Transgene Detection by Digital Droplet PCR

Somatic gene therapy is a promising tool for the treatment of severe diseases. Because of its abuse potential for performance enhancement in sports, the World Anti-Doping Agency (WADA) included the term ‘gene doping’ in the official list of banned substances and methods in 2004. Several nested PCR or qPCR-based strategies have been proposed that aim at detecting long-term presence of transgene in blood, but these strategies are hampered by technical limitations. We developed a digital droplet PCR (ddPCR) protocol for Insulin-Like Growth Factor 1 (IGF1) detection and demonstrated its applicability monitoring 6 mice injected into skeletal muscle with AAV9-IGF1 elements and 2 controls over a 33-day period. A duplex ddPCR protocol for simultaneous detection of Insulin-Like Growth Factor 1 (IGF1) and Erythropoietin (EPO) transgenic elements was created. A new DNA extraction procedure with target-orientated usage of restriction enzymes including on-column DNA-digestion was established. In vivo data revealed that IGF1 transgenic elements could be reliably detected for a 33-day period in DNA extracted from whole blood. In vitro data indicated feasibility of IGF1 and EPO detection by duplex ddPCR with high reliability and sensitivity. On-column DNA-digestion allowed for significantly improved target detection in downstream PCR-based approaches. As ddPCR provides absolute quantification, it ensures excellent day-to-day reproducibility. Therefore, we expect this technique to be used in diagnosing and monitoring of viral and bacterial infection, in detecting mutated DNA sequences as well as profiling for the presence of foreign genetic material in elite athletes in the future.     

逆转录-聚合酶链式反应检测果树RNA病毒

为调查主要果树携带苹果褪绿叶斑病毒(ACLSV)、苹果茎沟病毒(ASGV)、苹果茎痘病毒(ASPV)和李属坏死环斑病毒(PNRSV)的情况,以指示植物为试材,优化了四种病毒的逆转录-聚合酶链式反应(RT-PCR)检测体系.使用该优化体系检测了苹果树、樱桃树、桃树中携带上述四种病毒的情况.结果:建立了特异性强、灵敏度高、成本低的RT-PCR检测体系;同时又证明主要果树被这几种病毒单独感染和复合感染的程度均较高.     

双重数字PCR在转基因大豆检测中的应用

利用微滴式数字PCR(droplet digital PCR, ddPCR)平台建立针对MON87705、MON87769、DP356043三种转基因大豆中外源基因的双重PCR检测方法。利用双重数字PCR方法检测特异性、定量范围等参数,优化所用引物探针组合及实验体系程序,检测外源基因与内标准基因的拷贝数。结果表明,所用引物探针组合在数字PCR方法中仅对目标大豆品系有荧光信号,具有特异性,可用于转基因大豆品系的筛选与鉴别。检测了大豆的转基因成分含量,结果与材料标准品参数基本一致,并根据结果设定定量检测限为0.5%,定性检测限为0.05%,可满足低纯度样品检测的需求。双重数字PCR体系能够准确且稳定的满足实际检测需要,在实际应用上具有良好的发展前景。     

荧光定量RT-PCR技术快速检测SARS病毒核酸

建立以特异性荧光探针为特点的TaqMan荧光定量RTPCR方法用于检测严重急性呼吸道综合症病毒(severeacuterespiratorysyndromeassociatecoronavirus,SARSCoV)核酸。筛选针对SARS病毒基因保守区域设计的引物与TaqMan探针,并对荧光定量RTPCR反应体系与反应条件进行优化,验证本方法的特异性、敏感度与重复性。实验结果表明:本方法对SARS病毒核酸的检测具有高度特异性,与甲1型、甲3型、乙型流感病毒、禽流感病毒H5N1、麻疹及其他呼吸道病毒均无交叉反应;检测灵敏度达0.1TCID50;从病毒核酸提取至检测完成仅需3h左右,且操作简便,重复性好。本研究建立的TaqMan荧光定量RTPCR方法特异、敏感、快速,适合于临床实验室进行SARS病毒的早期快速检测。     

荧光定量RT-PCR技术快速检测SARS病毒核酸

建立以特异性荧光探针为特点的TaqMan荧光定量RT-PCR方法用于检测严重急性呼吸道综合症病毒(severe acute respiratory syndrome -associate coronavirus,SARS-CoV)核酸.筛选针对SARS病毒基因保守区域设计的引物与TaqMan探针,并对荧光定量RT-PCR反应体系与反应条件进行优化,验证本方法的特异性、敏感度与重复性. 实验结果表明本方法对SARS病毒核酸的检测具有高度特异性,与甲1型、甲3型、乙型流感病毒、禽流感病毒H5N1、麻疹及其他呼吸道病毒均无交叉反应;检测灵敏度达0.1TCID50;从病毒核酸提取至检测完成仅需3h左右,且操作简便,重复性好.本研究建立的TaqMan荧光定量RT-PCR方法特异、敏感、快速,适合于临床实验室进行SARS病毒的早期快速检测.     

链特异性RT-PCR方法检测口蹄疫病毒负链RNA

旨在建立一种快速、灵敏、特异的检测口蹄疫病毒在复制过程中产生的负链RNA的方法。根据口蹄疫病毒(foot-and-mouth disease virus,FMDV)病毒5’-非编码区(5’-UTR)基因序列,设计了5条引物链特异性RT-PCR引物,建立检测口蹄疫病毒负链RNA的链特异性RT-PCR方法。提取FMD病毒RNA,应用设计的正向引物T1-H1做反转录引物,经反转录和RNA酶A消化后,再经两轮链特异性PCR扩增,可特异性地检测FMDV在复制过程中产生的负链RNA。所建立的检测口蹄疫病毒负链RNA的链特异性RT-PCR方法是一种可靠的方法,在确定细胞培养物和动物感染FMDV的病毒复制和了解病毒的致病性研究中具有应用前景。     

食品中椰毒假单胞菌微滴式数字PCR定量检测方法的建立

椰毒假单胞菌可污染包括发酵米面等多种食物,其产生的两种毒素导致严重的食物中毒,威胁人体健康。基于数字PCR技术,建立了针对椰毒假单胞菌的定量测量方法。该方法具有较好的特异性与重复性,人工污染糯米汤样品中的检出限为361 CFU/mL。与平板培养法相比,基于数字PCR技术的测量方法缩短了测量时间,有利于椰毒假单胞菌的快速准确测量,未来可在食品安全监测与中毒分析等领域进行应用。     

Comparison of Droplet Digital PCR and Seminested Real-Time PCR for Quantification of Cell-Associated HIV-1 RNA

Cell-associated (CA) HIV-1 RNA is considered a potential marker for assessment of viral reservoir dynamics and antiretroviral therapy (ART) response in HIV-infected patients. Recent studies employed sensitive seminested real-time quantitative (q)PCR to quantify CA HIV-1 RNA. Digital PCR has been recently described as an alternative PCR-based technique for absolute quantification with higher accuracy compared to qPCR. Here, a comparison was made between the droplet digital PCR (ddPCR) and the seminested qPCR for quantification of unspliced (us) and multiply spliced (ms) CA HIV-1 RNA. Synthetic RNA standards and CA HIV-1 RNA from infected patients on and off ART (N = 34) were quantified with both methods. Correlations were observed between the methods both for serially diluted synthetic standards (usRNA: R² = 0.97, msRNA: R² = 0.92) and patient-derived samples (usRNA: R² = 0.51, msRNA: R² = 0.87). Seminested qPCR showed better quantitative linearity, accuracy and sensitivity in the quantification of synthetic standards than ddPCR, especially in the lower quantification ranges. Both methods demonstrated equally high detection rate of usRNA in patient samples on and off ART (91%), whereas ddPCR detected msRNA in larger proportion of samples from ART-treated patients (p = 0.13). We observed an average agreement between the methods for usRNA quantification in patient samples, albeit with a large standard deviation (bias = 0.05±0.75 log₁₀). However, a bias of 0.94±0.36 log₁₀ was observed for msRNA. No-template controls were consistently negative in the seminested qPCR, but yielded a positive ddPCR signal for some wells. Therefore, the false positive signals may have affected the detection power of ddPCR in this study. Digital PCR is promising for HIV nucleic acid quantification, but the false positive signals need further attention. Quantitative assays for CA HIV RNA have the potential to improve monitoring of patients on ART and to be used in clinical studies aimed at HIV eradication, but should be cross-validated by multiple laboratories prior to wider use.     

Development of the Droplet Digital PCR Method for the Detection and Quantification of Erwinia pyrifoliae

Black shoot blight disease caused by Erwinia pyrifoliae has serious impacts on quality and yield in pear production in Korea; therefore, rapid and accurate methods for its detection are needed. However, traditional detection methods require a great deal of time and fail to achieve absolute quantification. In the present study, we developed a droplet digital polymerase chain reaction (ddPCR) method for the detection and absolute quantification of E. pyrifoliae using a pair of species-specific primers. The detection range was 10³–10⁷ copies/ml (DNA templates) and cfu/ml (cell culture templates). This new method exhibited good linearity and repeatability and was validated by absolute quantification of E. pyrifoliae DNA copies from samples of artificially inoculated immature pear fruits. Here, we present the first study of ddPCR assay for the detection and quantification of E. pyrifoliae. This method has potential applications in epidemiology and for the early prediction of black shoot blight outbreaks.     

Detection of MET Gene Copy Number in Cancer Samples Using the Droplet Digital PCR Method

Purpose

The analysis of MET gene copy number (CN) has been considered to be a potential biomarker to predict the response to MET-targeted therapies in various cancers. However, the current standard methods to determine MET CN are SNP 6.0 in the genomic DNA of cancer cell lines and fluorescence in situ hybridization (FISH) in tumor models, respectively, which are costly and require advanced technical skills and result in relatively subjective judgments. Therefore, we employed a novel method, droplet digital PCR (ddPCR), to determine the MET gene copy number with high accuracy and precision.

Methods

The genomic DNA of cancer cell lines or tumor models were tested and compared with the MET gene CN and MET/CEN-7 ratio determined by SNP 6.0 and FISH, respectively.

Results

In cell lines, the linear association of the MET CN detected by ddPCR and SNP 6.0 is strong (Pearson correlation = 0.867). In tumor models, the MET CN detected by ddPCR was significantly different between the MET gene amplification and non-amplification groups according to FISH (mean: 15.4 vs 2.1; P = 0.044). Given that MET gene amplification is defined as MET CN >5.5 by ddPCR, the concordance rate between ddPCR and FISH was 98.0%, and Cohen''s kappa coefficient was 0.760 (95% CI, 0.498–1.000; P <0.001).

Conclusions

The results demonstrated that the ddPCR method has the potential to quantify the MET gene copy number with high precision and accuracy as compared with the results from SNP 6.0 and FISH in cancer cell lines and tumor samples, respectively.     

逆转录套式PCR检测粪便样本中的SARS冠状病毒

为了观察SARS冠状病毒在SARS患者粪便中的存在规律,建立了检测SARS冠状病毒RNA的逆转录-聚合酶链反应(RT-PCR)方法,并应用该方法检测了241份SARS患者粪便样本。部分PCR产物应用测序技术进行验证。RT-PCR的灵敏度为10^-10稀释度的病毒原液(原液为10^8TCID50／ml)。241份粪便样本的总体检出率为24．1％(58／241),其中发病后的前10d和20d的检出率均为50．0％。随着发病时间的延长,阳性检出率呈下降趋势。应用RT-PCR从粪便中检测SARS冠状病毒是可行的,在发病50d以后仍有17．0％左右的阳性检出率,提示SARS恢复期患者具有排毒的可能性,给后续的卫生防疫措施提供了一定的参考数据。     

Innate Immune Suppression Enables Frequent Transfection with RNA Encoding Reprogramming Proteins

Background

Generating autologous pluripotent stem cells for therapeutic applications will require the development of efficient DNA-free reprogramming techniques. Transfecting cells with in vitro-transcribed, protein-encoding RNA is a straightforward method of directly expressing high levels of reprogramming proteins without genetic modification. However, long-RNA transfection triggers a potent innate immune response characterized by growth inhibition and the production of inflammatory cytokines. As a result, repeated transfection with protein-encoding RNA causes cell death.

Methodology/Principal Findings

RNA viruses have evolved methods of disrupting innate immune signaling by destroying or inhibiting specific proteins to enable persistent infection. Starting from a list of known viral targets, we performed a combinatorial screen to identify siRNA cocktails that could desensitize cells to exogenous RNA. We show that combined knockdown of interferon-β (Ifnb1), Eif2ak2, and Stat2 rescues cells from the innate immune response triggered by frequent long-RNA transfection. Using this technique, we were able to transfect primary human fibroblasts every 24 hours with RNA encoding the reprogramming proteins Oct4, Sox2, Klf4, and Utf1. We provide evidence that the encoded protein is active, and we show that expression can be maintained for many days, through multiple rounds of cell division.

Conclusions/Significance

Our results demonstrate that suppressing innate immunity enables frequent transfection with protein-encoding RNA. This technique represents a versatile tool for investigating expression dynamics and protein interactions by enabling precise control over levels and timing of protein expression. Our finding also opens the door for the development of reprogramming and directed-differentiation methods based on long-RNA transfection.     

Digital Twin Enables Rational Design of Ultrahigh-Power Lithium-Ion Batteries

With the widespread applications of electric vehicles, power grid stabilization, and high-pulsed power loads, high-power lithium-ion batteries (LIBs) are in urgent demand. However, the existing experimental-based design of high-power batteries is usually costly and inefficient, and provides limited information on the complex physicochemical processes inside the batteries. Digital twin concept is promising for capturing the batteries’ electrochemical performance, and optimizing the power capability of LIBs. Here, an electrochemical-thermal coupled model is developed as a digital twin model for rational design of ultrahigh-power LiFePO₄/graphite LIBs. The model can accurately predict the batteries’ performance and help to predetermine the optimal parameters to achieve an ultrahigh power capability. After model-guided optimization, the battery shows a high energy density of 92.38 Wh kg⁻¹ at an ultrafast discharging current of 50 C and can withstand 150 C pulse discharging tests. Notably, the digital twin model can reveal experimentally inaccessible time- and space-resolved information and identify the rate-determining steps inside the battery. Hence, model-driven optimization of ultrahigh-power LiFePO₄/graphite batteries is successfully realized aiming at the critical factors in the rate-determining steps. The work provides an instructive design of ultrahigh-power LiFePO₄/graphite batteries, which might guide the future direction to boost the power capability of LIBs.     

Repeat cDNA synthesis and RT-PCR with the same source of RNA

A simple method has been developed that enables reextraction of RNA from an RNA-cDNA mixture. The reextracted RNA was converted to cDNA followed by polymerase chain reaction (PCR). Thus, cDNA synthesis (followed by PCR) was carried out two times on the same source of RNA. The method has been applied to 40 RNA samples of diverse tissue origin with a success rate of 100%. Thus, the method offers more versatile use of small but valuable RNA sources than currently possible.     

Picoinjection of Microfluidic Drops Without Metal Electrodes

Existing methods for picoinjecting reagents into microfluidic drops require metal electrodes integrated into the microfluidic chip. The integration of these electrodes adds cumbersome and error-prone steps to the device fabrication process. We have developed a technique that obviates the needs for metal electrodes during picoinjection. Instead, it uses the injection fluid itself as an electrode, since most biological reagents contain dissolved electrolytes and are conductive. By eliminating the electrodes, we reduce device fabrication time and complexity, and make the devices more robust. In addition, with our approach, the injection volume depends on the voltage applied to the picoinjection solution; this allows us to rapidly adjust the volume injected by modulating the applied voltage. We demonstrate that our technique is compatible with reagents incorporating common biological compounds, including buffers, enzymes, and nucleic acids.     

微滴数字PCR技术应用进展

微滴数字PCR技术是数字PCR技术的一种,是近年来分子生物学领域的新型革命性技术,其特点是高度灵敏、绝对定量及高效方便等。目前,该技术在微生物检测、转基因检测、疾病检测以及质检领域的研究和应用中已凸显优势,随着微滴数字PCR仪的普及,该技术必将广泛应用于生命科学的各个领域。     

RNA Interference Silencing of a Major Lipid Droplet Protein Affects Lipid Droplet Size in Chlamydomonas reinhardtii

Eukaryotic cells store oils in the chemical form of triacylglycerols in distinct organelles, often called lipid droplets. These dynamic storage compartments have been intensely studied in the context of human health and also in plants as a source of vegetable oils for human consumption and for chemical or biofuel feedstocks. Many microalgae accumulate oils, particularly under conditions limiting to growth, and thus have gained renewed attention as a potentially sustainable feedstock for biofuel production. However, little is currently known at the cellular or molecular levels with regard to oil accumulation in microalgae, and the structural proteins and enzymes involved in the biogenesis, maintenance, and degradation of algal oil storage compartments are not well studied. Focusing on the model green alga Chlamydomonas reinhardtii, the accumulation of triacylglycerols and the formation of lipid droplets during nitrogen deprivation were investigated. Mass spectrometry identified 259 proteins in a lipid droplet-enriched fraction, among them a major protein, tentatively designated major lipid droplet protein (MLDP). This protein is specific to the green algal lineage of photosynthetic organisms. Repression of MLDP gene expression using an RNA interference approach led to increased lipid droplet size, but no change in triacylglycerol content or metabolism was observed.Triacylglycerols (TAGs) are stored in lipid droplets which are subcellular structures in specialized cells ubiquitous to eukaryotes but have more recently also been identified in some prokaryotes (26). In plants and animals, lipid droplets are surrounded by cytosol and are believed to bud off the endoplasmic reticulum (ER) (15, 26). While traditionally considered merely as storage compartments, recent studies suggest that lipid droplets in animals play important additional roles in lipid homeostasis and protein storage (8). In oilseed plants, TAG accumulated in seeds is used as a reservoir of energy and membrane lipid building blocks to support rapid growth after germination (15). Many green algae are capable of accumulating large amounts of TAG in lipid droplets, particularly as a result of abiotic stresses, such as nutrient deprivation or high-light exposure. Although TAG metabolism in algae has not yet been extensively studied at the biochemical or molecular level, it is proposed that TAG turnover contributes primarily to the assembly of membrane lipids to facilitate rapid cell division after the cessation of nutrient limitation (14, 38).The general structure of lipid droplets is conserved in different species with a globular neutral lipid core enclosed by a membrane lipid monolayer (26). In addition, specific proteins are associated with lipid droplets and play important roles in lipid droplet structure and function. A number of recent proteomic studies of lipid droplets from different animals and tissues (8, 40), Saccharomyces cerevisiae (4), and plants (16, 17) have revealed that the lipid droplet-associated proteins of these organisms are quite distinct. For example, the abundant lipid droplet proteins in animals—the so-called “PAT” family of proteins comprised of perilipin, adipose differentiation-related protein (ADRP), and TIP47 (21)—have no apparent orthologs in the desiccating seed plant Arabidopsis thaliana; conversely, the oleosins which coat the oil bodies of Arabidopsis and many other seed plants are not found in animals (26). Reverse genetic studies of these proteins have helped to elucidate the role of A. thaliana oleosins in regulating lipid droplet size and preventing droplet fusion (35, 36) or that of mouse adipocyte perilipin in regulating lipolytic activity at the lipid droplet surface (37). Moreover, recent genomewide RNAi screens in Drosophila cells implicated 1.5 to 3.0% of all genes as directly or indirectly involved in lipid droplet formation and/or regulation and resulted in the identification of a new role for the Arf1-COPI vesicular transport machinery in regulating droplet morphology and lipid utilization (7, 12). In contrast, few molecular details are known about algal lipid droplet biogenesis although many TAG-rich algal species have been described (14).Our efforts to identify proteins related to the PAT protein family or oleosins in the Chlamydomonas reinhardtii genome (24) or genomes of other green algal and diatom species, including Thalassiosira pseudonana, Volvox carteri, and Chlorella sp. NC64A, revealed no putative algal orthologs. In order to identify both potentially novel and conserved proteins which function in algal lipid droplet biogenesis, we studied the accumulation of TAG in lipid droplets of nitrogen-limited C. reinhardtii cells and identified candidate lipid droplet-associated proteins by mass spectrometry.