A real-time quantitative optical method to characterize crack propagation in colloidal photonic crystal film(CPCF)is developed based on particle deformation models and previous real-time crack observations. The crac...A real-time quantitative optical method to characterize crack propagation in colloidal photonic crystal film(CPCF)is developed based on particle deformation models and previous real-time crack observations. The crack propagation process and temperature dependence of the crack propagation rate in CPCF are investigated. By this method, the crack propagation rate is found to slow down gradually to zero when cracks become more numerous and dense. Meanwhile, with the temperature increasing, the crack propagation rate constant decreases. The negative temperature dependence of the crack propagation rate is due to the increase of van der Waals attraction, which finally results in the decrease of resultant force. The findings provide new insight into the crack propagation process in CPCF.展开更多
[Objective] This study aimed to establish a Real-Time quantitative PCR method for the determination of transposon copy number in C. sakazakii. [ Method ] With single-copy housekeeping gene atpD as the reference gene, ...[Objective] This study aimed to establish a Real-Time quantitative PCR method for the determination of transposon copy number in C. sakazakii. [ Method ] With single-copy housekeeping gene atpD as the reference gene, recombinant plasmid containing both single-copy housekeeping gene atpD and EZ-TN5 transposon was constructed; based on the established standard curves for real-time quantitative detection of atpD gene and EZ-TN5 transposon, copy number of atpD gene and EZ-TN5 transpason in three C. sakazakii mutants was detected and the ratio was calculated. [ Result] Correlation coefficients of the standard curves for real-time quantitative detection of atpD gene and EZ-TN5 transposon were 0. 999 and 0.998, respectively ; the ratios of copy number of atpD gene and EZ-TN5 transposon in three C. sakazakii mutants were 0.98, 1.17 and 0.91, respectively, which indicates that EZ-TN5 transpeson in C. sakakii mutants is a single-copy. [ Conclusion] Real-time quantitative PCR method established in this study had high availability and could replace the Southern blot method to detect the copy num- ber of EZ-TN5 transposon in different bacteria.展开更多
TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybean...TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybeans as the endogenous reference standard, the gradient dilution method was used to separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies, and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation. The standard curve equation of endogenous reference gene was y =–3.422x+35.201, R2=0.998; the standard curve equation of exogenous gene was y =–3.495x+35.303, R2=0.999. The sample copies was got by putting Ct value into the standard curve equation, and it was the ratio of exogenous gene and reference gene. We found that CaMV35S gene in transgenic soy was single copy.展开更多
CT9 is a recently cloned cancer-testis antigen, which is a member of the bromodomain and extraterminal family. Each member of this protein family contains two N-terminal bromodomain motifs. We investigated the distrib...CT9 is a recently cloned cancer-testis antigen, which is a member of the bromodomain and extraterminal family. Each member of this protein family contains two N-terminal bromodomain motifs. We investigated the distribution of CT9 in different tissues and the possibility for it to be used as a potential therapeutic target in cancer treament. By using the real-time RT-PCR method and 18SrRNA as an internal standard, we analyzed the CT9 expression in several normal human tissues and in the tissues of patients suffering from cancer. The result of this study shows that the highest level of mRNA is only present in testis tissue because the CT9 expression has not been detected in other normal tissues. In 6 of 10 cases of gastric adenocarcinoma, in 3 of 10 cases of esophageal squamous cell carcinoma, in 2 of 9 cases of endometrial carcinoma and only in 1 of 12 cases of brain cancer, the low level expression of CT9 was detected. In none of the 12 cases of cervical squamous cell carcinoma, the expression of CT9 was detected. Since the high level expression of CT9 is only found in the normal testis tissue, but the low expression in cancer tissues, for example tissues of cervical squamous cell carcinoma, brain cancer, endometfial adenocarcinoma, esophageal squamous cell carcinoma, we conclude that CT9 cannot be used as a cancer therapeutic target molecule for cervical squamous cell carcinoma, brain cancer, endometrial adenocarcinoma, esophageal squamous cell carcinoma.展开更多
A plasmid containing target DNA and a standard curve for real-time quantitative PCR of the cold-induced Cor3 gene of Volvariella volvacea were constructed. These will provide the basis for further research on Cor3 gen...A plasmid containing target DNA and a standard curve for real-time quantitative PCR of the cold-induced Cor3 gene of Volvariella volvacea were constructed. These will provide the basis for further research on Cor3 gene expression at low temperature, and ultimately for assigning a role for the gene in the low temperature autolysis of V. volvacea.展开更多
AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl trans...AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl transferase(HPRT),ribosomal protein large P0(36B4)and terminal uridylyl transferase 1(U6)in the diabetic retinal tissue of rat model.METHODS:The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)in two groups;normal control rats and streptozotocininduced diabetic rats.The stability analysis of gene expression was investigated using geNorm,NormFinder,BestKeeper,and comparative delta-Ct(ΔCt)algorithms.RESULTS:The 36B4 gene was stably expressed in the retinal tissues of normal control animals;however,it was less stable in diabetic retinas.The 18s gene was expressed consistently in both normal control and diabetic rats’retinal tissue.That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats.Furthermore,there was no ideal gene stably expressed for use in all experimental settings.CONCLUSION:Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.展开更多
Quantitative real-time PCR(qRT-PCR)has been widely used for gene expression analysis,and selection of reference genes is a key point to obtain accurate results.To find out optimal reference genes for qRT-PCR in Manila...Quantitative real-time PCR(qRT-PCR)has been widely used for gene expression analysis,and selection of reference genes is a key point to obtain accurate results.To find out optimal reference genes for qRT-PCR in Manila clam Ruditapes philippinarum in response to hypoxia,different tissues were used and compared to evaluate the stability of candidate reference genes under low oxygen stress(DO 0.5mgL^(−1) and DO 2.0mgL^(−1))and normal condition(DO 7.5mgL^(−1)).Seven candidate reference genes were selected to evaluate the stability of their expression levels.The reference genes were evaluated by Delta Ct,BestKeeper,NormFinder and geNorm,and then screened by RefFinder calculation.Under hypoxic stress of 0.5mgL^(−1),the most suitable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were TUB and HIS,respectively.For hypoxic stress of 2.0mgL^(−1),the most stable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were RPS23 and EF1A,respectively.At the normal condition,HIS and EF1A were identified as the optimal internal reference genes in gill and hepatopancreas respectively,and GFRP2 was the best internal reference gene for axe foot and adductor muscle.The present findings will provide important basis for the selection of reference genes for qRT-PCR analysis of gene expression level in bivalves under hypoxic stress,which might be helpful for the analysis of other molluscs too.展开更多
Objective To establish and modify quantitative real-time polymerase chain reaction(qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes.Methods A database of capsular polysaccharide(cps)loci sequence...Objective To establish and modify quantitative real-time polymerase chain reaction(qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes.Methods A database of capsular polysaccharide(cps)loci sequences was generated,covering 97 pneumococcal serotypes.Bioinformatics analyses were performed to identify the cps loci structure and target genes related to different pneumococcal serotypes with specific SNPs.A total of 27 novel qPCR serotyping assay primers and probes were established based on qPCR,while 27 recombinant plasmids containing serotype-specific DNA sequence fragments were constructed as reference target sequences to examine the specificity and sensitivity of the qPCR assay.A panel of pneumococcal reference strains was employed to evaluate the capability of pneumococcal serotyping.Results A total of 97 pneumococcal serotyping assays based on qPCR were established and modified,which included 64 serotypes previously reported as well as an additional 33 serotypes.Twenty-seven novel qPCR serotyping target sequences were implemented in the pneumococcal qPCR serotyping system.A total of 97 pneumococcal serotypes,which included 52 individual serotypes and 45 serotypes belonging to 20 serogroups,could not be identified as individual serotypes.The sensitivity of qPCR assays based on 27 target sequences was 1–100 copies/μL.The specificity of the qPCR assays was 100%,which were tested by a panel of 90 serotypes of the pneumococcal reference strains.Conclusion A total of 27 novel qPCR assays were established and modified to analyze 97pneumococcal serotypes.展开更多
Background Real-time quantitative RT-PCR (RQ-PCR) assay has become a vital tool to monitor residual disease of leukemia, However, the complexity and standardization of RQ-PCR should never be overlooked and the resul...Background Real-time quantitative RT-PCR (RQ-PCR) assay has become a vital tool to monitor residual disease of leukemia, However, the complexity and standardization of RQ-PCR should never be overlooked and the results should be interpreted cautiously in clinical conditions. We aimed to assess the methodology of RQ-PCR and its clinical applications in monitoring molecular kinetics of 36 newly diagnosed cases of acute promyelocytic leukemia patients with t (15;17) from October 2004 to December 2005.Methods All the TaqMan probe-based RQ-PCR reactions and analysis were performed on an ABI-PRISM 7500 platform, The quantitation of PML-RARα transcripts was represented by the normalized quotient, that is, PML-RARα transcript copies divided by ABL transcript copies, According to induction therapy, the patients were classed into two groups: group 1 (n=23), three-drug combination including arsenics, all-trans retinoic acid and mitoxantrone; and group 2 (n=13).two-drug combination from all-trans retinoic acid, arsenics and mitoxantrone.Results The sensitivity of RQ-PCR was 1 per 105 cells and 5 copies of the PML-RARα transcript could be reproducibly detected, No false positive results occurred in 40 non-acute promyelocytic leukemia samples, Optimal amplification efficiency could be attained, which was determined by the slope of the standard curves (slope: -3.2 -- -3.7). The inter-assay and intra-assay variation coefficients of the method were 1.01% and 0.56% respectively. Although the time to attain hematological complete remission was similar in both groups, the time to achieve molecular remission of group 1 was significantly shorter than that of group 2 (61 days vs 75 days, P=0.034). The rate of molecular remission within 70 days was higher in group 1 than in group 2 (75.00% vs 38.46%, P=0.036), Compared with pretreatment, median reduction of the PML-RARα transcript before first consolidation therapy differed significantly between group 1 and group 2 (log scale, 3.15 vs 2.31, P=0.024), Interestingly, we found that PML-RARα transcript levels temporarily increased in bone marrow (7 patients) and peripheral blood (22 patients) samples of patients during induction therapy in both groups.Conclusions The RQ-PCR assay is reliable for the detection of PML-RARα transcripts. Arsenics, all-trans retinoic acid and mitoxantrone triad induction treatment of acute promyelocytic leukemia is superior to two-drug combination induction therapy in terms of the molecular response.展开更多
Real-time quantitative polymerase chain reaction(qPCR)has gained popularity as a technique to detect and quantify a specific group of target microorganisms from various environmental samples including soil,water,sedim...Real-time quantitative polymerase chain reaction(qPCR)has gained popularity as a technique to detect and quantify a specific group of target microorganisms from various environmental samples including soil,water,sediments,and sludge.Although qPCR is a very useful technique for nucleic acid quantification,accurately quantifying the target microbial group strongly depends on the quality of the primer and probe used.Many aspects of conducting qPCR assays have become increasingly routine and automated;however,one of the most important aspects,designing and selecting primer and probe sets,is often a somewhat arcane process.In many cases,failed or non-specific amplification can be attributed to improperly designed primer-probe sets.This paper is intended to provide guidelines and general principles for designing group-specific primers and probes for qPCR assays.We demonstrate the effectiveness of these guidelines by reviewing the use of qPCR to study anaerobic processes and biologic nutrient removal processes.qPCR assays using group-specific primers and probes designed with this method,have been used to successfully quantify 16S ribosomal Ribonucleic Acid(16S rRNA)gene copy numbers from target methanogenic and ammoniaoxidizing bacteria in various laboratory-and full-scale biologic processes.Researchers with a good command of primer and probe design can use qPCR as a valuable tool to study biodiversity and to develop more efficient control strategies for biologic processes.展开更多
Objective To establish a TaqMan real-time fluorescent quantitative PCR to detect Vibrio vulnificus based on the hemolysin gene (vvhA) coding cytolysin. Methods Primers and probes in the conserved region of the vvhA ...Objective To establish a TaqMan real-time fluorescent quantitative PCR to detect Vibrio vulnificus based on the hemolysin gene (vvhA) coding cytolysin. Methods Primers and probes in the conserved region of the vvhA gene sequence were designed for the TaqMan real-time PCR to detect 100 bp amplicon from V. vulnificus DNA. Recombinant plasmid pMD19-vvhA100 was constructed and used as a positive control during the detection. Minimal amplification cycles (Ct value) and fluorescence intensity enhancement (ARn value) were used as observing indexes to optimize the reaction conditions of TaqMan real-time PCR. The TaqMan assay for the detection of Vbirio vulnificus was evaluated in pure culture, mice tissue which artificially contaminated Vibrio vulnificus and clinical samples. Results The established TaqMan real-time PCR showed positive results only for Vibrio vulnificus DNA and pMD19-vvhA100. The standard curve was plotted and the minimum level of the vvhA target from the recombinant plasmid DNA was 103 copies with a Ct value of 37.94±0.19, as the equivalent of 0.01 ng purified genomic DNA of Vibrio vulnificus. The results detected by TaqMan PCR were positive for the 16 clinical samples and all the specimens of peripheral blood and subcutaneous tissue of mice which were infected with Vibrio vulnificus. Conclusion TaqMan real-time PCR is a rapid, effective, and quantitative tool to detect Vibro vulnificus, and can be used in clinical laboratory diagnosis of septicemia and wound infection caused by Vibrio vulnificus.展开更多
A real-time data processing system is designed for the carbon dioxide dispersion interferometer(CO_(2)-DI)on EAST.The system utilizes the parallel and pipelining capabilities of an fieldprogrammable gate array(FPGA)to...A real-time data processing system is designed for the carbon dioxide dispersion interferometer(CO_(2)-DI)on EAST.The system utilizes the parallel and pipelining capabilities of an fieldprogrammable gate array(FPGA)to digitize and process the intensity of signals from the detector.Finally,the real-time electron density signals are exported through a digital-to-analog converter(DAC)module in the form of analog signals.The system has been successfully applied in the CO_(2)-DI system to provide low-latency electron density input to the plasma control system on EAST.Experimental results of the latest campaign with long-pulse discharges on EAST(2022–2023)demonstrate that the system can respond effectively in the case of rapid density changes,proving its reliability and accuracy for future electron density calculation.展开更多
The co-frequency vibration fault is one of the common faults in the operation of rotating equipment,and realizing the real-time diagnosis of the co-frequency vibration fault is of great significance for monitoring the...The co-frequency vibration fault is one of the common faults in the operation of rotating equipment,and realizing the real-time diagnosis of the co-frequency vibration fault is of great significance for monitoring the health state and carrying out vibration suppression of the equipment.In engineering scenarios,co-frequency vibration faults are highlighted by rotational frequency and are difficult to identify,and existing intelligent methods require more hardware conditions and are exclusively time-consuming.Therefore,Lightweight-convolutional neural networks(LW-CNN)algorithm is proposed in this paper to achieve real-time fault diagnosis.The critical parameters are discussed and verified by simulated and experimental signals for the sliding window data augmentation method.Based on LW-CNN and data augmentation,the real-time intelligent diagnosis of co-frequency is realized.Moreover,a real-time detection method of fault diagnosis algorithm is proposed for data acquisition to fault diagnosis.It is verified by experiments that the LW-CNN and sliding window methods are used with high accuracy and real-time performance.展开更多
To address the impact of wind-power fluctuations on the stability of power systems,we propose a comprehensive approach that integrates multiple strategies and methods to enhance the efficiency and reliability of a sys...To address the impact of wind-power fluctuations on the stability of power systems,we propose a comprehensive approach that integrates multiple strategies and methods to enhance the efficiency and reliability of a system.First,we employ a strategy that restricts long-and short-term power output deviations to smoothen wind power fluctuations in real time.Second,we adopt the sliding window instantaneous complete ensemble empirical mode decomposition with adaptive noise(SW-ICEEMDAN)strategy to achieve real-time decomposition of the energy storage power,facilitating internal power distribution within the hybrid energy storage system.Finally,we introduce a rule-based multi-fuzzy control strategy for the secondary adjustment of the initial power allocation commands for different energy storage components.Through simulation validation,we demonstrate that the proposed comprehensive control strategy can smoothen wind power fluctuations in real time and decompose energy storage power.Compared with traditional empirical mode decomposition(EMD),ensemble empirical mode decomposition(EEMD),and complete ensemble empirical mode decomposition with adaptive noise(CEEMDAN)decomposition strategies,the configuration of the energy storage system under the SW-ICEEMDAN control strategy is more optimal.Additionally,the state-of-charge of energy storage components fluctuates within a reasonable range,enhancing the stability of the power system and ensuring the secure operation of the energy storage system.展开更多
To obtain more stable spectral data for accurate quantitative analysis of multi-element,especially for the large-area in-situ elements detection of soils, we propose a method for a multielement quantitative analysis o...To obtain more stable spectral data for accurate quantitative analysis of multi-element,especially for the large-area in-situ elements detection of soils, we propose a method for a multielement quantitative analysis of soils using calibration-free laser-induced breakdown spectroscopy(CF-LIBS) based on data filtering. In this study, we analyze a standard soil sample doped with two heavy metal elements, Cu and Cd, with a specific focus on the line of Cu I324.75 nm for filtering the experimental data of multiple sample sets. Pre-and post-data filtering,the relative standard deviation for Cu decreased from 30% to 10%, The limits of detection(LOD)values for Cu and Cd decreased by 5% and 4%, respectively. Through CF-LIBS, a quantitative analysis was conducted to determine the relative content of elements in soils. Using Cu as a reference, the concentration of Cd was accurately calculated. The results show that post-data filtering, the average relative error of the Cd decreases from 11% to 5%, indicating the effectiveness of data filtering in improving the accuracy of quantitative analysis. Moreover, the content of Si, Fe and other elements can be accurately calculated using this method. To further correct the calculation, the results for Cd was used to provide a more precise calculation. This approach is of great importance for the large-area in-situ heavy metals and trace elements detection in soil, as well as for rapid and accurate quantitative analysis.展开更多
The real-time detection and instance segmentation of strawberries constitute fundamental components in the development of strawberry harvesting robots.Real-time identification of strawberries in an unstructured envi-r...The real-time detection and instance segmentation of strawberries constitute fundamental components in the development of strawberry harvesting robots.Real-time identification of strawberries in an unstructured envi-ronment is a challenging task.Current instance segmentation algorithms for strawberries suffer from issues such as poor real-time performance and low accuracy.To this end,the present study proposes an Efficient YOLACT(E-YOLACT)algorithm for strawberry detection and segmentation based on the YOLACT framework.The key enhancements of the E-YOLACT encompass the development of a lightweight attention mechanism,pyramid squeeze shuffle attention(PSSA),for efficient feature extraction.Additionally,an attention-guided context-feature pyramid network(AC-FPN)is employed instead of FPN to optimize the architecture’s performance.Furthermore,a feature-enhanced model(FEM)is introduced to enhance the prediction head’s capabilities,while efficient fast non-maximum suppression(EF-NMS)is devised to improve non-maximum suppression.The experimental results demonstrate that the E-YOLACT achieves a Box-mAP and Mask-mAP of 77.9 and 76.6,respectively,on the custom dataset.Moreover,it exhibits an impressive category accuracy of 93.5%.Notably,the E-YOLACT also demonstrates a remarkable real-time detection capability with a speed of 34.8 FPS.The method proposed in this article presents an efficient approach for the vision system of a strawberry-picking robot.展开更多
In recent years,frequent fire disasters have led to enormous damage in China.Effective firefighting rescues can minimize the losses caused by fires.During the rescue processes,the travel time of fire trucks can be sev...In recent years,frequent fire disasters have led to enormous damage in China.Effective firefighting rescues can minimize the losses caused by fires.During the rescue processes,the travel time of fire trucks can be severely affected by traffic conditions,changing the effective coverage of fire stations.However,it is still challenging to determine the effective coverage of fire stations considering dynamic traffic conditions.This paper addresses this issue by combining the traveling time calculationmodelwith the effective coverage simulationmodel.In addition,it proposes a new index of total effective coverage area(TECA)based on the time-weighted average of the effective coverage area(ECA)to evaluate the urban fire services.It also selects China as the case study to validate the feasibility of the models,a fire station(FS-JX)in Changsha.FS-JX station and its surrounding 9,117 fire risk points are selected as the fire service supply and demand points,respectively.A total of 196 simulation scenarios throughout a consecutiveweek are analyzed.Eventually,1,933,815 sets of valid sample data are obtained.The results showed that the TECA of FS-JX is 3.27 km^(2),which is far below the standard requirement of 7.00 km^(2) due to the traffic conditions.The visualization results showed that three rivers around FS-JX interrupt the continuity of its effective coverage.The proposed method can provide data support to optimize the locations of fire stations by accurately and dynamically determining the effective coverage of fire stations.展开更多
基金Project supported by the National Basic Research Program of China(Grant Nos.2012CB932903 and 2012CB932904)the National Natural Science Foundation of China(Grant Nos.51372270,11474333,and 21173260)
文摘A real-time quantitative optical method to characterize crack propagation in colloidal photonic crystal film(CPCF)is developed based on particle deformation models and previous real-time crack observations. The crack propagation process and temperature dependence of the crack propagation rate in CPCF are investigated. By this method, the crack propagation rate is found to slow down gradually to zero when cracks become more numerous and dense. Meanwhile, with the temperature increasing, the crack propagation rate constant decreases. The negative temperature dependence of the crack propagation rate is due to the increase of van der Waals attraction, which finally results in the decrease of resultant force. The findings provide new insight into the crack propagation process in CPCF.
基金Supported by Key Project of Tianjin Municipal Education Commission(2010ZD01)
文摘[Objective] This study aimed to establish a Real-Time quantitative PCR method for the determination of transposon copy number in C. sakazakii. [ Method ] With single-copy housekeeping gene atpD as the reference gene, recombinant plasmid containing both single-copy housekeeping gene atpD and EZ-TN5 transposon was constructed; based on the established standard curves for real-time quantitative detection of atpD gene and EZ-TN5 transposon, copy number of atpD gene and EZ-TN5 transpason in three C. sakazakii mutants was detected and the ratio was calculated. [ Result] Correlation coefficients of the standard curves for real-time quantitative detection of atpD gene and EZ-TN5 transposon were 0. 999 and 0.998, respectively ; the ratios of copy number of atpD gene and EZ-TN5 transposon in three C. sakazakii mutants were 0.98, 1.17 and 0.91, respectively, which indicates that EZ-TN5 transpeson in C. sakakii mutants is a single-copy. [ Conclusion] Real-time quantitative PCR method established in this study had high availability and could replace the Southern blot method to detect the copy num- ber of EZ-TN5 transposon in different bacteria.
基金Supported by the Program of Technology Bureau of Harbin (2010RFQXN101)the Subproject of Transgenic Significant Specific Project (20112X08004-002-002-004)
文摘TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybeans as the endogenous reference standard, the gradient dilution method was used to separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies, and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation. The standard curve equation of endogenous reference gene was y =–3.422x+35.201, R2=0.998; the standard curve equation of exogenous gene was y =–3.495x+35.303, R2=0.999. The sample copies was got by putting Ct value into the standard curve equation, and it was the ratio of exogenous gene and reference gene. We found that CaMV35S gene in transgenic soy was single copy.
文摘CT9 is a recently cloned cancer-testis antigen, which is a member of the bromodomain and extraterminal family. Each member of this protein family contains two N-terminal bromodomain motifs. We investigated the distribution of CT9 in different tissues and the possibility for it to be used as a potential therapeutic target in cancer treament. By using the real-time RT-PCR method and 18SrRNA as an internal standard, we analyzed the CT9 expression in several normal human tissues and in the tissues of patients suffering from cancer. The result of this study shows that the highest level of mRNA is only present in testis tissue because the CT9 expression has not been detected in other normal tissues. In 6 of 10 cases of gastric adenocarcinoma, in 3 of 10 cases of esophageal squamous cell carcinoma, in 2 of 9 cases of endometrial carcinoma and only in 1 of 12 cases of brain cancer, the low level expression of CT9 was detected. In none of the 12 cases of cervical squamous cell carcinoma, the expression of CT9 was detected. Since the high level expression of CT9 is only found in the normal testis tissue, but the low expression in cancer tissues, for example tissues of cervical squamous cell carcinoma, brain cancer, endometfial adenocarcinoma, esophageal squamous cell carcinoma, we conclude that CT9 cannot be used as a cancer therapeutic target molecule for cervical squamous cell carcinoma, brain cancer, endometrial adenocarcinoma, esophageal squamous cell carcinoma.
基金Sponsored by the Shanghai Academy of Agricultural Science and Shanghai Science Committee (No .003912076)
文摘A plasmid containing target DNA and a standard curve for real-time quantitative PCR of the cold-induced Cor3 gene of Volvariella volvacea were constructed. These will provide the basis for further research on Cor3 gene expression at low temperature, and ultimately for assigning a role for the gene in the low temperature autolysis of V. volvacea.
基金Supported by grant from Fundamental Research Grant Scheme by Ministry of Higher Education(MoHE)600-IRMI/FRGS 5/3(101/2019).
文摘AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl transferase(HPRT),ribosomal protein large P0(36B4)and terminal uridylyl transferase 1(U6)in the diabetic retinal tissue of rat model.METHODS:The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)in two groups;normal control rats and streptozotocininduced diabetic rats.The stability analysis of gene expression was investigated using geNorm,NormFinder,BestKeeper,and comparative delta-Ct(ΔCt)algorithms.RESULTS:The 36B4 gene was stably expressed in the retinal tissues of normal control animals;however,it was less stable in diabetic retinas.The 18s gene was expressed consistently in both normal control and diabetic rats’retinal tissue.That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats.Furthermore,there was no ideal gene stably expressed for use in all experimental settings.CONCLUSION:Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.
基金supported by research grants from the Science and Technology Innovation Program of the Laoshan Laboratory(No.LSKJ202203803)the National Natural Science Foundation of China(No.32273107)+2 种基金supported by the Central Public-Interest Scientific Institution Basal Research Fund,Yellow Sea Fisheries Research Institute,CAFS(No.20603022022001)the project of Putian Science and Technology Department(No.2021NJJ002)the Shinan District Science and Technology Plan Project(No.2022-2-026-ZH).
文摘Quantitative real-time PCR(qRT-PCR)has been widely used for gene expression analysis,and selection of reference genes is a key point to obtain accurate results.To find out optimal reference genes for qRT-PCR in Manila clam Ruditapes philippinarum in response to hypoxia,different tissues were used and compared to evaluate the stability of candidate reference genes under low oxygen stress(DO 0.5mgL^(−1) and DO 2.0mgL^(−1))and normal condition(DO 7.5mgL^(−1)).Seven candidate reference genes were selected to evaluate the stability of their expression levels.The reference genes were evaluated by Delta Ct,BestKeeper,NormFinder and geNorm,and then screened by RefFinder calculation.Under hypoxic stress of 0.5mgL^(−1),the most suitable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were TUB and HIS,respectively.For hypoxic stress of 2.0mgL^(−1),the most stable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were RPS23 and EF1A,respectively.At the normal condition,HIS and EF1A were identified as the optimal internal reference genes in gill and hepatopancreas respectively,and GFRP2 was the best internal reference gene for axe foot and adductor muscle.The present findings will provide important basis for the selection of reference genes for qRT-PCR analysis of gene expression level in bivalves under hypoxic stress,which might be helpful for the analysis of other molluscs too.
基金supported by a grant from Beijing Municipal Natural Science Foundation [L212011]National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention [131031102000210003&102393230020020000002]。
文摘Objective To establish and modify quantitative real-time polymerase chain reaction(qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes.Methods A database of capsular polysaccharide(cps)loci sequences was generated,covering 97 pneumococcal serotypes.Bioinformatics analyses were performed to identify the cps loci structure and target genes related to different pneumococcal serotypes with specific SNPs.A total of 27 novel qPCR serotyping assay primers and probes were established based on qPCR,while 27 recombinant plasmids containing serotype-specific DNA sequence fragments were constructed as reference target sequences to examine the specificity and sensitivity of the qPCR assay.A panel of pneumococcal reference strains was employed to evaluate the capability of pneumococcal serotyping.Results A total of 97 pneumococcal serotyping assays based on qPCR were established and modified,which included 64 serotypes previously reported as well as an additional 33 serotypes.Twenty-seven novel qPCR serotyping target sequences were implemented in the pneumococcal qPCR serotyping system.A total of 97 pneumococcal serotypes,which included 52 individual serotypes and 45 serotypes belonging to 20 serogroups,could not be identified as individual serotypes.The sensitivity of qPCR assays based on 27 target sequences was 1–100 copies/μL.The specificity of the qPCR assays was 100%,which were tested by a panel of 90 serotypes of the pneumococcal reference strains.Conclusion A total of 27 novel qPCR assays were established and modified to analyze 97pneumococcal serotypes.
文摘Background Real-time quantitative RT-PCR (RQ-PCR) assay has become a vital tool to monitor residual disease of leukemia, However, the complexity and standardization of RQ-PCR should never be overlooked and the results should be interpreted cautiously in clinical conditions. We aimed to assess the methodology of RQ-PCR and its clinical applications in monitoring molecular kinetics of 36 newly diagnosed cases of acute promyelocytic leukemia patients with t (15;17) from October 2004 to December 2005.Methods All the TaqMan probe-based RQ-PCR reactions and analysis were performed on an ABI-PRISM 7500 platform, The quantitation of PML-RARα transcripts was represented by the normalized quotient, that is, PML-RARα transcript copies divided by ABL transcript copies, According to induction therapy, the patients were classed into two groups: group 1 (n=23), three-drug combination including arsenics, all-trans retinoic acid and mitoxantrone; and group 2 (n=13).two-drug combination from all-trans retinoic acid, arsenics and mitoxantrone.Results The sensitivity of RQ-PCR was 1 per 105 cells and 5 copies of the PML-RARα transcript could be reproducibly detected, No false positive results occurred in 40 non-acute promyelocytic leukemia samples, Optimal amplification efficiency could be attained, which was determined by the slope of the standard curves (slope: -3.2 -- -3.7). The inter-assay and intra-assay variation coefficients of the method were 1.01% and 0.56% respectively. Although the time to attain hematological complete remission was similar in both groups, the time to achieve molecular remission of group 1 was significantly shorter than that of group 2 (61 days vs 75 days, P=0.034). The rate of molecular remission within 70 days was higher in group 1 than in group 2 (75.00% vs 38.46%, P=0.036), Compared with pretreatment, median reduction of the PML-RARα transcript before first consolidation therapy differed significantly between group 1 and group 2 (log scale, 3.15 vs 2.31, P=0.024), Interestingly, we found that PML-RARα transcript levels temporarily increased in bone marrow (7 patients) and peripheral blood (22 patients) samples of patients during induction therapy in both groups.Conclusions The RQ-PCR assay is reliable for the detection of PML-RARα transcripts. Arsenics, all-trans retinoic acid and mitoxantrone triad induction treatment of acute promyelocytic leukemia is superior to two-drug combination induction therapy in terms of the molecular response.
基金This work was also supported by the Korea Research Foundation Grant funded by the Korean Government(MOEHRD)by New&Renewable Energy of the Korea Institute of Energy Technology Evaluation and Planning(KETEP)grant(No.20101T100100366).
文摘Real-time quantitative polymerase chain reaction(qPCR)has gained popularity as a technique to detect and quantify a specific group of target microorganisms from various environmental samples including soil,water,sediments,and sludge.Although qPCR is a very useful technique for nucleic acid quantification,accurately quantifying the target microbial group strongly depends on the quality of the primer and probe used.Many aspects of conducting qPCR assays have become increasingly routine and automated;however,one of the most important aspects,designing and selecting primer and probe sets,is often a somewhat arcane process.In many cases,failed or non-specific amplification can be attributed to improperly designed primer-probe sets.This paper is intended to provide guidelines and general principles for designing group-specific primers and probes for qPCR assays.We demonstrate the effectiveness of these guidelines by reviewing the use of qPCR to study anaerobic processes and biologic nutrient removal processes.qPCR assays using group-specific primers and probes designed with this method,have been used to successfully quantify 16S ribosomal Ribonucleic Acid(16S rRNA)gene copy numbers from target methanogenic and ammoniaoxidizing bacteria in various laboratory-and full-scale biologic processes.Researchers with a good command of primer and probe design can use qPCR as a valuable tool to study biodiversity and to develop more efficient control strategies for biologic processes.
文摘Objective To establish a TaqMan real-time fluorescent quantitative PCR to detect Vibrio vulnificus based on the hemolysin gene (vvhA) coding cytolysin. Methods Primers and probes in the conserved region of the vvhA gene sequence were designed for the TaqMan real-time PCR to detect 100 bp amplicon from V. vulnificus DNA. Recombinant plasmid pMD19-vvhA100 was constructed and used as a positive control during the detection. Minimal amplification cycles (Ct value) and fluorescence intensity enhancement (ARn value) were used as observing indexes to optimize the reaction conditions of TaqMan real-time PCR. The TaqMan assay for the detection of Vbirio vulnificus was evaluated in pure culture, mice tissue which artificially contaminated Vibrio vulnificus and clinical samples. Results The established TaqMan real-time PCR showed positive results only for Vibrio vulnificus DNA and pMD19-vvhA100. The standard curve was plotted and the minimum level of the vvhA target from the recombinant plasmid DNA was 103 copies with a Ct value of 37.94±0.19, as the equivalent of 0.01 ng purified genomic DNA of Vibrio vulnificus. The results detected by TaqMan PCR were positive for the 16 clinical samples and all the specimens of peripheral blood and subcutaneous tissue of mice which were infected with Vibrio vulnificus. Conclusion TaqMan real-time PCR is a rapid, effective, and quantitative tool to detect Vibro vulnificus, and can be used in clinical laboratory diagnosis of septicemia and wound infection caused by Vibrio vulnificus.
基金funded and supported by the Comprehensive Research Facility for Fusion Technology Program of China(No.2018-000052-73-01-001228)the HFIPS Director’s Fund(No.YZJJKX202301)+1 种基金the Anhui Provincial Major Science and Technology Project(No.2023z020004)Task JB22001 from the Anhui Provincial Department of Economic and Information Technology。
文摘A real-time data processing system is designed for the carbon dioxide dispersion interferometer(CO_(2)-DI)on EAST.The system utilizes the parallel and pipelining capabilities of an fieldprogrammable gate array(FPGA)to digitize and process the intensity of signals from the detector.Finally,the real-time electron density signals are exported through a digital-to-analog converter(DAC)module in the form of analog signals.The system has been successfully applied in the CO_(2)-DI system to provide low-latency electron density input to the plasma control system on EAST.Experimental results of the latest campaign with long-pulse discharges on EAST(2022–2023)demonstrate that the system can respond effectively in the case of rapid density changes,proving its reliability and accuracy for future electron density calculation.
基金Supported by National Natural Science Foundation of China(Grant Nos.51875031,52242507)Beijing Municipal Natural Science Foundation of China(Grant No.3212010)Beijing Municipal Youth Backbone Personal Project of China(Grant No.2017000020124 G018).
文摘The co-frequency vibration fault is one of the common faults in the operation of rotating equipment,and realizing the real-time diagnosis of the co-frequency vibration fault is of great significance for monitoring the health state and carrying out vibration suppression of the equipment.In engineering scenarios,co-frequency vibration faults are highlighted by rotational frequency and are difficult to identify,and existing intelligent methods require more hardware conditions and are exclusively time-consuming.Therefore,Lightweight-convolutional neural networks(LW-CNN)algorithm is proposed in this paper to achieve real-time fault diagnosis.The critical parameters are discussed and verified by simulated and experimental signals for the sliding window data augmentation method.Based on LW-CNN and data augmentation,the real-time intelligent diagnosis of co-frequency is realized.Moreover,a real-time detection method of fault diagnosis algorithm is proposed for data acquisition to fault diagnosis.It is verified by experiments that the LW-CNN and sliding window methods are used with high accuracy and real-time performance.
基金supported by the National Natural Science Foundation of China(Grant No.51677058)。
文摘To address the impact of wind-power fluctuations on the stability of power systems,we propose a comprehensive approach that integrates multiple strategies and methods to enhance the efficiency and reliability of a system.First,we employ a strategy that restricts long-and short-term power output deviations to smoothen wind power fluctuations in real time.Second,we adopt the sliding window instantaneous complete ensemble empirical mode decomposition with adaptive noise(SW-ICEEMDAN)strategy to achieve real-time decomposition of the energy storage power,facilitating internal power distribution within the hybrid energy storage system.Finally,we introduce a rule-based multi-fuzzy control strategy for the secondary adjustment of the initial power allocation commands for different energy storage components.Through simulation validation,we demonstrate that the proposed comprehensive control strategy can smoothen wind power fluctuations in real time and decompose energy storage power.Compared with traditional empirical mode decomposition(EMD),ensemble empirical mode decomposition(EEMD),and complete ensemble empirical mode decomposition with adaptive noise(CEEMDAN)decomposition strategies,the configuration of the energy storage system under the SW-ICEEMDAN control strategy is more optimal.Additionally,the state-of-charge of energy storage components fluctuates within a reasonable range,enhancing the stability of the power system and ensuring the secure operation of the energy storage system.
基金supported by the Major Science and Technology Project of Gansu Province(No.22ZD6FA021-5)the Industrial Support Project of Gansu Province(Nos.2023CYZC-19 and 2021CYZC-22)the Science and Technology Project of Gansu Province(Nos.23YFFA0074,22JR5RA137 and 22JR5RA151).
文摘To obtain more stable spectral data for accurate quantitative analysis of multi-element,especially for the large-area in-situ elements detection of soils, we propose a method for a multielement quantitative analysis of soils using calibration-free laser-induced breakdown spectroscopy(CF-LIBS) based on data filtering. In this study, we analyze a standard soil sample doped with two heavy metal elements, Cu and Cd, with a specific focus on the line of Cu I324.75 nm for filtering the experimental data of multiple sample sets. Pre-and post-data filtering,the relative standard deviation for Cu decreased from 30% to 10%, The limits of detection(LOD)values for Cu and Cd decreased by 5% and 4%, respectively. Through CF-LIBS, a quantitative analysis was conducted to determine the relative content of elements in soils. Using Cu as a reference, the concentration of Cd was accurately calculated. The results show that post-data filtering, the average relative error of the Cd decreases from 11% to 5%, indicating the effectiveness of data filtering in improving the accuracy of quantitative analysis. Moreover, the content of Si, Fe and other elements can be accurately calculated using this method. To further correct the calculation, the results for Cd was used to provide a more precise calculation. This approach is of great importance for the large-area in-situ heavy metals and trace elements detection in soil, as well as for rapid and accurate quantitative analysis.
基金funded by Anhui Provincial Natural Science Foundation(No.2208085ME128)the Anhui University-Level Special Project of Anhui University of Science and Technology(No.XCZX2021-01)+1 种基金the Research and the Development Fund of the Institute of Environmental Friendly Materials and Occupational Health,Anhui University of Science and Technology(No.ALW2022YF06)Anhui Province New Era Education Quality Project(Graduate Education)(No.2022xscx073).
文摘The real-time detection and instance segmentation of strawberries constitute fundamental components in the development of strawberry harvesting robots.Real-time identification of strawberries in an unstructured envi-ronment is a challenging task.Current instance segmentation algorithms for strawberries suffer from issues such as poor real-time performance and low accuracy.To this end,the present study proposes an Efficient YOLACT(E-YOLACT)algorithm for strawberry detection and segmentation based on the YOLACT framework.The key enhancements of the E-YOLACT encompass the development of a lightweight attention mechanism,pyramid squeeze shuffle attention(PSSA),for efficient feature extraction.Additionally,an attention-guided context-feature pyramid network(AC-FPN)is employed instead of FPN to optimize the architecture’s performance.Furthermore,a feature-enhanced model(FEM)is introduced to enhance the prediction head’s capabilities,while efficient fast non-maximum suppression(EF-NMS)is devised to improve non-maximum suppression.The experimental results demonstrate that the E-YOLACT achieves a Box-mAP and Mask-mAP of 77.9 and 76.6,respectively,on the custom dataset.Moreover,it exhibits an impressive category accuracy of 93.5%.Notably,the E-YOLACT also demonstrates a remarkable real-time detection capability with a speed of 34.8 FPS.The method proposed in this article presents an efficient approach for the vision system of a strawberry-picking robot.
基金support from the National Natural Science Foundation of China (No.52204202)the Hunan Provincial Natural Science Foundation of China (No.2023JJ40058)the Science and Technology Program of Hunan Provincial Departent of Transportation (No.202122).
文摘In recent years,frequent fire disasters have led to enormous damage in China.Effective firefighting rescues can minimize the losses caused by fires.During the rescue processes,the travel time of fire trucks can be severely affected by traffic conditions,changing the effective coverage of fire stations.However,it is still challenging to determine the effective coverage of fire stations considering dynamic traffic conditions.This paper addresses this issue by combining the traveling time calculationmodelwith the effective coverage simulationmodel.In addition,it proposes a new index of total effective coverage area(TECA)based on the time-weighted average of the effective coverage area(ECA)to evaluate the urban fire services.It also selects China as the case study to validate the feasibility of the models,a fire station(FS-JX)in Changsha.FS-JX station and its surrounding 9,117 fire risk points are selected as the fire service supply and demand points,respectively.A total of 196 simulation scenarios throughout a consecutiveweek are analyzed.Eventually,1,933,815 sets of valid sample data are obtained.The results showed that the TECA of FS-JX is 3.27 km^(2),which is far below the standard requirement of 7.00 km^(2) due to the traffic conditions.The visualization results showed that three rivers around FS-JX interrupt the continuity of its effective coverage.The proposed method can provide data support to optimize the locations of fire stations by accurately and dynamically determining the effective coverage of fire stations.