evaluated nine candidate reference genes from M. persicae under diverse experimental conditions. The tested candidate genes were comprehensively ranked based on five alternative methods (RefFinder, geNorm, Normfinder, BestKeeper and the comparative ΔCt method). 18s, Actin and ribosomal protein L27 (L27) were recommended as the most stable reference genes for M. persicae, whereas ribosomal protein L27 (L27) was found to be the least stable reference genes for abiotic studies (photoperiod, temperature and insecticide susceptibility). Our finding not only sheds light on establishing an accurate and reliable normalization of real time qRT‐PCR data in M. persicae but also lays a solid foundation for further studies of M. persicae involving RNA interference and functional gene research. 相似文献
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Evaluation of consistency in quantification of gene copy number by real‐time reverse transcription quantitative polymerase chain reaction and virus titer by plaque‐forming assay for human respiratory syncytial virus 下载免费PDF全文
Keisuke Yamamoto Noriko Ogasawara Soh Yamamoto Kenichi Takano Tsukasa Shiraishi Toyotaka Sato Hiroyuki Tsutsumi Tetsuo Himi Shin‐ichi Yokota 《Microbiology and immunology》2018,62(2):90-98
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YeongHo Kim YiSeul Kim Donghun Kim SeYeon Kim GyeongJin Seo SooHyun Shin JaeYoung Lee Young Ho Kim 《Entomological Research》2019,49(6):277-283
Drosophila melanogaster is attracted to chemicals produced by fermentation and it is abundantly found in rotten fruits. Considering its habitat, the fruit fly is reported to be tolerant to environmental chemicals. Quantitative real‐time polymerase chain reaction was employed to investigate the expression pattern and physiological function of genes putatively involved in chemical detoxification. In quantitative real‐time polymerase chain reaction assays, normalization of target gene expression with internal reference genes is required. These reference genes should be stably expressed during chemical exposure and in chemical‐free conditions. In this study, therefore, we used two programs (geNorm and BestKeeper) to evaluate the expression stability of five reference genes (nd, rpL18, ef1β, hsp22 and tbp) in female adult flies exposed to various concentrations of methanol and ethyl acetate. Four genes (nd, rpL18, ef1β and tbp) were found to be suitable for use as reference genes in methanol‐treated flies and three genes (ef1β, nd, tbp) were found to be suitable for use as reference genes in ethyl acetate‐treated flies. These results suggested that a combination of two genes among these stably expressed genes can be used for accurate normalization of target gene expression in quantitative real‐time polymerase chain reaction‐based determination of gene expression profiles in D. melanogaster treated with both chemicals. 相似文献
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Junqi Zhang Ran Wang Jizhen Song Zhaopeng Luo Jun Yang Fucheng Lin 《Journal of Phytopathology》2013,161(2):92-97
A one‐step multiplex RT‐PCR method has been developed for the simultaneous detection of four viruses frequently occurring in tobacco (Cucumber mosaic virus, Tobacco mosaic virus, Tobacco etch virus and Potato virus Y). Four sets of specific primers were designed to work with the same reaction reagents and cycling conditions, resulting in four distinguishable amplicons representative of the four viruses independently. This one‐step multiplex RT‐PCR is consistently specific using different combinations of virus RNA as templates, and no non‐specific band was observed. It has high sensitivity compared to single RT‐PCR. Moreover, field samples in China can be tested by this method for virus detection. Our results show that one‐step multiplex RT‐PCR is a high‐throughput, specific, sensitive method for tobacco virus detection. 相似文献
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建立的一步PCR方法即反转录和PCR在同一管中进行,同时检测甲型肝炎和脊髓灰质炎病毒病毒RNA。实验中对不同的反转录温度以及一步多重PCR的特异性和灵敏度进行了探讨。结果表明:42℃、50℃反转录时polio有非特异性条带出现,60℃反转录特异性较好,而HAV在三种不同的反转录温度下均得到牧场划性较好的条带;应用一步PCR同时检测两种病毒与检测单一病毒的灵敏度基本一致,但在同等反应条件下后者的反应效率高于前者,特别是在检测HAV时。 相似文献
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Detection of Helicobacter pylori in stool samples of young children using real‐time polymerase chain reaction 下载免费PDF全文
Background
The aims of this study were to develop and validate a multiplex real‐time polymerase chain reaction (q‐PCR) assay of Helicobacter pylori in stool samples of healthy children. Additionally, we determined the prevalence of clarithromycin resistance and cagA gene in H. pylori‐positive samples.Materials and methods
Archived stool samples from 188 children aged 6‐9 years and 272 samples of 92 infants aged 2‐18 months were tested for H. pylori antigens using enzyme immunoassay (EIA). A multiplex q‐PCR assay was designed to detect H. pylori 16S rRNA and urease and the human RNase P gene as an internal control. Kappa coefficient was calculated to assess the agreement between q‐PCR and EIA.Results
Laboratory validation of the q‐PCR assay using quantitated H. pylori ATCC 43504 extracted DNA showed S‐shaped amplification curves for all genes; the limit of detection was 1 CFU/reaction. No cross‐reactivity with other bacterial pathogens was noted. Applying the multiplex q‐PCR to DNA extracted from fecal samples showed clear amplification curves for urease gene, but not for 16S rRNA. The prevalence of H. pylori infection was 50% (95% CI 43%‐57%) by q‐PCR (urease cycle threshold <44) vs 59% (95% CI 52%‐66%) by EIA. Kappa coefficient was .80 (P < .001) and .44 (P < .001) for children aged 6‐9 years and 2‐18 months, respectively. Sixteen samples were positive for cagA and three were positive for clarithromycin resistance mutation (A2143G) as confirmed by sequencing.Conclusions
The developed q‐PCR can be used as a cotechnique to enhance the accuracy of H. pylori detection in epidemiological studies and in clinical settings. 相似文献11.
Shoichi Niwa Hiroyuki Tsukagoshi Taisei Ishioka Yoshiko Sasaki Masakazu Yoshizumi Yukio Morita Hirokazu Kimura Kunihisa Kozawa 《Microbiology and immunology》2014,58(1):68-71
To improve detection of norovirus (NoVGI, NoVGII) and sapovirus (SaV), a simultaneous quantitative RT‐PCR method was established. This triplex real‐time PCR method was evaluated using a combination of optimized specific primers and probes. The performance of the developed PCR assay was equivalent to that of monoplex real‐time PCR across a broad dynamic range of 102–107 copies/assay using plasmid DNA standards. The limit of detection was 102 copies/assay. The quantitative value was comparable with that of monoplex real‐time PCR of stool samples. Our triplex real‐time PCR is useful for detection of NoV and SaV infections. 相似文献
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- 1 Neodiprion sertifer nucleopolyhedrovirus (NeseNPV) is widely used as a viral bio‐insecticide against larvae of the European pine sawfly N. sertifer (Geoff.) (Hymenoptera: Diprionidae), which is one of the most harmful defoliators of pines in Northern Europe. A major obstacle to studying this pathogenic virus in nature is the difficulty of confirming and quantifying the presence of NeseNPV.
- 2 In the present study, we developed real‐time polymerase chain reaction (PCR) primers, based on the caspid gene 39 sequence, for the specific and quantitative detection of NeseNPV. The quantitative real‐time PCR (qPCR) assay can detect virus from any substrate tested, including different insect life stages (egg, larval, adult), pine foliage, and litter or ground vegetation. The reproducible detection limit for the real‐time assay is 0.013 pg of viral DNA (0.013×10?12 g), corresponding to 136 viral genomes or approximately one to seven virus occlusion bodies per sample.
- 3 qPCR is a specific, quantitative, sensitive, reliable and flexible procedure, and is a good supplement to conventional microscopy‐ or bioassay‐based methods for detection of the virus. We have used qPCR to quantify the level of NeseNPV in samples collected in the field after aerial application of the virus, and demonstrated significantly higher virus levels in sawfly larvae from sprayed areas compared with unsprayed control areas 4 weeks after spraying.
- 4 This qPCR assay can be used to determine important aspects of the biology of NeseNPV (e.g. virus levels in different insect life stages and in their microhabitats on pine foliage and in forest litter).
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In this study, Streptococcus gordonii‐specific quantitative real‐time polymerase chain reaction (qPCR) primers, RTSgo‐F2/RTSgo‐R2, were developed based on the nucleotide sequences of RNA polymerase β‐subunit gene (rpoB). The specificity of the RTSgo‐F2/RTSgo‐R2 primers was assessed by conventional PCR on 99 strains comprising 63 oral bacterial species, including the type strain and eight clinical isolates of S. gordonii. PCR products were amplified from the genomic DNAs of only S. gordonii strains. The qPCR primers were able to detect as little as 40 fg of S. gordonii genomic DNA at a cycle threshold value of 33. These findings suggest that these qPCR primers detect S. gordonii with high specificity and sensitivity. 相似文献
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Jasmin Heidepriem Verena Krhling Christine Dahlke Timo Wolf Florian Klein Marylyn M. Addo Stephan Becker Felix F. Loeffler 《Biotechnology journal》2020,15(9)
The Ebola virus (EBOV) can cause severe infections in humans, leading to a fatal outcome in a high percentage of cases. Neutralizing antibodies against the EBOV surface glycoprotein (GP) can prevent infections, demonstrating a straightforward way for an efficient vaccination strategy. Meanwhile, many different anti‐EBOV antibodies have been identified, whereas the exact binding epitopes are often unknown. Here, the analysis of serum samples from an EBOV vaccine trial with the recombinant vesicular stomatitis virus‐Zaire ebolavirus (rVSV‐ZEBOV) and an Ebola virus disease survivor, using high‐density peptide arrays, is presented. In this proof‐of‐principle study, distinct IgG and IgM antibodies binding to different epitopes of EBOV GP is detected: By mapping the whole GP as overlapping peptide fragments, new epitopes and confirmed epitopes from the literature are found. Furthermore, the highly selective binding epitope of a neutralizing monoclonal anti‐EBOV GP antibody could be validated. This shows that peptide arrays can be a valuable tool to study the humoral immune response to vaccines in patients and to support Ebola vaccine development. 相似文献
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A method for detecting rash and fever illness‐associated viruses using multiplex reverse transcription polymerase chain reaction 下载免费PDF全文
Yuki Matsushima Tomomi Shimizu Ikuko Doi Fuminori Mizukoshi Koo Nagasawa Akihide Ryo Hideaki Shimizu Masae Kobayashi Keiji Funatogawa Noriko Nagata Mariko Ishikawa Ayako Komane Nobuhiko Okabe Yoshio Mori Makoto Takeda Hirokazu Kimura 《Microbiology and immunology》2017,61(8):337-344
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The polymerase chain reaction (PCR) method readily detected bean yellow mosaic virus (BYMV) in gladioli leaves, but in initial tests PCR did not detect virus in corm tissue. Extracts of RN A from corm tissue were shown to inhibit the amplification of viral sequences when added to a PCR reaction. An additional purification step for the RNA extracts using a Sephadex G-50 column eliminated the inhibitory effect and enabled PCR to amplify and detect viral RNA in corm tissue preparations. 相似文献
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