Heterodera filipjevi continues to be a major threat to wheat production worldwide.Rapid detection and quantification of cyst nematodes are essential for more effective control against this nematode disease.In the pres...Heterodera filipjevi continues to be a major threat to wheat production worldwide.Rapid detection and quantification of cyst nematodes are essential for more effective control against this nematode disease.In the present study,a TaqManminor groove binder(TaqMan-MGB)probe-based fluorescence quantitative real-time PCR(qPCR)was successfully developed and used for quantifying H.filipjevi from DNA extracts of soil.The primers and probe designed from the obtained RAPD-SCAR marker fragments of H.filipjevi showed high specificity to H.filipjevi using DNA from isolatesconfirmed species of 23 Heterodera spp.,1 Globodera spp.and 3 Pratylenchus spp.The qPCR assay is highly sensitive and provides improved H.filipjevi detection sensitivity of as low as 4^(-3) single second-stage juvenile(J2)DNAs,10^(-3) female DNAs,and 0.01μgμL^(-1) genomic DNAs.A standard curve relating to the threshold cycle and log values of nematode numbers was generated and validated from artificially infested soils and was used to quantify H.filipjevi in naturally infested field soils.There was a high correlation between the H.filipjevi numbers estimated from 32 naturally infested field soils by both conventional methods and the numbers quantified using the qPCR assay.qPCR potentially provides a useful platform for the efficient detection and quantification of H.filipjevi directly from field soils and to quantify this species directly from DNA extracts of field soils.展开更多
Summary:The novel coronavirus SARS-CoV-2 caused an outbreak of pneumonia in Wuhan,Hubei province of China in January 2020.This study aims to investigate the effects of different temperature and time durations of virus...Summary:The novel coronavirus SARS-CoV-2 caused an outbreak of pneumonia in Wuhan,Hubei province of China in January 2020.This study aims to investigate the effects of different temperature and time durations of virus inactivation on the results of PCR testing for SARS-CoV-2.Twelve patients at the Renmin Hospital of Wuhan University suspected of being infected with SARS-CoV-2 were selected on February 13,2020 and throat swabs were taken.The swabs were stored at room tempcrature(20-25℃),then divided into aliquots and subjected to different temperature for different periods in order to inactivate the viruses(56℃for 30,45,60 min;65,70,80℃for 10,15,20 min).Control aliquots were stored at room temperature for 60 min.Then all aliquots were tested in a real-time fluorescence PCR using primers against SARS-CoV-2.Regardless of inactivation temperature and time,7 of 12 cases(58.3%)tested were positive for SARS-CoV-2 by PCR,and cycle threshold values were similar.These results suggest that virus inactivation parameters exert minimal infuence on PCR test results.Inactivation at 65℃for 10 min may be sufficient to ensure safe,reliable testing.展开更多
Objective Establishing a highly sensitive real-time fluorescence quantitative PCR (qPCR) method for universal testing of epidemic African swine fever virus (ASFV) strains. Methods The ASFV p72 gene was targeted to des...Objective Establishing a highly sensitive real-time fluorescence quantitative PCR (qPCR) method for universal testing of epidemic African swine fever virus (ASFV) strains. Methods The ASFV p72 gene was targeted to design primer probes covering 24 p72 genotypes. The optimal amount of dimethylsulphoxide (DMSO) for qPCR amplification was determined, Various sensitivity and limit of detection (LOD) tests were performed, and clinical samples from China and imported goods were tested. Results The optimal primer-probe combination could specifically detect ASFV, 1.5% DMSO was optimal for qPCR, and LOD reached 3.2 copies/μL with good reproducibility (n = 20, p = 0.369). The method was employed to test 142 clinically suspected samples, of which 30 pig blood and 37 pig tissue samples were ASFV-positive. Moreover, the positive testing rate for ASFV was higher than for the standard qPCR method recommended by the Office International Des Epizooties (OIE), and for the commercially available kit. Thus, our method is superior for testing weakly positive samples with low virus titre, and epidemic strains present in imported goods. Conclusion Our method could be employed for universal testing of epidemic ASFV strains worldwide, ensuring wider coverage of hosts and ASFV strains/endemic strains, reducing false<span style="font-family:;" "=""> </span><span style="font-family:Verdana;">negatives, and benefitting early diagnosis.</span>展开更多
Green fluorescent protein (GFP) and its variants /homolog proteins are generally called as GFP-like fluorescent proteins (FPs), which are widely used as visible molecular tools for monitoring a wide range of biologica...Green fluorescent protein (GFP) and its variants /homolog proteins are generally called as GFP-like fluorescent proteins (FPs), which are widely used as visible molecular tools for monitoring a wide range of biological processes due to their capability of simple, accurate and real time quantification. The FPs-based molecular and visible quantification tools are giving more impact on bioprocess engineering, enabling the biomolecule-level dynamic information to be linked with the process-level events. In this review, different applications of FPs in biological engineering with emphasis on rapid molecular bioprocess quantification, such as quantification of the transcription efficiency, the protein production, the protein folding efficiency, the cell concentration, the intracellular microenvironments and so on, would be first introduced. The challenges of using FPs with respect to actual bioprocess applications for the precise quantification including the interaction of FPs and the fused partner proteins, the maturation of FPs, the inner filter effect and sensing technology were then discussed. Finally, the future development for the FPs used in molecular bioprocess quantification would be proposed.展开更多
Peanut,sesame and other raw materials of food are allergens for special populations.In this study,specific primers and TaqMan probes labeled by different fluorescences were designed targeting Ara h 2 gene of peanut an...Peanut,sesame and other raw materials of food are allergens for special populations.In this study,specific primers and TaqMan probes labeled by different fluorescences were designed targeting Ara h 2 gene of peanut and Ses i 1 gene of sesame.After the optimization of reaction conditions,a real-time fluorescent PCR method was established for simultaneous detection of allergenic ingredients of peanut and sesame in food.Genomic DNA samples of peanut,sesame,rice,wheat,barley,soybean,celery,maize,potato,tomato,walnut,groundnut in shell,cashew nut,sunflower seed,almond,apple,pear and strawberry,pork,beef,mutton and fish were used as templates for PCR amplification with deionized water as negative control template.Results indicated that the established real-time fluorescent PCR method could specifically identify allergenic ingredients of peanut and sesame simultaneously.Sensitivity test showed that the minimum detection limit of this method was 0.01%.Therefore,the established real-time fluorescent PCR method is a specific,sensitive and effective assay for simultaneously detecting allergenic ingredients of peanut and sesame in food.展开更多
In order to improve the standardized technical systems of quantitative analyses for genetically modified organisms(GMOs) and products,ensure bio-safety and reduce ecological risk in China,a real-time fluorescent quant...In order to improve the standardized technical systems of quantitative analyses for genetically modified organisms(GMOs) and products,ensure bio-safety and reduce ecological risk in China,a real-time fluorescent quantitative PCR assay was established for detection of genetically modified maize line MON88017.The established method was evaluated based on the specificity,sensitivity,accuracy and measurement uncertainty.The results showed that the established method had strong specificity in detection of genetically modified maize line MON88017.1.50%MON88017 sample was detected with 29 replications.The average measured value(1.541%) was close to the actual value(1.50%) and the relative deviation was 2.70%.The variation coefficient of the measured value was 0.110 4;the recovery was 100.00%and the measurement uncertainty was 0.096.The limit of detection for genetically modified maize line MON88017 with the established method was 5 copies at the 97.5%confidence level.Thus,the real-time fluorescent quantitative PCR assay established in this study exhibited high specificity,accuracy and sensitivity,which could provide technical support for the safety supervision of genetically modified organisms and products in China.展开更多
To establish a rapid quantification method for heparinase I during its production in recombinant Es-cherichia coli, a translational fusion vector was constructed by fusing the N terminus of heparinase I to the C ter-m...To establish a rapid quantification method for heparinase I during its production in recombinant Es-cherichia coli, a translational fusion vector was constructed by fusing the N terminus of heparinase I to the C ter-minus of a green fluorescent protein mutant (GFPmut1). As a result, not only was the functional recombinant ex-pression of heparinase I in E. coli accomplished, but also a linear correlation was obtained between the GFP fluores-cence intensity and heparinase I activity, allowing enzyme activity to be quantified rapidly during the fermentation.展开更多
Even though multispectral imaging is considered very significant in biological imaging,it is only commonly used in microscopy in a 2D approach.Here,we present a Fluorescence Molecular Tomography system capable of reco...Even though multispectral imaging is considered very significant in biological imaging,it is only commonly used in microscopy in a 2D approach.Here,we present a Fluorescence Molecular Tomography system capable of recording simultaneously tomographic data at several spectral windows,enabling multispectral tomography.3D reconstructed data from several spectral windows is used to construct a linear unmixing algorithm for multispectral deconvolution of overlapping fluorescence signals.The method is applied on tomographic 3D fluorescence concentration maps in tissue-mimicking phantoms,yielding absolute quantification of the concentration of each individual fluorophore.Results are compared to the case when unmixing is performed in the raw 2D data instead of the reconstructed 3D concentration map,showing greater accuracy when unmixing algorithms are applied in the reconstructed data.Both the reflection and transmission geometries are considered.展开更多
There is an urgent need for developing a procedure for biomarker standardization and relative quantificationin clinical laboratories. Measuring the expression levels of cell antigens is critical for the diagnosis of m...There is an urgent need for developing a procedure for biomarker standardization and relative quantificationin clinical laboratories. Measuring the expression levels of cell antigens is critical for the diagnosis of many diseases, e.g. leukemia, lymphoma and immunodeficiency diseases. One of the most significant challenges in flow cytometry is obtaining inter-laboratory and intra-laboratory consistent and reproducible results across multiple cytometer platforms and locations longitudinally over time. To obtain measurement consistency, the target flow cytometer voltages should be optimized to segregate the negative population from the electronic noise, and to keep the brightest positive population within the dynamic range of each detector. Then target values should be determined and transferred to selected cytometers. In this study, we optimized a procedure for instrument standardization across three different flow cytometer platforms from the same vendor and in two different locations. The biomarker quantification was implemented on standardized instruments using CD4 expression on T lymphocytes with a known amount of antibody bound per cell as a quantification standard. Our results on blood cell subset typing and CD19 quantification demonstrated that consistent and reliable results could be accomplished between instruments using the developed procedure. Quantitating the expression levels of certain cell biomarkers relative to a known reference marker before, during, and after therapy would provide important information for monitoring antibody-based therapy and could be potentially used to adjust dosing. Presently, we are implementing this protocol to quantify critical disease biomarkers, and making necessary modifications to the procedure to include instruments from different instrument manufacturers.展开更多
A quantitative analysis method of molybdenum in FeMo alloys by X-ray spectrometry using borate fusion technique was compared with that with pressed pellet. The complete pre-oxidation of FeMo alloys for the preparation...A quantitative analysis method of molybdenum in FeMo alloys by X-ray spectrometry using borate fusion technique was compared with that with pressed pellet. The complete pre-oxidation of FeMo alloys for the preparation of homogeneous fused discs was achieved by employing an automated fusion machine equipped with specially designed O2-blowing nozzles, which used lithium tetra-borate as flux with the addition of lithium nitrate (LiNO3) as oxidizer. The calibration curves of Mo and Fe were used in the quantitative analysis of standard materials and unknown plant samples with satisfactory accuracy and precision, utilizing the corrections of the matrix effects and line overlap. It was confirmed that the newly proposed method of preparing fused glass discs of FeMo alloys can replace the conventional wet chemical analyses requiring the labor intensive and time consuming procedure.展开更多
[Objectives]To verify the specificity,sensitivity,precision and negative-positive deviation of the foodproof gluten component de-tection kit for the detection of gluten allergens in milk powder matrix,and to establish...[Objectives]To verify the specificity,sensitivity,precision and negative-positive deviation of the foodproof gluten component de-tection kit for the detection of gluten allergens in milk powder matrix,and to establish a real-time fluorescent PCR legal method for the detec-tion of gluten allergens in milk powder.[Methods]The specificity,sensitivity,precision and negative-positive deviation of the detection method of foodproof gluten component detection kit(PCR-probe method)were verified by artificially adding different concentrations of wheat bran and extracting sample DNA by kit method,and applied to sample detection.[Results] The specific detection results of two kinds of milk powder with wheat bran and buckwheat added showed that the foodproof gluten component detection kit(PCR-probe method)had good speci-ficity for wheat gluten.The results of artificially added wheat bran positive samples showed that the false positive rate and false negative rate of the kit in the milk powder matrix were O,and the sensitivity and precision were high.[Conclusions]The kit is simple to operate and has high accuracy,which is suitable for the detection of gluten allergen components in milk powder.展开更多
Noroviruses are the leading cause of acute gastroenteritis in humans. Real-time reverse transcription-polymerase chain reaction(real-time RT-PCR) is a promising molecular method for the detection of noroviruses. In th...Noroviruses are the leading cause of acute gastroenteritis in humans. Real-time reverse transcription-polymerase chain reaction(real-time RT-PCR) is a promising molecular method for the detection of noroviruses. In this study, the performance of three Taq Man real-time RT-PCR assays was assessed, which were one commercially available real-time RT-PCR kit(assay A:Norovirus Real Time RT-PCR kit) and two in-house real-time RT-PCR assays(assay B: Light Cycler RNA Master Hybprobe and assay C: Real Time ready RNA Virus Master). Assays A and B showed higher sensitivity than assay C for norovirus GI, while they all had the same sensitivity(103 DNA copies/m L) for GII DNA standard controls. Assay B had the highest efficiency for both genogroups.No cross-reactivity was observed among GI and GII noroviruses, rotavirus, hepatitis A virus, and poliovirus. The detection rates of these assays in GI and GII norovirus-positive fecal samples were not significantly different. However, the mean quantification cycle(Cq) value of assay B for GII was lower than assays A and C with statistical significance(P-value, 0.000). All three real-time RT-PCR assays could detect a variety of noroviruses including GI.2, GII.2, GII.3, GII.4, GII.6, GII.12, GII.17,and GII.21. This study suggests assay B as a suitable assay for the detection and quantification of noroviruses GI and GII due to good analytical sensitivity and higher performance to amplify norovirus on DNA standard controls and clinical samples.展开更多
The molecular techniques including Northern blot, dot blot, in situ hybridization, etc. have been successfully used to estimate semi-quantitatively mRNA levels in plant samples. In this study, we employed a real-time ...The molecular techniques including Northern blot, dot blot, in situ hybridization, etc. have been successfully used to estimate semi-quantitatively mRNA levels in plant samples. In this study, we employed a real-time reverse transcription-PCR (RT-PCR) assay using SYBR Green I fluorescence methodology to evaluate accurate quantitation and sequence specific detection of Aux/IAA mRNA levels in Arabidopsis. Results obtained indicate a linear dynamic range of 102-106 Aux/IAA mRNA copies with standard deviations of generally less than 15%. As a model experiment, the outcome of analysis of expression patterns of five Aux/IAA genes in Arabidopsis under various chemical and temperature treatments is presented. The method presented here provides a sensitive and rapid technique to evaluate plant Aux/IAA mRNA expression levels in nanogram order.展开更多
Understanding the fate and toxicity of microplastics(MPs,<5 mm plastic particles)is limited by quantification methods.This paper summarizes the methods in use and presents new ones.First,sampling and pretreatment p...Understanding the fate and toxicity of microplastics(MPs,<5 mm plastic particles)is limited by quantification methods.This paper summarizes the methods in use and presents new ones.First,sampling and pretreatment processes ofMPs,including sample collection,digestion,density separation,and quality control are reviewed.Then the promising and convenient staining procedures and quantification methods for MPs using fluorescence dyes are reviewed.The factors that influence the staining of MPs,including their physicochemical properties,are summarized to provide an optimal operation procedure.In general,the digestion step is crucial to eliminate natural organic matter(NOM)to avoid interference in quantification.Chloroform was reported to be the most appropriate solvent,and 10–20μg/mL are recommended as optimal dye concentrations.In addition,a heating and cooling procedure is recommended to maintain the fluorescence intensity of MPs for two months.After staining,a fluorescence microscope is usually used to characterize the morphology,mass,or number of MPs,but compositional analysis cannot be determined with it.These fluorescence staining methods have been implemented to study MP abundance,transport,and toxicity and have been combined with other chemical characterization techniques,such as Fourier transform infrared spectroscopy and Raman spectroscopy.More studies are needed to focus on the synthesis of novel dyes to avoid NOM’s interference.They need to be combined with other spectroscopic techniques to characterize plastic composition and to develop image-analysis methods.The stability of stained MPs needs to be improved.展开更多
Nuclear RNA export into the cytoplasm is one of the key steps in protein expression to realize biological functions.Despite the broad availability of nucleic acid dyes,tracking and quantifying the highly dynamic proce...Nuclear RNA export into the cytoplasm is one of the key steps in protein expression to realize biological functions.Despite the broad availability of nucleic acid dyes,tracking and quantifying the highly dynamic process of RNA export in live cells is challenging.When dye-labeled RNA enters the cytoplasm,the dye molecules are released upon degradation of the RNA,allowing them to re-enter the cell nucleus.As a result,the ratio between the dye exported with RNA into the cytoplasm and the portion staying inside the nucleus cannot be determined.To address this common limitation,we report the design of a smart probe that can only check into the nucleus once.When adding to cells,this probe rapidly binds with nuclear RNAs in live cells and reacts with intrinsic H_(2)S.This reaction not only activates the fluorescence for RNA tracking but also changes the structure of probe and consequently its intracellular localization.After disassociating from exported RNAs in cytoplasm,the probe preferentially enters lysosomes rather than cell nucleus,enabling real-time quantitative measurement of nuclear RNA exports.Using this probe,we successfully evaluated the effects of hormones and cancer drugs on nuclear RNA export in live cells.Interestingly,we found that hormones inhibiting RNA exports can partially offset the effect of chemotherapy.展开更多
基金financially supported by the National Natural Science Foundation of China(31972247)the Science and Technology Innovation Project of the Chinese Academy of Agricultural Sciences(ASTIP-2016-IPP-04)the Special Fund for Agro-scientific Research in the Public Interest,China(201503114)。
文摘Heterodera filipjevi continues to be a major threat to wheat production worldwide.Rapid detection and quantification of cyst nematodes are essential for more effective control against this nematode disease.In the present study,a TaqManminor groove binder(TaqMan-MGB)probe-based fluorescence quantitative real-time PCR(qPCR)was successfully developed and used for quantifying H.filipjevi from DNA extracts of soil.The primers and probe designed from the obtained RAPD-SCAR marker fragments of H.filipjevi showed high specificity to H.filipjevi using DNA from isolatesconfirmed species of 23 Heterodera spp.,1 Globodera spp.and 3 Pratylenchus spp.The qPCR assay is highly sensitive and provides improved H.filipjevi detection sensitivity of as low as 4^(-3) single second-stage juvenile(J2)DNAs,10^(-3) female DNAs,and 0.01μgμL^(-1) genomic DNAs.A standard curve relating to the threshold cycle and log values of nematode numbers was generated and validated from artificially infested soils and was used to quantify H.filipjevi in naturally infested field soils.There was a high correlation between the H.filipjevi numbers estimated from 32 naturally infested field soils by both conventional methods and the numbers quantified using the qPCR assay.qPCR potentially provides a useful platform for the efficient detection and quantification of H.filipjevi directly from field soils and to quantify this species directly from DNA extracts of field soils.
基金This work was supported by grants from the Special Science and Technology Cooperation Project of Ningxia Hui Autonomous Region Key R&D Program(No.2018BFG02008)the National Science and Technology Key Projects on"Major Infectious Diseases such as HIV/AIDS,Viral Hepatitis Prevention and Treatment"(No.2017ZX10103005).
文摘Summary:The novel coronavirus SARS-CoV-2 caused an outbreak of pneumonia in Wuhan,Hubei province of China in January 2020.This study aims to investigate the effects of different temperature and time durations of virus inactivation on the results of PCR testing for SARS-CoV-2.Twelve patients at the Renmin Hospital of Wuhan University suspected of being infected with SARS-CoV-2 were selected on February 13,2020 and throat swabs were taken.The swabs were stored at room tempcrature(20-25℃),then divided into aliquots and subjected to different temperature for different periods in order to inactivate the viruses(56℃for 30,45,60 min;65,70,80℃for 10,15,20 min).Control aliquots were stored at room temperature for 60 min.Then all aliquots were tested in a real-time fluorescence PCR using primers against SARS-CoV-2.Regardless of inactivation temperature and time,7 of 12 cases(58.3%)tested were positive for SARS-CoV-2 by PCR,and cycle threshold values were similar.These results suggest that virus inactivation parameters exert minimal infuence on PCR test results.Inactivation at 65℃for 10 min may be sufficient to ensure safe,reliable testing.
文摘Objective Establishing a highly sensitive real-time fluorescence quantitative PCR (qPCR) method for universal testing of epidemic African swine fever virus (ASFV) strains. Methods The ASFV p72 gene was targeted to design primer probes covering 24 p72 genotypes. The optimal amount of dimethylsulphoxide (DMSO) for qPCR amplification was determined, Various sensitivity and limit of detection (LOD) tests were performed, and clinical samples from China and imported goods were tested. Results The optimal primer-probe combination could specifically detect ASFV, 1.5% DMSO was optimal for qPCR, and LOD reached 3.2 copies/μL with good reproducibility (n = 20, p = 0.369). The method was employed to test 142 clinically suspected samples, of which 30 pig blood and 37 pig tissue samples were ASFV-positive. Moreover, the positive testing rate for ASFV was higher than for the standard qPCR method recommended by the Office International Des Epizooties (OIE), and for the commercially available kit. Thus, our method is superior for testing weakly positive samples with low virus titre, and epidemic strains present in imported goods. Conclusion Our method could be employed for universal testing of epidemic ASFV strains worldwide, ensuring wider coverage of hosts and ASFV strains/endemic strains, reducing false<span style="font-family:;" "=""> </span><span style="font-family:Verdana;">negatives, and benefitting early diagnosis.</span>
基金Supported by the National Natural Science Foundation of China (20836004 20806046) the Special Fund for Major State Basic Research Program of China (2009CB724702) the National High Technology Research and Development Program ofChina (2009AA062903)
文摘Green fluorescent protein (GFP) and its variants /homolog proteins are generally called as GFP-like fluorescent proteins (FPs), which are widely used as visible molecular tools for monitoring a wide range of biological processes due to their capability of simple, accurate and real time quantification. The FPs-based molecular and visible quantification tools are giving more impact on bioprocess engineering, enabling the biomolecule-level dynamic information to be linked with the process-level events. In this review, different applications of FPs in biological engineering with emphasis on rapid molecular bioprocess quantification, such as quantification of the transcription efficiency, the protein production, the protein folding efficiency, the cell concentration, the intracellular microenvironments and so on, would be first introduced. The challenges of using FPs with respect to actual bioprocess applications for the precise quantification including the interaction of FPs and the fused partner proteins, the maturation of FPs, the inner filter effect and sensing technology were then discussed. Finally, the future development for the FPs used in molecular bioprocess quantification would be proposed.
基金Supported by Scientific Research Project of Anhui Bureau of Quality and Technical Supervision(13zj370033)
文摘Peanut,sesame and other raw materials of food are allergens for special populations.In this study,specific primers and TaqMan probes labeled by different fluorescences were designed targeting Ara h 2 gene of peanut and Ses i 1 gene of sesame.After the optimization of reaction conditions,a real-time fluorescent PCR method was established for simultaneous detection of allergenic ingredients of peanut and sesame in food.Genomic DNA samples of peanut,sesame,rice,wheat,barley,soybean,celery,maize,potato,tomato,walnut,groundnut in shell,cashew nut,sunflower seed,almond,apple,pear and strawberry,pork,beef,mutton and fish were used as templates for PCR amplification with deionized water as negative control template.Results indicated that the established real-time fluorescent PCR method could specifically identify allergenic ingredients of peanut and sesame simultaneously.Sensitivity test showed that the minimum detection limit of this method was 0.01%.Therefore,the established real-time fluorescent PCR method is a specific,sensitive and effective assay for simultaneously detecting allergenic ingredients of peanut and sesame in food.
基金Supported by Project of Standardization Technical System from the Administration of Quality and Technology Supervision of Sichuan Province(ZYBZ2013-39)
文摘In order to improve the standardized technical systems of quantitative analyses for genetically modified organisms(GMOs) and products,ensure bio-safety and reduce ecological risk in China,a real-time fluorescent quantitative PCR assay was established for detection of genetically modified maize line MON88017.The established method was evaluated based on the specificity,sensitivity,accuracy and measurement uncertainty.The results showed that the established method had strong specificity in detection of genetically modified maize line MON88017.1.50%MON88017 sample was detected with 29 replications.The average measured value(1.541%) was close to the actual value(1.50%) and the relative deviation was 2.70%.The variation coefficient of the measured value was 0.110 4;the recovery was 100.00%and the measurement uncertainty was 0.096.The limit of detection for genetically modified maize line MON88017 with the established method was 5 copies at the 97.5%confidence level.Thus,the real-time fluorescent quantitative PCR assay established in this study exhibited high specificity,accuracy and sensitivity,which could provide technical support for the safety supervision of genetically modified organisms and products in China.
基金Supported by the National Natural Science Foundation of China (No.20336010 and No.20176025).
文摘To establish a rapid quantification method for heparinase I during its production in recombinant Es-cherichia coli, a translational fusion vector was constructed by fusing the N terminus of heparinase I to the C ter-minus of a green fluorescent protein mutant (GFPmut1). As a result, not only was the functional recombinant ex-pression of heparinase I in E. coli accomplished, but also a linear correlation was obtained between the GFP fluores-cence intensity and heparinase I activity, allowing enzyme activity to be quantified rapidly during the fermentation.
基金E.U.FP6 Integrated Project“Molecular Imaging”LSHG-CT-2003-503259E.U.FP7 Collaborative Project“FMT-XCT”.R.F.acknowledges support from the Marie Curie Program EST-MolecImag Early Stage Training MEST-CT-2004-007643.
文摘Even though multispectral imaging is considered very significant in biological imaging,it is only commonly used in microscopy in a 2D approach.Here,we present a Fluorescence Molecular Tomography system capable of recording simultaneously tomographic data at several spectral windows,enabling multispectral tomography.3D reconstructed data from several spectral windows is used to construct a linear unmixing algorithm for multispectral deconvolution of overlapping fluorescence signals.The method is applied on tomographic 3D fluorescence concentration maps in tissue-mimicking phantoms,yielding absolute quantification of the concentration of each individual fluorophore.Results are compared to the case when unmixing is performed in the raw 2D data instead of the reconstructed 3D concentration map,showing greater accuracy when unmixing algorithms are applied in the reconstructed data.Both the reflection and transmission geometries are considered.
文摘There is an urgent need for developing a procedure for biomarker standardization and relative quantificationin clinical laboratories. Measuring the expression levels of cell antigens is critical for the diagnosis of many diseases, e.g. leukemia, lymphoma and immunodeficiency diseases. One of the most significant challenges in flow cytometry is obtaining inter-laboratory and intra-laboratory consistent and reproducible results across multiple cytometer platforms and locations longitudinally over time. To obtain measurement consistency, the target flow cytometer voltages should be optimized to segregate the negative population from the electronic noise, and to keep the brightest positive population within the dynamic range of each detector. Then target values should be determined and transferred to selected cytometers. In this study, we optimized a procedure for instrument standardization across three different flow cytometer platforms from the same vendor and in two different locations. The biomarker quantification was implemented on standardized instruments using CD4 expression on T lymphocytes with a known amount of antibody bound per cell as a quantification standard. Our results on blood cell subset typing and CD19 quantification demonstrated that consistent and reliable results could be accomplished between instruments using the developed procedure. Quantitating the expression levels of certain cell biomarkers relative to a known reference marker before, during, and after therapy would provide important information for monitoring antibody-based therapy and could be potentially used to adjust dosing. Presently, we are implementing this protocol to quantify critical disease biomarkers, and making necessary modifications to the procedure to include instruments from different instrument manufacturers.
文摘A quantitative analysis method of molybdenum in FeMo alloys by X-ray spectrometry using borate fusion technique was compared with that with pressed pellet. The complete pre-oxidation of FeMo alloys for the preparation of homogeneous fused discs was achieved by employing an automated fusion machine equipped with specially designed O2-blowing nozzles, which used lithium tetra-borate as flux with the addition of lithium nitrate (LiNO3) as oxidizer. The calibration curves of Mo and Fe were used in the quantitative analysis of standard materials and unknown plant samples with satisfactory accuracy and precision, utilizing the corrections of the matrix effects and line overlap. It was confirmed that the newly proposed method of preparing fused glass discs of FeMo alloys can replace the conventional wet chemical analyses requiring the labor intensive and time consuming procedure.
文摘[Objectives]To verify the specificity,sensitivity,precision and negative-positive deviation of the foodproof gluten component de-tection kit for the detection of gluten allergens in milk powder matrix,and to establish a real-time fluorescent PCR legal method for the detec-tion of gluten allergens in milk powder.[Methods]The specificity,sensitivity,precision and negative-positive deviation of the detection method of foodproof gluten component detection kit(PCR-probe method)were verified by artificially adding different concentrations of wheat bran and extracting sample DNA by kit method,and applied to sample detection.[Results] The specific detection results of two kinds of milk powder with wheat bran and buckwheat added showed that the foodproof gluten component detection kit(PCR-probe method)had good speci-ficity for wheat gluten.The results of artificially added wheat bran positive samples showed that the false positive rate and false negative rate of the kit in the milk powder matrix were O,and the sensitivity and precision were high.[Conclusions]The kit is simple to operate and has high accuracy,which is suitable for the detection of gluten allergen components in milk powder.
基金supported by research grant from the Thailand Research Fund (TRF) through the Royal Golden Jubilee Ph.D. program (Grant No. PHD/0085/2554)the Thai Government Budget through Mahidol University, fiscal year 2015-2017
文摘Noroviruses are the leading cause of acute gastroenteritis in humans. Real-time reverse transcription-polymerase chain reaction(real-time RT-PCR) is a promising molecular method for the detection of noroviruses. In this study, the performance of three Taq Man real-time RT-PCR assays was assessed, which were one commercially available real-time RT-PCR kit(assay A:Norovirus Real Time RT-PCR kit) and two in-house real-time RT-PCR assays(assay B: Light Cycler RNA Master Hybprobe and assay C: Real Time ready RNA Virus Master). Assays A and B showed higher sensitivity than assay C for norovirus GI, while they all had the same sensitivity(103 DNA copies/m L) for GII DNA standard controls. Assay B had the highest efficiency for both genogroups.No cross-reactivity was observed among GI and GII noroviruses, rotavirus, hepatitis A virus, and poliovirus. The detection rates of these assays in GI and GII norovirus-positive fecal samples were not significantly different. However, the mean quantification cycle(Cq) value of assay B for GII was lower than assays A and C with statistical significance(P-value, 0.000). All three real-time RT-PCR assays could detect a variety of noroviruses including GI.2, GII.2, GII.3, GII.4, GII.6, GII.12, GII.17,and GII.21. This study suggests assay B as a suitable assay for the detection and quantification of noroviruses GI and GII due to good analytical sensitivity and higher performance to amplify norovirus on DNA standard controls and clinical samples.
基金J. Liu was supported by the JAERI Research Fellow Program.
文摘The molecular techniques including Northern blot, dot blot, in situ hybridization, etc. have been successfully used to estimate semi-quantitatively mRNA levels in plant samples. In this study, we employed a real-time reverse transcription-PCR (RT-PCR) assay using SYBR Green I fluorescence methodology to evaluate accurate quantitation and sequence specific detection of Aux/IAA mRNA levels in Arabidopsis. Results obtained indicate a linear dynamic range of 102-106 Aux/IAA mRNA copies with standard deviations of generally less than 15%. As a model experiment, the outcome of analysis of expression patterns of five Aux/IAA genes in Arabidopsis under various chemical and temperature treatments is presented. The method presented here provides a sensitive and rapid technique to evaluate plant Aux/IAA mRNA expression levels in nanogram order.
基金This study was supported by the National Key R&D Program of China(Grant No.2017YFA0605001)the National Natural Science Foundation of China(Grant Nos.52170024,21677015 and 22006031)+1 种基金the Natural Science Foundation of Hebei Province(No.B2019204315)the Sponsored Research Overhead Fund(Grant No.472120)from Kansas State University.
文摘Understanding the fate and toxicity of microplastics(MPs,<5 mm plastic particles)is limited by quantification methods.This paper summarizes the methods in use and presents new ones.First,sampling and pretreatment processes ofMPs,including sample collection,digestion,density separation,and quality control are reviewed.Then the promising and convenient staining procedures and quantification methods for MPs using fluorescence dyes are reviewed.The factors that influence the staining of MPs,including their physicochemical properties,are summarized to provide an optimal operation procedure.In general,the digestion step is crucial to eliminate natural organic matter(NOM)to avoid interference in quantification.Chloroform was reported to be the most appropriate solvent,and 10–20μg/mL are recommended as optimal dye concentrations.In addition,a heating and cooling procedure is recommended to maintain the fluorescence intensity of MPs for two months.After staining,a fluorescence microscope is usually used to characterize the morphology,mass,or number of MPs,but compositional analysis cannot be determined with it.These fluorescence staining methods have been implemented to study MP abundance,transport,and toxicity and have been combined with other chemical characterization techniques,such as Fourier transform infrared spectroscopy and Raman spectroscopy.More studies are needed to focus on the synthesis of novel dyes to avoid NOM’s interference.They need to be combined with other spectroscopic techniques to characterize plastic composition and to develop image-analysis methods.The stability of stained MPs needs to be improved.
基金supported by the National Natural Science Foundation of China(Nos.21775001,21874137,21974001,21976183and 22074001)Natural Science Foundation of Anhui Province(No.1808085MB32)Nature and Science Foundation from Anhui Province Ministry of Education(No.KJ2019A0011)。
文摘Nuclear RNA export into the cytoplasm is one of the key steps in protein expression to realize biological functions.Despite the broad availability of nucleic acid dyes,tracking and quantifying the highly dynamic process of RNA export in live cells is challenging.When dye-labeled RNA enters the cytoplasm,the dye molecules are released upon degradation of the RNA,allowing them to re-enter the cell nucleus.As a result,the ratio between the dye exported with RNA into the cytoplasm and the portion staying inside the nucleus cannot be determined.To address this common limitation,we report the design of a smart probe that can only check into the nucleus once.When adding to cells,this probe rapidly binds with nuclear RNAs in live cells and reacts with intrinsic H_(2)S.This reaction not only activates the fluorescence for RNA tracking but also changes the structure of probe and consequently its intracellular localization.After disassociating from exported RNAs in cytoplasm,the probe preferentially enters lysosomes rather than cell nucleus,enabling real-time quantitative measurement of nuclear RNA exports.Using this probe,we successfully evaluated the effects of hormones and cancer drugs on nuclear RNA export in live cells.Interestingly,we found that hormones inhibiting RNA exports can partially offset the effect of chemotherapy.