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Application of digital pcr in the Analysis of Transgenic Soybean Plants

Advances in Bioscience and Biotechnology 2016年第10期7卷 403-417页

作者： Jinrong Wan Li Song Yalei Wu Pius Brzoska David Keys Caifu Chen Babu Valliyodan J. Grover Shannon Henry T. Nguyen Division of Plant Sciences University of Missouri Columbia MO USA Genetic Medical & Applied Sciences Division Thermo Fisher Scientific South San Francisco CA USA Integrated DNA Technologies Redwood City CA USA

Detection and quantification of transgenes are important in analyzing genetically modified organisms (GMOs). Quantitative polymerase chain reaction (qpcr) is commonly utilized for such purposes. However, qpcr has certain limitations in detecting and quantifying transgenes in GMOs, such as the need of certified reference materials, a standard curve, and possible affection by inhibitors. Therefore, alternative and possibly better methods are needed. Recent advances in digital pcr technologies have promised to allow accurate quantification of nucleic acids and therefore provided another useful technique to analyze GMOs. Thermo Fisher ScientificTM has recently commercialized the Applied BiosystemsTM QuantStudioTM 3D digital pcr system that can be used for a wide range of applications involving nucleic acids. It will be beneficial to the scientific community to show the applicability of this digital pcr system in detecting and quantifying transgenes in GMOs. In the present study, the transgenes present in the Roundup Ready Soybean (RR1, event 40-3-2) and Roundup Ready Soybean 2 (RR2, event MON89788) developed by Monsanto Corporation were analyzed by using this digital pcr system. The qpcr analysis results were included for comparison. Using specifically designed TaqMan assays, as low as 1% of the RR1 or RR2 soybean material was reliably detected and quantified on the dpcr platform. Therefore, digital pcr is a sensitive and reliable method to analyze the RR transgenic soybeans, and should be another useful tool for analyzing other transgenic plants.

关键词： digital pcr GMO qpcr Soybean TaqMan

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Comparative analysis of QS3D and droplet digital pcr for detection of plasma EGFR T790M mutation at disease progression

Comparative analysis of QS3D and droplet digital PCR for det...

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2021年中国肿瘤标志物学术大会暨第十五届肿瘤标志物青年科学家论坛

作者： Qiaomei Guo Xia Ahung Mingna Zhao Mengyan Wang Jiatao Lou Department of Laboratory Medicine Shanghai Chest Hospital

Objectives: Numerous studies have demonstrated the ability of QuantStudioTM 3 D(QS3 D) and droplet digital pcr(dpcr) platforms in the successful detection of EGFR mutations from plasma. However,no prospective data on quantitative comparative analysis of the two methods are available. Here we directly compared the performance of the two platforms in the detection of the EGFR T790 M mutation in cell-free DNA(cfDNA) samples from lung cancer ***: cfDNA was isolated from plasma samples of 72 non-small cell lung cancer patients who initially received tyrosine kinase inhibitor treatment and acquired drug resistance. Identical cfDNA samples were subjected to T790 M mutation analysis using QS3 D and droplet dpcr in ***: The T790 M mutation was detected in 15/72(20.8%) cfDNA samples by QS3 D dpcr and 21/72(29.2%) cfDNA samples by droplet dpcr. The overall concordance between the two methods was91.7%(66/72). The median allele frequency of the T790 M mutation detected by QS3 D dpcr and droplet dpcr was 2.01% and 2.62%, respectively. The partition number of QS3 D dpcr is much higher than that of droplet dpcr, while the mutation abundance detected by the two platforms is similar. Furthermore, a strong correlation was observed in allele frequencies and copy numbers between the two methods, with R2 of 0.98 and 0.97, ***: Our study demonstrated a high level of concordance between the QS3 D and droplet dpcr platforms for the detection and absolute quantification of the EGFR T790 M mutation in cfDNA.

关键词： cfDNA T790M digital pcr concordance mutation abundance

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Sphere-forming corneal cells repopulate dystrophic keratoconic stroma:Implications for potential therapy

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World Journal of Stem Cells 2020年第1期12卷 35-54页

作者： Himanshu Wadhwa Salim Ismail Jennifer J McGhee Bert Van der Werf Trevor Sherwin Department of Ophthalmology Faculty of Medical and Health SciencesThe University of AucklandAuckland 1023New Zealand Department of Epidemiology and Biostatistics School of Population HealthFaculty of Medical and Health SciencesThe University of AucklandAuckland 1023New Zealand

BACKGROUND Keratoconus is a degenerative corneal disease characterised by aberrant cell behaviour and loss of matrix that can result in vision *** extracted from peripheral corneas can form stem cell-enriched spheres,which have shown the potential to repopulate the normal peripheral corneal stroma in vitro upon sphere implantation but have not been previously studied in keratoconic *** To investigate the therapeutic potential of stem cell-enriched spheres formed from extracted peripheral human corneal cells when introduced to keratoconic *** Stem cell-enriched spheres were formed from extracts of normal cadaveric human peripheral corneal *** spheres were implanted into incisions created in full thickness and onto the surface of 10μm thin sections of keratoconic and normal stromal tissues in *** sections were used to maximise use of limited keratoconic tissue available for *** cells were stained with Calcein-AM and visualised with stereo and fluorescence microscopy to assess survival and behaviours between the time of implantation day 0 and 14 d(D14)from *** cells in implanted tissues were characterised for stem cell and differentiation markers using immunohistochemistry and droplet digital pcr to assess the potential implications of these characteristics in the use of spheres in keratoconus *** Spheres were successfully implanted into full-thickness central corneal tissue and onto the surface of 10μm thin en face tissue *** observable differences were seen in sphere migration,proliferation or differentiation in keratoconic tissue compared to normal between day 0 and *** stained positively with Calcein-AM up to *** migration increased from day 0 to D14,occurring radially in three dimensions from the sphere and in alignment with tissue *** proliferation marker,EdU,was detected at day *** spheres stained positively for putative stem cell markersΔNp63

关键词： Keratoconus Cell culture Immunohistochemistry Quantitative pcr digital pcr Spheroid Holoclone Neurosphere Regeneration

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Integrated photothermal microcarriers for precise exosome-secreted microRNA profiling in breast cancer diagnosis

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Quantitative Biology 2024年第4期12卷 389-399页

作者： Yunjie Shi Yun Cheng Peiyu Chen Lexiang Zhang Fangfu Ye School of Pharmaceutical Sciences Wenzhou Medical UniversityWenzhouChina Oujiang Laboratory(Zhejiang Lab for Regenerative Medicine Vision and Brain Health)Wenzhou InstituteUniversity of Chinese Academy of SciencesWenzhouChina Department of Pediatrics The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical UniversityWenzhouChina Joint Centre of Translational Medicine the First Affiliated Hospital of Wenzhou Medical UniversityWenzhouChina Beijing National Laboratory for Condensed Matter Physics Institute of PhysicsChinese Academy of SciencesBeijingChina

Breast cancer constitutes a significant global health burden,while conventional diagnosis approaches may lack precision and can be discomforting for *** have emerged as promising biomarkers for breast cancer due to their participation in diverse pathological processes,and a convenient analysis platform is believed to greatly promote its *** this study,we propose a novel digital pcr approach utilizing near-infrared(NIR)photo-responsive thermosensitive microcarriers integrated with black phosphorus for quantifying microRNA(miRNA)biomarkers within ***-like biomimetic nanomaterials were firstly assembled for nonspecific exosome capture based on the affinity effect of avidin and ***-responsive microcarriers,fabricated using gelatin-based substrates blended with photothermal nanocomposite,exhibited NIRinduced heating and reversible phase transition *** optimized synthesis parameters on thermal response and established a programmable and controllable NIR light source *** results indicated a significant elevation in the levels of biomarkers miRNA-1246 and miRNA-122,with fold increases ranging from 6.2 to 23.6 and 5.9 to 13.0,respectively,in breast cancer cell lines MCF-7 and MDA-MB-231 compared to healthy control cells *** study offers broad prospects for utilizing exosomes to resolve predictive biomarkers.

关键词： breast cancer digital pcr exosomes MicroRNA photothermal

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Absolute quantitative detection of genetically modified soybean MON87708×MON89788 with stacked traits by digital polymerase chain reaction

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Oil Crop Science 2022年第4期7卷 180-188页

作者： Junyi Xu Xin Li Jinglian Bai Ying Liu Shaojie Wang Yueting Liu Chunguang Yang Technology Center of Dalian Customs District DalianChina Logistics Management Center of Dalian Customs District DalianChina

The main advantage of digital pcr(dpcr) is that it facilitates absolute quantification of the target without reference to the standard/calibration *** droplet dpcr has a three-color staining detection function,which enables multiplex pcr *** this study,this technique was used to establish triple dpcr detection for the genetically modified soybean MON87708 × MON89788 with stacked *** absolute quantitative detection was accomplished for the genomic DNA extracted from the homogenized seeds of GM stack MON87708× MON89788 *** results can serve as a reference for the absolute quantitative detection of stacked events of genetically modified crops.

关键词： Absolute quantification digital pcr Genetically modified soybean MON87708×MON89788 Stacked event

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An Automatic Implementation of Oropharyngeal Swab Sampling for Diagnosing Respiratory Infectious Diseases via Soft Robotic End-Effectors

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Chinese Journal of Mechanical Engineering 2024年第2期37卷 55-67页

作者： Yafeng Cui Wenjie Yu Jingjing Li Qi Shao Ding Weng Guoping Yin Xiaohao Zhang Xinjun Liu Jingying Ye Jiadao Wang Huichan Zhao Department of Mechanical Engineering Tsinghua UniversityBeijing 100084China Beijing Huakang Tongbang Technology Company Beijing 102609China Department of Otorhinolaryngology Beijing Tsinghua Changgung HospitalSchool of Clinical MedicineTsinghua UniversityBeijing 102218China Tianjin Research Institute for Advanced Equipment Tsinghua UniversityTianjin 300300China

The most widely adopted method for diagnosing respiratory infectious diseases is to conduct polymerase chain reaction(pcr)assays on patients’respiratory specimens,which are collected through either nasal or oropharyngeal *** manual swab sampling process poses a high risk to the examiner and may cause false-negative results owing to improper *** this paper,we propose a pneumatically actuated soft end-effector specifically designed to achieve all of the tasks involved in swab *** soft end-effector utilizes circumferential instability to ensure grasping stability,and exhibits several key properties,including high load-to-weight ratio,error tolerance,and variable swab-tip stiffness,leading to successful automatic robotic oropharyngeal swab sampling,from loosening and tightening the transport medium tube cap,holding the swab,and conducting sampling,to snapping off the swab tail and sterilizing *** an industrial collaborative robotic arm,we integrated the soft end-effector,force sensor,camera,lights,and remote-control stick,and developed a robotic oropharyngeal swab sampling *** this swab sampling system,we conducted oropharyngeal swab-sampling tests on 20 *** digital pcr assay results(RNase P RNA gene absolute copy numbers for the samples)revealed that our system successfully collected sufficient numbers of cells from the pharyngeal wall for respiratory disease *** summary,we have developed a pharyngeal swab-sampling system based on an“enveloping”soft actuator,studied the sampling process,and imple-mented whole-process robotic oropharyngeal swab-sampling.

关键词： Diagnosis Medical robot Soft end-effector Swab-sampling digital pcr

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The Mitochondrial DNA copy number acts as a biomarker in the pathological state

The Mitochondrial DNA copy number acts as a biomarker in the...

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2021海峡两岸暨港澳精准医学青年博士论坛

作者： Zhang Tong Zhao Guoqiang Liu Yanan Wei Fengxiang The Genetics Laboratory Longgang District Maternity and Child Healthcare Hospital School of Clinical Medical Jiamusi University

Mitochondria are vital organelles in cells. Mitochondrial DNA is the only genetic material that exists outside the nucleus, and there are hundreds to thousands of copies in each cell in a tissue-specific way. When mitochondrial DNA is subjected to oxidative damage and carcinogen attack, it causes overproduction of reactive oxygen species, damage to some large molecules, insufficient energy supply and production of essential molecules such as heme and nucleotides, and abnormal mitochondrial signaling function. It can easily lead to mutations in mitochondrial DNA, which can lead to abnormalities in energy metabolism and oxidative stress. This is also one of the important reasons leading to mitochondrial dysfunction. At present, the change of mitochondrial DNA copy number is very important in various pathological conditions in the body such as diabetes, cardiovascular disease, mental disease, cancer, and aging, and reflects the biomarkers of various pathological conditions in the human body. Therefore, the regulation and detection of mitochondrial DNA copy number is an important aspect of mitochondrial genetics and biogenesis. And it is helpful for targeted disease prevention, early diagnosis and individualized treatment. It is of great significance for applications in clinical medicine, genetics, and life sciences. Nowadays, digital pcr technology can be used to identify diseases in the early stage of disease progression. Continue to track the existence, expansion and disappearance of pathogenic microorganisms and genetic variations in cancer, infectious diseases, and other human diseases. Nucleic acid detection with high resolution and low target level has a significant impact in the field of diagnosis, providing great advantages for diagnosis and preventive medicine. In short, changes in plasma mitochondrial DNA content have been associated with a variety of disease types, suggesting that the mitochondrial genome may be a key contributor to the occurrence of some di

关键词： mitochondrial DNA copy number mitochondrial dysfunction hereditary diseases digital pcr

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digital Polymerase Chain Reaction and its application in biological detection

Digital Polymerase Chain Reaction and its application in bio...

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2021海峡两岸暨港澳精准医学青年博士论坛

作者： Liu Yannan Zhang Tong Zhao Guoqiang Wei Feixiang Longgang District Maternity&Child Healthcare Hospital of Shenzhen City Department of Basic Medicine Jiamusi University

digital polymerase chain reaction is a high-sensitivity and high-specificity nucleic acid absolute quantitative analysis technology developed after real-time quantitative polymerase chain reaction, which does not depend on the standard curve. The reaction system is divided into a large number of independent reaction molecule pools for pcr amplification, and the number of nucleic acid copies is corrected according to the Poisson distribution. It can achieve absolute quantitative analysis of low-concentration samples. In recent years, based on the development of microfluidic technology, digital pcr is mainly divided into three types: microfluidic digital pcr, microdroplet digital pcr and chip digital pcr. digital polymerase chain reaction detection technology is very suitable for the detection of circulating nucleic acid, circulating tumor cell DNA or microribonucleic acid in blood or other body fluids. It not only realizes detection and quantification, but also discovers new tumor markers and studies the function of nucleic acid And prognosis, such as: detecting early diagnosis markers of oral cancer in oral saliva samples to predict the risk of oral cancer. Simultaneously detect a variety of molecular aberrations, improve the early diagnosis rate of lung cancer, and improve the detection and quantification of copy number variation. In gynecological prenatal diagnosis, traditional prenatal diagnosis is carried out through amniocentesis and chorionic villus sampling. This invasive sampling method is likely to cause trauma to the fetus and increase the risk of miscarriage for pregnant women. dpcr can accurately analyze free fetal DNA in maternal plasma and is a safe, fast and accurate new technology for prenatal diagnosis. For example: It has strong applicability in the risk management of large gestational babies with singleton *** ddpcr method has high sensitivity and strong specificity, and is suitable for the screening of chromosomal genetic diseases such a

关键词： digital pcr absolute quantification liquid biopsy

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A Centrifugal-Driven Droplet Generation Method with Minimal Waste for Single-Cell Whole Genome Amplification

A Centrifugal-Driven Droplet Generation Method with Minimal ...

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第十一届全国化学生物学学术会议

作者： Xingrui Li Dongfeng Zhang Weidong Ruan Weizhi Liu Kun Yin Tian Tian Yunpeng Bi Qingyu Ruan Yuan Zhao Zhi Zhu Chaoyong Yang Department of Chemical Biology College of Chemistry and Chemical EngineeringPen-Tung Sah Institute of Micro-Nano Science and TechnologyXiamen University Institute of Molecular Medicine Renji HospitalShanghai Jiao Tong University School of Medicine

High-quality whole-genome amplification（WGA） of individual cells is the primary step for characterizing the genetic information of single cells in biology and *** the most popular single-cell WGA method,multiple displacement amplification（MDA） is often plagued by the nonuniform *** droplet MDA has been an innovative tool to solve this dilemma by mitigating the amplification bias and increasing the genomic *** these advantages,the time-consuming droplet generation process,the waste of small volume samples and the difficulty of parallel operation for multiple single-cell samples remain major ***,we introduce a centrifugal-driven droplet generation method for rapid and convenient generation of uniform droplets from a relatively small volume sample（5 μL） in 60 s with more than 98% sample ***-cell centrifugal-driven droplet MDA（cd-MDA） has been conducted for single-cell sequencing,achieving uniform amplification and broad genomic *** the single-molecule sensitivity,minimum sample waste,high genomic coverage as well as excellent sequencing evenness,this centrifugal-driven droplet generation method is promising for convenient and scalable use in digital pcr and single-cell whole-genome research.

关键词： single cell WGA droplet generation digital pcr centrifugal driven microchip

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