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Development and evaluation of a sybr green-based RT-qPCR assay with a specific primer set for tomato seed testing against tomato brown rugose fruit virus

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JOURNAL OF GENERAL PLANT PATHOLOGY 2025年第3期91卷 160-170页

作者： Ota, Emi Shinosaka, Hibiki Ishibashi, Kazuhiro Takeyama, Sawana Matsuyama, Momoko Tomitaka, Yasuhiro Matsushita, Yosuke Osaki, Kohei Kubota, Kenji NARO Inst Plant Protect Div Core Technol Pest Control Res 2-1-18 Kannondai Tsukuba Ibaraki 3058666 Japan NARO Ctr Seeds & Seedlings Dept DUS Test & Seed Inspect 2-2 Fujimoto Tsukuba Ibaraki 3050852 Japan NARO Inst Agrobiol Sci Div Plant Mol Regulat Res 2-1-2 Kannondai Tsukuba Ibaraki 3058602 Japan NARO Inst Plant Protect Div Crop Pest Control Res 2-1-18 Kannondai Tsukuba Ibaraki 3058666 Japan

Since its first occurrence in Israel and Jordan in 2014 and 2015, the tomato brown rugose fruit virus (ToBRFV) has become one of the most concerning pathogens affecting tomatoes and other crops worldwide. Its rapid spread is believed to result from the international trade of contaminated seeds and its seed transmissibility, underscoring the critical importance of seed health testing for ToBRFV to prevent further dissemination of the virus. To this end, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) protocols employing TaqMan probe chemistry have been widely adopted. However, the development of RT-qPCR protocols for ToBRFV seed testing using sybr green chemistry remains limited. The sybr green method offers the advantage of distinguishing ToBRFV from other tobamoviruses through melt curve analysis. In this study, we developed a sybr green-based RT-qPCR detection method using newly designed primer sets, which demonstrated high specificity for ToBRFV and sufficient sensitivity. While this protocol requires further optimization and validation for application in routine seed testing, it establishes a foundational approach for sybr green-based RT-qPCR seed testing. Additionally, this study raises an important question regarding the relationship between RT-qPCR results in seed tests and the likelihood of virus contamination or transmission via seeds.

关键词： ToBRFV Tobamoviruses sybr green RT-qPCR Melt curve Tomato

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Optimization of a Conventional Assay into sybr green Real-Time PCR for the Detection of the Nc5 Segment from Neospora caninum

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ACTA PARASITOLOGICA 2025年第1期70卷 1-12页

作者： da Fonseca, Flavia Moreira Koch, Marilia de Oliveira Sato, Ana Paula Rodriguez, Maria Constanza Locatelli-Dittrich, Rosangela Univ Fed Parana Dept Vet Med Rua Funcionarios 1540 BR-80035050 Curitiba Parana Brazil Agr Def Agcy Parana Marcos Enrietti Diagnost Ctr R Jaime Balao 575 BR-80040340 Curitiba Parana Brazil Univ Zurich Vetsuisse Fak Vet Lab Winterthurerstr260 CH-8057 Zurich Switzerland

Purpose The aim of the present study was to establish a sybr green-based real-time PCR assay for detection of the Nc5 segment from the Neospora caninum genome. Methods The oligonucleotides sequences targeting the Nc5 gene previously reported and designed in-house were validated. Two Primer sets were evaluated and tested in four different combinations. The NP7/NP10 assay was selected and reaction conditions optimized. Efficiency, analytical sensitivity, precision and specificity were assessed. The assay was evaluated in triplicate, in three independent PCR runs performed by two technicians to generate robust results. Results The standard curve determined by tenfold serial dilutions (1 to 1 x 10(-7)) established a reaction efficiency (E) of 102.34%, a correlation coefficient (R2) of 0.999 and a slope of -3.267. LOD of the real-time PCR assay was 0.456 tachyzoites DNA per reaction, as compared to 45.62 for the conventional method. sybr green real-time PCR was 100 times more sensitive than the conventional method. Precision analysis showed 100% intra- and inter-assay repeatability at the minimum detection limit. The mean assay coefficient of variation (CV%) was 4.19% and standard deviation (SD) 1.67%. No significant differences between the means of Cq in the replicates and technicians (P > 0.05) was found, indicating that the assay is robust and accurate. The applicability of the assay was tested and N. caninum DNA was detected in milk, blood, amniotic fluid, placenta and different tissue samples. Conclusion The protocol had high specificity, confirmed by melting curve analysis and no cross-reactions with other tested microorganisms. The sybr green-based PCR protocol standardized in this study is a highly sensitive and specific method, reproducible and applicable for the detection of N. caninum in different biological samples.

关键词： Neosporosis Diagnosis Nc5 Real-time PCR sybr green

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猫传染性腹膜炎病毒sybr green实时荧光定量PCR检测方法的建立与临床应用

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中国兽医杂志 2025年第3期61卷 44-49页

作者：李星颖谢梓民原耀贤孙铭澮江文康赵明明何诗何静赖健仪白银山陈胜锋陈志胜马骏王丙云佛山大学生命科学与工程学院广东佛山528225

为了提高猫传染性腹膜炎(FIP)的临床检出率,本试验根据猫传染性腹膜炎病毒(FIPV)M基因设计特异性引物,将扩增的目的基因连接到p MD19-T载体上,获得重组质粒并作为标准阳性模板,建立针对FIPV的sybr green实时荧光定量PCR(q PCR)检测方法... 详细信息

为了提高猫传染性腹膜炎(FIP)的临床检出率,本试验根据猫传染性腹膜炎病毒(FIPV)M基因设计特异性引物,将扩增的目的基因连接到p MD19-T载体上,获得重组质粒并作为标准阳性模板,建立针对FIPV的sybr green实时荧光定量PCR(q PCR)检测方法,并对其特异性、敏感性、重复性和临床检出率进行验证。结果显示,本方法梯度稀释的标准品与Ct值呈良好的线性关系,R^(2)=0.999 9,扩增效率为92.4%;与其他猫常见病毒未发生交叉反应;最低检测限为3.48×10^(2) copies/μL,敏感性是普通PCR检测方法的100倍;该方法重复性好,批内和批间变异系数均小于2.4%;应用该方法对临床阳性样本的检出率为100%,阴性样本检出率为0。结果表明,本试验建立的sybr green q PCR检测方法特异性强、敏感性高、重复性好、临床检测效果佳,可用于FIPV的临床诊断。

关键词：猫传染性腹膜炎病毒 M基因 sybr green 实时荧光定量PCR(qPCR)

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鸽疱疹病毒gB基因sybr greenⅠ荧光定量PCR检测方法的建立与应用

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中国家禽 2025年第3期47卷 93-99页

作者：李鹏苏和韩慧敏孟海王昊王凤雪温永俊内蒙古农业大学兽医学院农业农村部动物疾病临床诊断与治疗重点实验室内蒙古呼和浩特010018 呼和浩特市动物疫病预防控制中心内蒙古呼和浩特010018

为建立检测鸽疱疹病毒(PiHV)的sybr greenⅠ荧光定量PCR(qPCR)方法,研究根据PiHV gB基因的高度保守区域设计特异性引物,构建质粒作为标准品建立PiHV qPCR检测方法,并进行敏感性和特异性试验。结果显示:建立的PiHV qPCR方法最低DNA检出... 详细信息

为建立检测鸽疱疹病毒(PiHV)的sybr greenⅠ荧光定量PCR(qPCR)方法,研究根据PiHV gB基因的高度保守区域设计特异性引物,构建质粒作为标准品建立PiHV qPCR检测方法,并进行敏感性和特异性试验。结果显示:建立的PiHV qPCR方法最低DNA检出浓度为8.62×10^(2)拷贝/μL,敏感性比普通PCR方法高100倍,可特异性区分鸽疱疹病毒与其他多种常见感染鸽的细菌和病毒,其批内变异系数小于1%,批间变异系数小于2%;应用该方法对呼和浩特市及周边地区赛鸽公棚和鸽养殖场采集的30份疑似病鸽肝脏样品进行检测,可快速准确地检出样品中的PiHV,并且检出率比普通PCR方法高。研究表明,建立的PiHV qPCR检测方法特异性强,敏感性较高,能够应用于赛鸽、肉鸽等的鸽疱疹病毒快速检测。

关键词：鸽疱疹病毒 sybr green 荧光定量PCR 聚合酶链式反应 gB基因

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sybr green real time-polymerase chain reaction as a rapid and alternative assay for the efficient identification of all existing Escherichia coli biotypes approved directly in wastewater samples

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BIOTECHNOLOGY PROGRESS 2012年第4期28卷 1106-1113页

作者： Chetta, Massimiliano Bafunno, Valeria Grillo, Rosalba Mele, Antonio Lo Perfido, Pietro Notarnicola, Michele Cellini, Francesco Cifarelli, Rosa Anna Univ Foggia Dipartimento Sci Biomed Foggia Italy Euroqual Lab Bari Italy Dept Mol Biol FBK Trento Italy Metapontum Agrobios I-75010 Metaponto Matera Italy Politecn Bari Bari Italy

Escherichia coli has been recognized as the principal indicator of fecal contamination of water. Indeed, E. coli is the only species in the coliform group found in relationship with gastrointestinal tract of human and warm-blooded animals and subsequently excreted in large numbers in the human feces. To obtain a complete picture of water quality and therefore, a better protection of public health, different techniques for water analysis have been proposed. In this article, we describe an alternative method that uses sybr green real time-polymerase chain reaction (RT-PCR) technology to identify and quantify all E. coli biotypes in a group of wastewater samples collected from a wastewater depurator located in South of Italy. This new RT-PCR protocol is accurate in measuring the concentration of chromosomal E. coli DNA using the amplification of three new specific fragments of the following bacteria genes: CadC, HNS, and Allan whose sequence is specific for E. coli family and conserved in all E. coli subtypes. This method allowed us to detect the presence of all E. coli biotypes directly in wastewater samples and estimated the correspondence between colony forming units and bacterial DNA concentrations. The availability of a rapid and sensitive method may be useful to monitor the persistence of E. coli in water, to evaluate the efficiency of wastewater purification treatments and the possible recycle for agricultural use. Furthermore, the development of a simple and routine method to monitor water quality with RT-PCR analysis can encourage the testing of a higher number of samples. (c) 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 11061113, 2012

关键词： RT-PCR sybr green Escherichia coli CFU wastewater water contamination

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sybr green dye-based probe-free SNP genotyping: Introduction of T-Plex real-time PCR assay

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ANALYTICAL BIOCHEMISTRY 2013年第2期441卷 225-231页

作者： Baris, Ibrahim Etlik, Ozdal Koksal, Vedat Ocak, Zeynep Baris, Saniye Tugba Koc Univ Dept Mol Biol & Genet TR-34450 Istanbul Turkey Merter Is Merkezi BURC Mol Diagnost Labs TR-34169 Istanbul Turkey Sifa Univ Fac Med Dept Histol & Embryol Izmir Turkey Abant Izzet Baysal Univ Sch Med Dept Med Genet Bolu Turkey

Single-nucleotide polymorphism (SNP) genotyping is widely used in genetic association studies to characterize genetic factors underlying inherited traits. Despite many recent advances in high-throughput SNP genotyping, inexpensive and flexible methods with reasonable throughput levels are still needed. Real-time PCR methods for discovering and genotyping SNPs are becoming increasingly important in various fields of biology. In this study, we introduce a new, single-tube strategy that combines the tetra-primer ARMS PCR assay, sybr green I-based real-time PCR, and melting-point analysis with primer design strategies to detect the SNP of interest. This assay, T-Plex real-time PCR, is based on the T. discrimination of the amplified allele-specific amplicons in a single tube. The specificity, sensitivity, and robustness of the assay were evaluated for common mutations in the FV, PII, MTHFR, and FGFR3 genes. We believe that T-Plex real-time PCR would be a useful alternative for either individual genotyping requests or large epidemiological studies. (C) 2013 Elsevier Inc. All rights reserved.

关键词： Tetra-primer ARMS PCR SNP FVL MTHFR sybr green T-Plex real-time PCR

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sybr green as a fluorescent probe to evaluate the biofilm physiological state of Staphylococcus epidermidis, using flow cytometry

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CANADIAN JOURNAL OF MICROBIOLOGY 2011年第10期57卷 850-856页

作者： Cerca, Filipe Trigo, Gabriela Correia, Alexandra Cerca, Nuno Azeredo, Joana Vilanova, Manuel ICBAS UP Lab Imunol Dept Imunofisiol & Farmacol IMFF P-4099003 Oporto Portugal IBMC Oporto Portugal Univ Minho Dept Biol CMBA P-4710057 Braga Portugal Univ Minho CEB IBB P-4710057 Braga Portugal

Staphylococcus epidermidis biofilms with different proportions of viable but nonculturable bacteria were used to show that sybr green (sybr) may be used as a probe to evaluate the bacterial physiological state using flow cytometry. Biofilms grown in excess glucose presented significantly higher proportions of dormant bacteria than biofilms grown in excess glucose with buffered pH conditions or with exponential-phase planktonic cultures. Bacteria obtained from biofilms with high or low proportions of viable but nonculturable cells were further cultured in broth medium and stained with sybr at different time points. An association between bacterial growth and sybr staining intensity was observed. In addition, bacteria presenting higher sybr fluorescence intensity also stained more intensely with cyanoditolyl tetrazolium chloride, used as a probe to evaluate cellular metabolism. Accordingly, planktonic bacteria treated with rifampicin, an inhibitor of bacterial RNA transcription, presented lower sybr and cyanoditolyl tetrazolium chloride staining intensity than non-treated bacteria. Overall, our results indicate that sybr, in addition to being used as a component of LIVE/DEAD stain, may also be used as a probe to evaluate the physiological state of S. epidermidis cells.

关键词： Staphylococcus epidermidis biofilm flow cytometry viable but nonculturable bacteria VBNC sybr green cyanoditolyl tetrazolium chloride CTC LIVE/DEAD

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sybr green-activated sorting of Arabidopsis pollen nuclei based on different DNA/RNA content

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PLANT REPRODUCTION 2015年第1期28卷 61-72页

作者： Schoft, Vera K. Chumak, Nina Bindics, Janos Slusarz, Lucyna Twell, David Kohler, Claudia Tamaru, Hisashi Austrian Acad Sci Gregor Mendel Inst A-1030 Vienna Austria Univ Leicester Dept Biol Leicester LE1 7RH Leics England Swedish Univ Agr Sci Dept Plant Biol S-75007 Uppsala Sweden Linnean Ctr Plant Biol S-75007 Uppsala Sweden

Key message Purification of pollen nuclei. Germ cell epigenetics is a critical topic in plants and animals. The male gametophyte (pollen) of flowering plants is an attractive model to study genetic and epigenetic reprogramming during sexual reproduction, being composed of only two sperm cells contained within, its companion, vegetative cell. Here, we describe a simple and efficient method to purify sybr green-stained sperm and vegetative cell nuclei of Arabidopsis thaliana pollen using fluorescence-activated cell sorting to analyze chromatin and RNA profiles. The method obviates generating transgenic lines expressing cell-type-specific fluorescence reporters and facilitates functional genomic analysis of various mutant lines and accessions. We evaluate the purity and quality of the sorted pollen nuclei and analyze the technique's molecular basis. Our results show that both DNA and RNA contents contribute to sybr green-activated nucleus sorting and RNA content differences impact on the separation of sperm and vegetative cell nuclei. We demonstrate the power of the approach by sorting wild-type and polyploid mutant sperm and vegetative cell nuclei from mitotic and meiotic mutants, which is not feasible using cell-type-specific transgenic reporters. Our approach should be applicable to pollen nuclei of crop plants and possibly to cell/nucleus types and cell cycle phases of different species containing substantially different amounts of DNA and/or RNA.

关键词： sybr green Fluorescence-activated cell sorting (FACS) Pollen Sperm nuclei Male gametophyte Arabidopsis

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Simple and fast colorimetric detection of lipopolysaccharide based on aptamer and sybr green I mediated aggregation of gold nanoparticles

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INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES 2022年第PartA期223卷 231-239页

作者： Jiang, Jiajun Huang, Bingna Li, Ningjun An, Changcheng Sun, Changjiao Shen, Yue Gooneratne, Ravi Cui, Haixin Zhan, Shenshan Wang, Yan Chinese Acad Agr Sci Inst Environm & Sustainable Dev Agr Beijing 100081 Peoples R China Lincoln Univ Fac Agr & Life Sci Dept Wine Food & Mol Biosci Lincoln 7647 New Zealand

Lipopolysaccharide (LPS) poses a considerable threat to food safety and human health. A colorimetric assay for LPS detection based on LPS binding aptamer (LBA) and sybr green I (SG) mediated aggregation of gold nanoparticles (AuNPs) was established. In the absence of LPS, the LBA was absorbed onto the AuNPs surface which prevented SG-induced aggregation of AuNPs, and the sensing system exhibited red color. When LPS was added, it interacted with the LBA, forming a complex. At higher LPS concentration, many LBAs were exhausted resulting in SG-induced aggregation of AuNPs, and color change from red to blue. The range of colorimetric detection of LPS was linear in 0-12 EU/mL, with a limit of detection of 0.1698 EU/mL. Spiked LPS in real samples and interfering substances were also identified. This assay ingeniously using the fluorescent dye SG as an effective trigger of AuNPs aggregation, is rapid and facile than most of those earlier reported LBA-based LPS assays, and there is potential to be modified to construct assays for other targets.

关键词： Aptamer Colorimetric detection Lipopolysaccharide Endotoxin Gold nanoparticle sybr green

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Development of a sybr green I real-time PCR assay for specific identification of the fish pathogen Aeromonas salmonicida subspecies salmonicida

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APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 2016年第24期100卷 10585-10595页

作者： Fernandez-Alvarez, Clara Gonzalez, Santiago F. Santos, Ysabel Univ Santiago de Compostela Dept Microbiol & Parasitol Fac Biol Edificio CIBUS Santiago De Compostela 15782 Spain Univ Santiago de Compostela Inst Invest & Anal Alimentario Edificio CIBUS Santiago De Compostela 15782 Spain Univ Suvizzera Italiana Inst Res Biomed CH-6500 Bellinzona Switzerland

A sybr green I real-time polymerase chain reaction protocol for specific detection of the fish pathogen Aeromonas salmonicida subsp. salmonicida was developed and validated for rapid diagnosis of typical furunculosis. The sequence of the aopO gene of A. salmonicida subsp. salmonicida, which encodes for a serine/threonine protein kinase linked to virulence, was chosen for primer design. The selected primers amplified a 119-bp internal fragment of the aopO gene. The specificity test proved that 100 % (40/40) of the A. salmonicida subsp. salmonicida strains tested showed a positive amplification with subspecies-specific melting temperatures (Tm) of 80.75 +/- 0.35 A degrees C. Atypical A. salmonicida subspecies and other non-related bacterial fish pathogens did not amplify or showed unspecific melting profiles, except for one strain of A. salmonicida subsp. achromogenes and one strain of A. salmonicida subsp. smithia. The detection sensitivity was 21 fg of purified bacterial DNA per reaction, corresponding to 1-2 bacterial cells and 6-60 bacteria per reaction for seeded kidney and blood. The assay was highly reproducible with low variation coefficient values for intra-run and inter-run assays. The assay also allowed the specific detection of A. salmonicida subsp. salmonicida in tissues of fish naturally and experimentally infected. No amplification was detected when tissues from healthy fish or fish affected by other diseases were tested. The sybr green real-time PCR and melt curve analysis developed in this study is a rapid and accurate method for the specific identification of A. salmonicida subsp. salmonicida isolates and its detection on tissues of fish affected by furunculosis.

关键词： Aeromonas salmonicida subsp salmonicida aopO gene Diagnosis Real-time PCR sybr green Typical furunculosis

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