[Objective]To investigate the expression of zebrafish vascular endothelial growth factor-2（VEGFR-2） at different developmental stages.[Method]Total RNAs were extracted from 12,24,48,72 and 96 hpf stage zebrafish emb...[Objective]To investigate the expression of zebrafish vascular endothelial growth factor-2（VEGFR-2） at different developmental stages.[Method]Total RNAs were extracted from 12,24,48,72 and 96 hpf stage zebrafish embryos and larvae.Real-time quantitative RT-PCR was performed to examine the expression of VEGFR-2.The data were analyzed by 2^-△△Ct method.[Result]The expression level of VEGFR-2 gene increased gradually from 12 to 72 hpf,and subsequently decreased at 96 hpf.The expression level was lowest at 12 hpf,highest at 72 hpf,and had significant differences when compared with that of other developmental stages.[Conclusion]The expression level of VEGFR-2 increases gradually before blood vessel maturation and decreases as blood vessels mature.展开更多
[Objective] To explore the feasibility of using SYBR Green real-time quantitative PCR technique to estimate the copy numbers of exogenous gene in a transgenic plant.[Methods] Using SYBR Green real-time quantitative PC...[Objective] To explore the feasibility of using SYBR Green real-time quantitative PCR technique to estimate the copy numbers of exogenous gene in a transgenic plant.[Methods] Using SYBR Green real-time quantitative PCR technique,we have determined the copy numbers of the exogenous CYCD3;1 in transgenic Arabidopsis by comparing an endogenous single copy reference gene with CYCD3;1 copy numbers in transgenic plant,meanwhile comparing CYCD3;1 copy numbers between wild plant and transgenic plant.[Results]The exogenous CYCD3;1 copy numbers calculated by this method is identical with results of traditional Southern blot analysis which is highly accurate.[Conclusion]This method is simple,effective and safe for estimating transgene copy numbers.展开更多
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
The establishment of highly sensitive diagnostic methods is critical in the early diagnosis and control of Zika virus(ZIKV)and in preventing serious neurological complications of ZIKV infection. In this study, we esta...The establishment of highly sensitive diagnostic methods is critical in the early diagnosis and control of Zika virus(ZIKV)and in preventing serious neurological complications of ZIKV infection. In this study, we established micro-droplet digital polymerase chain reaction(ddPCR) and real-time quantitative PCR(RT-qPCR) protocols for the detection of ZIKV based on the amplification of the NS5 gene. For the ZIKV standard plasmid, the RT-qPCR results showed that the cycle threshold(Ct) value was linear from 10~1 to 10~8 copy/l L, with a standard curve R^2 of 0.999 and amplification efficiency of 92.203%;however, a concentration as low as 1 copy/l L could not be detected. In comparison with RT-qPCR, the dd PCR method resulted in a linear range of 10~1–10~4 copy/l L and was able to detect concentrations as low as 1 copy/l L. Thus, for detecting ZIKV from clinical samples, RT-qPCR is a better choice for high-concentration samples(above 10~1 copy/l L),while ddPCR has excellent accuracy and sensitivity for low-concentration samples. These results indicate that the ddPCR method should be of considerable use in the early diagnosis, laboratory study, and monitoring of ZIKV.展开更多
Objective:The aim of this study was to detect the expression level of Metadherin (MTDH) in peripheral blood of the breast cancer patients by real-time fluorescence quantitative polymerase chain reaction (PCR),and to e...Objective:The aim of this study was to detect the expression level of Metadherin (MTDH) in peripheral blood of the breast cancer patients by real-time fluorescence quantitative polymerase chain reaction (PCR),and to explore the relationship between expression of Metadherin gene in the patients peripheral blood and the clinic-pathological features in breast cancer. Methods:Real-time fluorescence quantitative polymerase chain reaction was employed to determine the expression level of Metadherin gene in 80 peripheral blood samples of breast cancer patients and healthy donors. Results:The expression of Metadherin gene in breast cancer patients peripheral blood were positive,in which 34 breast cancer patients were highly expressed,accounting for 55.7%,while the expression of Metadherin gene in normal females peripheral blood were negative,there was statistical significance (Ratio = 2.02±0.81,P < 0.05); Ratio of the Metadherin expression in breast cancer patients peripheral blood and the glyceraldehyde-3-phosphate dehydrogenase expression was 1.15 ± 0.36. REST software analysis showed that the expression of Metadherin gene was significantly up-regulated in breast cancer. Conclusion:The SYBR Green I quantitative real-time polymerase chain reaction method can successfully detect the expression level of Metadherin gene. Expression level of Metadherin gene in breast cancer patients peripheral blood is closely related to survival,and it maybe involved in the development of breast cancer and used as an indicator of prognosis.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
OBJECTIVE To explore the relationship between multimarker detection of MAGE-1,MAGE-3 and AFP mRNAs in the peripheral blood of patients with hepatocellular carcinoma and micrometastasis using a realtime quantitative-PC...OBJECTIVE To explore the relationship between multimarker detection of MAGE-1,MAGE-3 and AFP mRNAs in the peripheral blood of patients with hepatocellular carcinoma and micrometastasis using a realtime quantitative-PCR(real-time Q-PCR)assay. METHODS Peripheral blood samples were obtained from control subjects and 86 patients with hepatocellular carcinoma (HCC).Real-time Q-PCR was used to detect MAGE-1,MAGE-3, and AFP mRNAs in the blood cells. RESULTS In 86 tumor specimens,the positivity for MAGE-1, MAGE-3,and AFP genes was respectively 34.9%(30/86),60.5% (52/86)and 69.8%(60/86).All specimens expressed at least one marker.MAGE-1,MAGE-3,and AFP transcripts were detected respectively in 12(14.0%),18(20.1%)and 29(33.7%)of the 86 blood specimens from hepatocellular carcinoma patients,while 45 specimens(52.3%)were positive for at least one marker.In addition,MAGE-1,MAGE-3 and AFP gene transcripts were not detected in any peripheral blood specimens from 25 chronic liver disease patients and 28 normal healthy volunteers.The positive rate correlated with the TNM clinical stages,extrahepatic metastasis and portal vein carcinothrombosis(P<0.05).No correlation was found between tumor size,tumor number, differentiation,serum a-fetoprotein(AFP)and the positive rate. CONCLUSION Our results indicate that a multimarker real- time Q-PCR assay with cancer-specific markers such as MAGE-1 and MAGE-3 in combination with a hepatocyte-specific AFP marker may be a promising diagnostic tool for monitoring hepatocellular carcinoma patients with better sensitivity and specificity.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
AIM:Normalizing the results of real-time quantitative reverse transcription polymerase chain reaction(RT-q PCR)is essential for the accuracy of analysis.Commonly used approaches include input nucleic acid standardizat...AIM:Normalizing the results of real-time quantitative reverse transcription polymerase chain reaction(RT-q PCR)is essential for the accuracy of analysis.Commonly used approaches include input nucleic acid standardization(ΔCt method),normalization against a single internal reference gene(ΔΔCt method),and geometric averaging of multiple reference gene abundance using statistical software.We evaluated these approaches in the liver of db/db mice,a typical model of fatty liver disease.METHODS:Seven reference genes,β-actin(ACTB),eukaryotic initiation factor(e IF)5,glyceraldehyde-3-phosphate dehydrogenase(GAPDH),hydroxymethylbilane synthase(HMBS),hypoxanthineguanine phosphoribosyltransferase(HPRT)1,polymerase(RNA)II(DNA directed)polypeptide A(Polr2A)and ribosomal protein P〈0(RPLP〈0),were evaluated using software of ge Norm and Norm Finder.Hepatic lipogenesis genes,such as thyroid hormone-responsive protein(Thrsp),stearoyl-Co A desaturase(SCD)1,sterol regulatory element-binding protein(SREBP)1c and fatty acid synthase(FAS),were used as target genes of interest.RESULTS:The expression levels of all target genes and GAPDH were significantly elevated(P〈0.05)in db/db mouse livers by theΔCt method.ACTB and HMBS were the most stable genes calculated by the software of ge Norm.Norm Finder analysis indicated that ACTB was the most stable gene,and the best combination of 2 genes was GAPDH and RPLP〈0.Normalization against a single internal reference gene of ACTB or RPLP〈0,the geometric mean of ACTB and HMBS,or GAPDH and RPLP〈0 showed similar results that the expression levels of Thrsp,SCD1 and FAS,but not SREBP1 c increased(P〈0.05)in the liver of db/db mice.CONCLUSION:TheΔCt approach ensures a meaningful and biologically significant appraisal of gene expression.Use of the software like ge Norm or Norm Finder should be integrated withΔCt method.展开更多
Real-time fluorescent quantitative PCR （RQ-PCR） is a detection method by adding fluorescent dye or fluorescent probe into the PCR reaction system, using fluorescent signal accumulation to monitor amplification react...Real-time fluorescent quantitative PCR （RQ-PCR） is a detection method by adding fluorescent dye or fluorescent probe into the PCR reaction system, using fluorescent signal accumulation to monitor amplification reactions of PCR reaction process, and finally the unknown template can be quantitatively analyzed through the standard curve. So the detection level of PCR has improved from the qualitative to the quantitative. In order to provide a theoretical reference for further application, the principle, classification, advantages and disadvantages of RQ-PCR were intro- duced, and its application and progress in plants in recent years were reviewed.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
As a fundamental component in computer vision,edges can be categorized into four types based on discontinuities in reflectance,illumination,surface normal,or depth.While deep CNNs have significantly advanced generic e...As a fundamental component in computer vision,edges can be categorized into four types based on discontinuities in reflectance,illumination,surface normal,or depth.While deep CNNs have significantly advanced generic edge detection,real-time multi-class semantic edge detection under resource constraints remains challenging.To address this,we propose a lightweight framework based on PiDiNet that enables fine-grained semantic edge detection.Our model simultaneously predicts background and four edge categories from full-resolution inputs,balancing accuracy and efficiency.Key contributions include:a multi-channel output structure expanding binary edge prediction to five classes,supported by a deep supervision mechanism;a dynamic class-balancing strategy combining adaptive weighting with physical priors to handle extreme class imbalance;and maintained architectural efficiency enabling real-time inference.Extensive evaluations on BSDS-RIND show our approach achieves accuracy competitive with state-of-the-art methods while operating in real time.展开更多
Vehicle-induced response separation is a crucial issue in structural health monitoring(SHM).This paper proposes a block-wise sliding recursive wavelet transform algorithm to meet the real-time processing requirements ...Vehicle-induced response separation is a crucial issue in structural health monitoring(SHM).This paper proposes a block-wise sliding recursive wavelet transform algorithm to meet the real-time processing requirements of monitoring data.To extend the separation target from a fixed dataset to a continuously updating data stream,a block-wise sliding framework is first developed.This framework is further optimized considering the characteristics of real-time data streams,and its advantage in computational efficiency is theoretically demonstrated.During the decomposition and reconstruction processes,information from neighboring data blocks is fully utilized to reduce algorithmic complexity.In addition,a delay-setting strategy is introduced for each processing window to mitigate boundary effects,thereby balancing accuracy and efficiency.Simulated signal experiments are conducted to determine the optimal delay configuration and to verify the algorithm’s superior performance,achieving a lower Root Mean Square Error(RMSE)and only 0.0249 times the average computational time compared with the original algorithm.Furthermore,strain signals from the Lieshi River Bridge are employed to validate the method.The proposed algorithm successfully separates the static trend from vehicle-induced responses in real time across different sampling frequencies,demonstrating its effectiveness and applicability in real-time bridge monitoring.展开更多
An innovative real-time monitoring method for surrounding rock damage based on microseismic time-lapse double-difference tomography is proposed for delayed dynamic damage identification and insufficient detection of a...An innovative real-time monitoring method for surrounding rock damage based on microseismic time-lapse double-difference tomography is proposed for delayed dynamic damage identification and insufficient detection of adverse geological conditions in deep-buried tunnel construction.The installation techniques for microseismic sensors were optimized by mounting sensors at bolt ends which significantly improves signal-to-noise ratio(SNR)and anti-interference capability compared to conventional borehole placement.Subsequently,a 3D wave velocity evolution model that incorporates construction-induced disturbances was established,enabling the first visualization of spatiotemporal variations in surrounding rock wave velocity.It finds significant wave velocity reduction near the tunnel face,with roof and floor damage zones extending 40–50 m;wave velocities approaching undisturbed levels at 15 m ahead of the working face and on the laterally undisturbed side;pronounced spatial asymmetry in wave velocity distribution—values on the left side exceed those on the right,with a clear stress concentration or transition zone located 10–15 m;and systematically lower velocities behind the face than in front,indicating asymmetric rock damage development.These results provide essential theoretical support and practical guidance for optimizing dynamic construction strategies,enabling real-time adjustment of support parameters,and establishing safety early warning systems in deep-buried tunnel engineering.展开更多
Sulfur dioxide(SO_(2)) and its derivatives have been recognized as harmful environmental pollutants.However,they are often produced during the processing of traditional Chinese medicines,potentially compromising the q...Sulfur dioxide(SO_(2)) and its derivatives have been recognized as harmful environmental pollutants.However,they are often produced during the processing of traditional Chinese medicines,potentially compromising the quality of these medicinal materials and contributing to various health issues.Due to a lack of effective monitoring and imaging tools,the physiological effects of excessive SO_(2) residues in traditional Chinese medicine remain unclear.Therefore,developing a rapid and effective tool for detecting SO_(2) is crucial for understanding its metabolic pathways and effects in vivo.In this study,we developed a near infrared(NIR) and ratiometric fluorescent probe,NIR-RS,which exhibits high sensitivity,selectivity,and rapid response for SO_(2) detection.Notably,NIR-RS accurately quantifies SO_(2) contents in Pinelliae rhizoma(P.rhizoma) samples,with recovery rates from 98.46 % to 102.40 %,and relative standard deviations(RSDs)< 5.0 %.For bioimaging applications,NIR-RS has low cytotoxicity and good mitochondrial-targeting ability,making it suitable for imaging exogenous and endogenous SO_(2) in mitochondria.Additionally,NIR-RS was successfully applied to image SO_(2) content of P.rhizoma samples within cells,revealing that high SO_(2) residue elevated mitochondria adenosine triphosphate(ATP) content,these findings reveal that P.rhizoma with excessive SO_(2) can affect the organism's growth mechanisms through alterations in ATP pathways.In vivo,SO_(2) was found to predominantly accumulate in the liver following gavage with P.rhizoma solution,with accumulation levels increasing in proportion to SO_(2) residue concentration.High SO_(2) concentrations in P.rhizoma can cause pulmonary fibrosis and gastric mucosal damage.This work provides a valuable tool for regulating SO_(2) content in P.rhizoma and may help researcher better understand the metabolism of SO_(2) derivatives and explore their physiological roles in biological systems.展开更多
基金Supported by National Natural Science Foundation of Shandong Province (No. SY2008C179)~~
文摘[Objective]To investigate the expression of zebrafish vascular endothelial growth factor-2（VEGFR-2） at different developmental stages.[Method]Total RNAs were extracted from 12,24,48,72 and 96 hpf stage zebrafish embryos and larvae.Real-time quantitative RT-PCR was performed to examine the expression of VEGFR-2.The data were analyzed by 2^-△△Ct method.[Result]The expression level of VEGFR-2 gene increased gradually from 12 to 72 hpf,and subsequently decreased at 96 hpf.The expression level was lowest at 12 hpf,highest at 72 hpf,and had significant differences when compared with that of other developmental stages.[Conclusion]The expression level of VEGFR-2 increases gradually before blood vessel maturation and decreases as blood vessels mature.
基金Supported by National Natural Science Foundation Project(30270086)~~
文摘[Objective] To explore the feasibility of using SYBR Green real-time quantitative PCR technique to estimate the copy numbers of exogenous gene in a transgenic plant.[Methods] Using SYBR Green real-time quantitative PCR technique,we have determined the copy numbers of the exogenous CYCD3;1 in transgenic Arabidopsis by comparing an endogenous single copy reference gene with CYCD3;1 copy numbers in transgenic plant,meanwhile comparing CYCD3;1 copy numbers between wild plant and transgenic plant.[Results]The exogenous CYCD3;1 copy numbers calculated by this method is identical with results of traditional Southern blot analysis which is highly accurate.[Conclusion]This method is simple,effective and safe for estimating transgene copy numbers.
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 National Natural Science Foundation of China (Nos. 31470271 and 81730110)Guangzhou Science and Technology Program key projects (No. 201803040006)
文摘The establishment of highly sensitive diagnostic methods is critical in the early diagnosis and control of Zika virus(ZIKV)and in preventing serious neurological complications of ZIKV infection. In this study, we established micro-droplet digital polymerase chain reaction(ddPCR) and real-time quantitative PCR(RT-qPCR) protocols for the detection of ZIKV based on the amplification of the NS5 gene. For the ZIKV standard plasmid, the RT-qPCR results showed that the cycle threshold(Ct) value was linear from 10~1 to 10~8 copy/l L, with a standard curve R^2 of 0.999 and amplification efficiency of 92.203%;however, a concentration as low as 1 copy/l L could not be detected. In comparison with RT-qPCR, the dd PCR method resulted in a linear range of 10~1–10~4 copy/l L and was able to detect concentrations as low as 1 copy/l L. Thus, for detecting ZIKV from clinical samples, RT-qPCR is a better choice for high-concentration samples(above 10~1 copy/l L),while ddPCR has excellent accuracy and sensitivity for low-concentration samples. These results indicate that the ddPCR method should be of considerable use in the early diagnosis, laboratory study, and monitoring of ZIKV.
文摘Objective:The aim of this study was to detect the expression level of Metadherin (MTDH) in peripheral blood of the breast cancer patients by real-time fluorescence quantitative polymerase chain reaction (PCR),and to explore the relationship between expression of Metadherin gene in the patients peripheral blood and the clinic-pathological features in breast cancer. Methods:Real-time fluorescence quantitative polymerase chain reaction was employed to determine the expression level of Metadherin gene in 80 peripheral blood samples of breast cancer patients and healthy donors. Results:The expression of Metadherin gene in breast cancer patients peripheral blood were positive,in which 34 breast cancer patients were highly expressed,accounting for 55.7%,while the expression of Metadherin gene in normal females peripheral blood were negative,there was statistical significance (Ratio = 2.02±0.81,P < 0.05); Ratio of the Metadherin expression in breast cancer patients peripheral blood and the glyceraldehyde-3-phosphate dehydrogenase expression was 1.15 ± 0.36. REST software analysis showed that the expression of Metadherin gene was significantly up-regulated in breast cancer. Conclusion:The SYBR Green I quantitative real-time polymerase chain reaction method can successfully detect the expression level of Metadherin gene. Expression level of Metadherin gene in breast cancer patients peripheral blood is closely related to survival,and it maybe involved in the development of breast cancer and used as an indicator of prognosis.
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.
基金This work was supported by a grant from the Tianjin Natural Science Foundation of China(No.023610811)
文摘OBJECTIVE To explore the relationship between multimarker detection of MAGE-1,MAGE-3 and AFP mRNAs in the peripheral blood of patients with hepatocellular carcinoma and micrometastasis using a realtime quantitative-PCR(real-time Q-PCR)assay. METHODS Peripheral blood samples were obtained from control subjects and 86 patients with hepatocellular carcinoma (HCC).Real-time Q-PCR was used to detect MAGE-1,MAGE-3, and AFP mRNAs in the blood cells. RESULTS In 86 tumor specimens,the positivity for MAGE-1, MAGE-3,and AFP genes was respectively 34.9%(30/86),60.5% (52/86)and 69.8%(60/86).All specimens expressed at least one marker.MAGE-1,MAGE-3,and AFP transcripts were detected respectively in 12(14.0%),18(20.1%)and 29(33.7%)of the 86 blood specimens from hepatocellular carcinoma patients,while 45 specimens(52.3%)were positive for at least one marker.In addition,MAGE-1,MAGE-3 and AFP gene transcripts were not detected in any peripheral blood specimens from 25 chronic liver disease patients and 28 normal healthy volunteers.The positive rate correlated with the TNM clinical stages,extrahepatic metastasis and portal vein carcinothrombosis(P<0.05).No correlation was found between tumor size,tumor number, differentiation,serum a-fetoprotein(AFP)and the positive rate. CONCLUSION Our results indicate that a multimarker real- time Q-PCR assay with cancer-specific markers such as MAGE-1 and MAGE-3 in combination with a hepatocyte-specific AFP marker may be a promising diagnostic tool for monitoring hepatocellular carcinoma patients with better sensitivity and specificity.
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Supported by National Natural Science Foundation of China(No.81370477No.81370918)+2 种基金Science and Technology Research Outstanding Youth Fund of Hebei College(No.Y2011117)Undergraduate Innovation Project(No.201410081061No.X2014026)
文摘AIM:Normalizing the results of real-time quantitative reverse transcription polymerase chain reaction(RT-q PCR)is essential for the accuracy of analysis.Commonly used approaches include input nucleic acid standardization(ΔCt method),normalization against a single internal reference gene(ΔΔCt method),and geometric averaging of multiple reference gene abundance using statistical software.We evaluated these approaches in the liver of db/db mice,a typical model of fatty liver disease.METHODS:Seven reference genes,β-actin(ACTB),eukaryotic initiation factor(e IF)5,glyceraldehyde-3-phosphate dehydrogenase(GAPDH),hydroxymethylbilane synthase(HMBS),hypoxanthineguanine phosphoribosyltransferase(HPRT)1,polymerase(RNA)II(DNA directed)polypeptide A(Polr2A)and ribosomal protein P〈0(RPLP〈0),were evaluated using software of ge Norm and Norm Finder.Hepatic lipogenesis genes,such as thyroid hormone-responsive protein(Thrsp),stearoyl-Co A desaturase(SCD)1,sterol regulatory element-binding protein(SREBP)1c and fatty acid synthase(FAS),were used as target genes of interest.RESULTS:The expression levels of all target genes and GAPDH were significantly elevated(P〈0.05)in db/db mouse livers by theΔCt method.ACTB and HMBS were the most stable genes calculated by the software of ge Norm.Norm Finder analysis indicated that ACTB was the most stable gene,and the best combination of 2 genes was GAPDH and RPLP〈0.Normalization against a single internal reference gene of ACTB or RPLP〈0,the geometric mean of ACTB and HMBS,or GAPDH and RPLP〈0 showed similar results that the expression levels of Thrsp,SCD1 and FAS,but not SREBP1 c increased(P〈0.05)in the liver of db/db mice.CONCLUSION:TheΔCt approach ensures a meaningful and biologically significant appraisal of gene expression.Use of the software like ge Norm or Norm Finder should be integrated withΔCt method.
基金Supported by National Natural Science Foundation of China(31260406)Natural Science Fund Project of Inner Mongolia(2012MS0502)~~
文摘Real-time fluorescent quantitative PCR （RQ-PCR） is a detection method by adding fluorescent dye or fluorescent probe into the PCR reaction system, using fluorescent signal accumulation to monitor amplification reactions of PCR reaction process, and finally the unknown template can be quantitatively analyzed through the standard curve. So the detection level of PCR has improved from the qualitative to the quantitative. In order to provide a theoretical reference for further application, the principle, classification, advantages and disadvantages of RQ-PCR were intro- duced, and its application and progress in plants in recent years were reviewed.
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supported by the National Natural Science Foundation of China 62402171.
文摘As a fundamental component in computer vision,edges can be categorized into four types based on discontinuities in reflectance,illumination,surface normal,or depth.While deep CNNs have significantly advanced generic edge detection,real-time multi-class semantic edge detection under resource constraints remains challenging.To address this,we propose a lightweight framework based on PiDiNet that enables fine-grained semantic edge detection.Our model simultaneously predicts background and four edge categories from full-resolution inputs,balancing accuracy and efficiency.Key contributions include:a multi-channel output structure expanding binary edge prediction to five classes,supported by a deep supervision mechanism;a dynamic class-balancing strategy combining adaptive weighting with physical priors to handle extreme class imbalance;and maintained architectural efficiency enabling real-time inference.Extensive evaluations on BSDS-RIND show our approach achieves accuracy competitive with state-of-the-art methods while operating in real time.
基金the support of the Major Science and Technology Project of Yunnan Province,China(Grant No.202502AD080007)the National Natural Science Foundation of China(Grant No.52378288)。
文摘Vehicle-induced response separation is a crucial issue in structural health monitoring(SHM).This paper proposes a block-wise sliding recursive wavelet transform algorithm to meet the real-time processing requirements of monitoring data.To extend the separation target from a fixed dataset to a continuously updating data stream,a block-wise sliding framework is first developed.This framework is further optimized considering the characteristics of real-time data streams,and its advantage in computational efficiency is theoretically demonstrated.During the decomposition and reconstruction processes,information from neighboring data blocks is fully utilized to reduce algorithmic complexity.In addition,a delay-setting strategy is introduced for each processing window to mitigate boundary effects,thereby balancing accuracy and efficiency.Simulated signal experiments are conducted to determine the optimal delay configuration and to verify the algorithm’s superior performance,achieving a lower Root Mean Square Error(RMSE)and only 0.0249 times the average computational time compared with the original algorithm.Furthermore,strain signals from the Lieshi River Bridge are employed to validate the method.The proposed algorithm successfully separates the static trend from vehicle-induced responses in real time across different sampling frequencies,demonstrating its effectiveness and applicability in real-time bridge monitoring.
基金support of the National Natural Science Foundation of China(No.52274176)the Guangdong Province Key Areas R&D Program(No.2022B0101070001)+5 种基金Chongqing Elite Innovation and Entrepreneurship Leading talent Project(No.CQYC20220302517)the Chongqing Natural Science Foundation Innovation and Development Joint Fund(No.CSTB2022NSCQ-LZX0079)the National Key Research and Development Program Young Scientists Project(No.2022YFC2905700)the Chongqing Municipal Education Commission“Shuangcheng Economic Circle Construction in Chengdu-Chongqing Area”Science and Technology Innovation Project(No.KJCX2020031)the Fundamental Research Funds for the Central Universities(No.2024CDJGF-009)the Key Project for Technological Innovation and Application Development in Chongqing(No.CSTB2025TIAD-KPX0029).
文摘An innovative real-time monitoring method for surrounding rock damage based on microseismic time-lapse double-difference tomography is proposed for delayed dynamic damage identification and insufficient detection of adverse geological conditions in deep-buried tunnel construction.The installation techniques for microseismic sensors were optimized by mounting sensors at bolt ends which significantly improves signal-to-noise ratio(SNR)and anti-interference capability compared to conventional borehole placement.Subsequently,a 3D wave velocity evolution model that incorporates construction-induced disturbances was established,enabling the first visualization of spatiotemporal variations in surrounding rock wave velocity.It finds significant wave velocity reduction near the tunnel face,with roof and floor damage zones extending 40–50 m;wave velocities approaching undisturbed levels at 15 m ahead of the working face and on the laterally undisturbed side;pronounced spatial asymmetry in wave velocity distribution—values on the left side exceed those on the right,with a clear stress concentration or transition zone located 10–15 m;and systematically lower velocities behind the face than in front,indicating asymmetric rock damage development.These results provide essential theoretical support and practical guidance for optimizing dynamic construction strategies,enabling real-time adjustment of support parameters,and establishing safety early warning systems in deep-buried tunnel engineering.
基金supported by the Natural Science Foundation of Hubei Province (Nos.2023AFB376 and 2024AFD287)National Key Research and Development Program (No.2023YFC3503804)the National Natural Science Foundation of China (No.22077044)。
文摘Sulfur dioxide(SO_(2)) and its derivatives have been recognized as harmful environmental pollutants.However,they are often produced during the processing of traditional Chinese medicines,potentially compromising the quality of these medicinal materials and contributing to various health issues.Due to a lack of effective monitoring and imaging tools,the physiological effects of excessive SO_(2) residues in traditional Chinese medicine remain unclear.Therefore,developing a rapid and effective tool for detecting SO_(2) is crucial for understanding its metabolic pathways and effects in vivo.In this study,we developed a near infrared(NIR) and ratiometric fluorescent probe,NIR-RS,which exhibits high sensitivity,selectivity,and rapid response for SO_(2) detection.Notably,NIR-RS accurately quantifies SO_(2) contents in Pinelliae rhizoma(P.rhizoma) samples,with recovery rates from 98.46 % to 102.40 %,and relative standard deviations(RSDs)< 5.0 %.For bioimaging applications,NIR-RS has low cytotoxicity and good mitochondrial-targeting ability,making it suitable for imaging exogenous and endogenous SO_(2) in mitochondria.Additionally,NIR-RS was successfully applied to image SO_(2) content of P.rhizoma samples within cells,revealing that high SO_(2) residue elevated mitochondria adenosine triphosphate(ATP) content,these findings reveal that P.rhizoma with excessive SO_(2) can affect the organism's growth mechanisms through alterations in ATP pathways.In vivo,SO_(2) was found to predominantly accumulate in the liver following gavage with P.rhizoma solution,with accumulation levels increasing in proportion to SO_(2) residue concentration.High SO_(2) concentrations in P.rhizoma can cause pulmonary fibrosis and gastric mucosal damage.This work provides a valuable tool for regulating SO_(2) content in P.rhizoma and may help researcher better understand the metabolism of SO_(2) derivatives and explore their physiological roles in biological systems.
