The purpose of this study is to detect the plasmid copies encoding HBsAg in recombinant yeast Saccharomyces cerevisiae by real-time PCR, and provide the data for setting up an optimal process of manufacturing hepatiti...The purpose of this study is to detect the plasmid copies encoding HBsAg in recombinant yeast Saccharomyces cerevisiae by real-time PCR, and provide the data for setting up an optimal process of manufacturing hepatitis B vaccine in future. A single copy URA3 gene in the yeast genome was employed as internal control to test the plasmid copies by real-time PCR. The yeast cells carrying plasmids encoding HBsAg were cultured in regular non-feeding process and fed-batch process in shaking flasks at laboratory level and industrial fermentor. The cells at different stages of cultivation were collected and broken. Then the total DNAs of the cells were extracted and the plasmid copies were detected by real-time PCR with the method of relative quantification. The method of the real-time relative quantitative PCR used in this study was reliable with good accuracy and quickness. The plasmid copies in non-feeding and fed-batch flasks were 39.22±12.00 and 43.06±5.70, while the copies in fermentor were 96.16±21.00 and 82.50±7.78, respectively. The data show that the variation of the result is very small and the data precision is quite good. To prolong the fermentation time may increase the cell density without influence on the stability of the plasmid, and the method of relative quantification can also be applied to test the copy number of interest target gene in any host cell with certain known copy gene.展开更多
AIM: To preliminarily test proteomics in aqueous humor in patients with dry age-related macular degeneration(AMD) by using the proteomic technology.METHODS: Aqueous humor samples were collected from patients with or w...AIM: To preliminarily test proteomics in aqueous humor in patients with dry age-related macular degeneration(AMD) by using the proteomic technology.METHODS: Aqueous humor samples were collected from patients with or without dry AMD, who underwent cataract surgery. The aqueous samples were analyzed with isobaric tags for relative and absolute quantification(i TRAQ) combined with liquid chromatography tandem mass spectrometry(LC-MS/MS) technology. The differential expressed proteins were analyzed with gene ontology(GO) enrichment, Kyoto Encyclopedia of Genes and Genomes(KEGG) and protein-protein interaction(PPI) network analysis. The data were partly validated by ELISA and Western blot. False discovery rate(FDR) was used for statistical analysis. RESULTS: A total of 244 proteins were detected, in which 38 proteins were up-regulated and 51 were down-regulated significantly in patients with dry AMD compared with that in control groups(FDR value <1.0%). Several proteins, e.g., protein S100-A8(S10 A8), dystroglycan(DAG1), Ig alpha-1 chain C region(IGHA1), carbonic anhydrase 3(CAH3) and alpha-1-acid glycoprotein(A1 AG1) were increased more than 5 times of that in control group. The bioinformatics analysis showed that dry AMD is closely associated with inflammation or immune reaction, oxidative stress, blood coagulation and remodeling of extracellular matrix.CONCLUSION: i TRAQ-based proteomic analysis of aqueous humor demonstrate the differential expressions of proteins between dry AMD and control groups, providing the clues to understand the mechanisms and possible treatments of dry AMD.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
随着资源短缺以及环境问题的日益加剧,道路工程逐渐由单一交通属性向多功能性转变,将能源与路面相结合的“能源相关路面”逐渐成为交通运输领域研究的热点。为快捷有效地掌握能源相关路面的研究进展及存在的问题,采用Citespace文献可视...随着资源短缺以及环境问题的日益加剧,道路工程逐渐由单一交通属性向多功能性转变,将能源与路面相结合的“能源相关路面”逐渐成为交通运输领域研究的热点。为快捷有效地掌握能源相关路面的研究进展及存在的问题,采用Citespace文献可视化计量分析软件,以Web of Science数据库为基础,以energy pavement为主题词,检索得到1997—2020年的2337篇文献为分析对象,分别从文献共被引、作者合作网络及机构合作网络3个维度进行研究,分析能源相关路面的学科分布特点及各学科间联系,梳理并对比各作者和机构的研究趋势。研究表明:与能源相关路面主题相关的研究主要集中在中国和美国。从2009年开始,研究论文数量急剧增加。现阶段研究侧重点主要集中在能源路面领域,即对全寿命周期评估、热岛效应、温拌沥青和水损害等主题进行研究;对路面能源这一新型领域的研究还处于初级探索阶段,涉及能源收集、沥青太阳能集热器等研究主题。围绕以上两个维度的热点开展研究,并相互渗透与融合是加深能源相关路面研究的有效途径。展开更多
文摘The purpose of this study is to detect the plasmid copies encoding HBsAg in recombinant yeast Saccharomyces cerevisiae by real-time PCR, and provide the data for setting up an optimal process of manufacturing hepatitis B vaccine in future. A single copy URA3 gene in the yeast genome was employed as internal control to test the plasmid copies by real-time PCR. The yeast cells carrying plasmids encoding HBsAg were cultured in regular non-feeding process and fed-batch process in shaking flasks at laboratory level and industrial fermentor. The cells at different stages of cultivation were collected and broken. Then the total DNAs of the cells were extracted and the plasmid copies were detected by real-time PCR with the method of relative quantification. The method of the real-time relative quantitative PCR used in this study was reliable with good accuracy and quickness. The plasmid copies in non-feeding and fed-batch flasks were 39.22±12.00 and 43.06±5.70, while the copies in fermentor were 96.16±21.00 and 82.50±7.78, respectively. The data show that the variation of the result is very small and the data precision is quite good. To prolong the fermentation time may increase the cell density without influence on the stability of the plasmid, and the method of relative quantification can also be applied to test the copy number of interest target gene in any host cell with certain known copy gene.
基金Supported by National Natural Science Foundation of China(No.81570852)the Shanghai Municipal Health and Planning Commission Foundation(No.201540046)
文摘AIM: To preliminarily test proteomics in aqueous humor in patients with dry age-related macular degeneration(AMD) by using the proteomic technology.METHODS: Aqueous humor samples were collected from patients with or without dry AMD, who underwent cataract surgery. The aqueous samples were analyzed with isobaric tags for relative and absolute quantification(i TRAQ) combined with liquid chromatography tandem mass spectrometry(LC-MS/MS) technology. The differential expressed proteins were analyzed with gene ontology(GO) enrichment, Kyoto Encyclopedia of Genes and Genomes(KEGG) and protein-protein interaction(PPI) network analysis. The data were partly validated by ELISA and Western blot. False discovery rate(FDR) was used for statistical analysis. RESULTS: A total of 244 proteins were detected, in which 38 proteins were up-regulated and 51 were down-regulated significantly in patients with dry AMD compared with that in control groups(FDR value <1.0%). Several proteins, e.g., protein S100-A8(S10 A8), dystroglycan(DAG1), Ig alpha-1 chain C region(IGHA1), carbonic anhydrase 3(CAH3) and alpha-1-acid glycoprotein(A1 AG1) were increased more than 5 times of that in control group. The bioinformatics analysis showed that dry AMD is closely associated with inflammation or immune reaction, oxidative stress, blood coagulation and remodeling of extracellular matrix.CONCLUSION: i TRAQ-based proteomic analysis of aqueous humor demonstrate the differential expressions of proteins between dry AMD and control groups, providing the clues to understand the mechanisms and possible treatments of dry AMD.
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.
文摘随着资源短缺以及环境问题的日益加剧,道路工程逐渐由单一交通属性向多功能性转变,将能源与路面相结合的“能源相关路面”逐渐成为交通运输领域研究的热点。为快捷有效地掌握能源相关路面的研究进展及存在的问题,采用Citespace文献可视化计量分析软件,以Web of Science数据库为基础,以energy pavement为主题词,检索得到1997—2020年的2337篇文献为分析对象,分别从文献共被引、作者合作网络及机构合作网络3个维度进行研究,分析能源相关路面的学科分布特点及各学科间联系,梳理并对比各作者和机构的研究趋势。研究表明:与能源相关路面主题相关的研究主要集中在中国和美国。从2009年开始,研究论文数量急剧增加。现阶段研究侧重点主要集中在能源路面领域,即对全寿命周期评估、热岛效应、温拌沥青和水损害等主题进行研究;对路面能源这一新型领域的研究还处于初级探索阶段,涉及能源收集、沥青太阳能集热器等研究主题。围绕以上两个维度的热点开展研究,并相互渗透与融合是加深能源相关路面研究的有效途径。
