目的建立 SYBR Green I实时荧光定量 PCR检测乙型肝炎病毒 DNA的快速方法，并探讨其临床应用价值。方法根据GenBank公布的Hepatitis B virus gp1基因序列设计引物，建立SYBR Green I实时荧光定量PCR，并对反应体系和扩增程序进行优化。...目的建立 SYBR Green I实时荧光定量 PCR检测乙型肝炎病毒 DNA的快速方法，并探讨其临床应用价值。方法根据GenBank公布的Hepatitis B virus gp1基因序列设计引物，建立SYBR Green I实时荧光定量PCR，并对反应体系和扩增程序进行优化。定量标准品通过基因克隆方法获得，同时以浙江省夸克公司 HBV DNA定量检测试剂盒作对照，应用于随机选取的100份乙型肝炎患者血清检测。结果 SYBR Green I 实时荧光定量 PCR 检出限范围为5＆#215;102 copies／ml～5＆#215;108 copies／ml，HBV DNA浓度与CT值有良好线性关系，无交叉反应，整个过程仅需2.5 h。在随机抽取的100份临床标本应用中，与浙江省夸克公司的 HBV荧光定量 PCR检测试剂相比，建立的 SYBR Green I实时荧光定量PCR体系灵敏度100％，特异度92.5％。结论 SYBR Green实时荧光定量PCR快速、简便、灵敏度高、特异度强，可用于乙型肝炎患者病情监测，有效指导临床用药，准确评价 HBV感染者的病情。展开更多
Background: Psoriasis has been shown to highly accumulate protoporphyrin IX (PpIX), but a variable distribution within plaques after fluorescence diagnosis is seen. It is unknown what causes this heterogeneity of fluo...Background: Psoriasis has been shown to highly accumulate protoporphyrin IX (PpIX), but a variable distribution within plaques after fluorescence diagnosis is seen. It is unknown what causes this heterogeneity of fluorescence in psoriatic skin, despite adequate keratolytic treatment. Variations in fluorescence might explain the variable and the mostly partial clinical response of psoriasis seen after photodynamic therapy (PDT).Objectives: This study examines morphological and immunohistochemical differences in inhomogeneous PpIX-induced fluorescence in stable plaque psoriasis. Materials and methods: Fourteen patients with stable plaque psoriasis were included in this study. In each patient one psoriatic plaque was incubated with 20%5-aminolaevulinic acid (ALA) ointment for 3 h after keratolytic treatment. Fluorescence diagnosis with ALA induced porphyrins (FDAP) was performed and subsequently high-and low-fluorescent psoriatic skin samples were biopsied. Biopsies were investigated with respect to histologicalhyperkeratosis (thickness of stratum corneum), proliferation (Ki-67 antigen), keratinization (K10, filaggrin) and inflammation (CD3). Digital images acquired with FDAP were analysed using image analysis software. Results: Inhomogeneous fluorescence was seen in 12 of the 14 plaques. A significantly thicker stratum corneum was found in low-fluorescent psoriatic skin compared with highly fluorescent skin. Fluorescence intensity and thickness of the stratum corneum proved to be negatively correlated. The variable-fluorescent parts of the lesional psoriatic skin showed no differences in epidermal proliferation, keratinization or inflammation. Conclusions: Heterogeneous ALA-induced fluorescence in psoriasis plaques related to inhomogeneous distribution of PpIX in the epidermis may result from differences in penetration of ALA and/or light within a plaque caused by differences in stratum corneum thickness. The variable clinical response seen after PDT in psoriasis could be explained by this. These findings are consistent with the general assumption that optimal desquamation prior to FDAP or PDT is required for the most favourable results.展开更多
The gut microbiota is a complex ecosystem composed of many bacteria and their metabolites.It plays an irreplaceable role in human digestion,nutrient absorption,energy supply,fat metabolism,immune regulation,and many o...The gut microbiota is a complex ecosystem composed of many bacteria and their metabolites.It plays an irreplaceable role in human digestion,nutrient absorption,energy supply,fat metabolism,immune regulation,and many other aspects.Exploring the structure and function of the gut microbiota,as well as their key genes and metabolites,will enable the early diagnosis and auxiliary diagnosis of diseases,new treatment methods,better effects of drug treatments,and better guidance in the use of antibiotics.The identification of gut microbiota plays an important role in clinical diagnosis and treatment,as well as in drug research and development.Therefore,it is necessary to conduct a comprehensive review of this rapidly evolving topic.Traditional identification methods cannot comprehensively capture the diversity of gut microbiota.Currently,with the rapid development of molecular biology,the classification and identification methods for gut microbiota have evolved from the initial phenotypic and chemical identification to identification at the molecular level.This review integrates the main methods of gut microbiota identification and evaluates their application.We pay special attention to the research progress on molecular biological methods and focus on the application of high-throughput sequencing technology in the identification of gut microbiota.This revolutionary method for intestinal flora identification heralds a new chapter in our understanding of the microbial world.展开更多
Partial cDNA sequence of rabbit BMP15 was cloned by RT-PCR from rabbit ovaries, showing a similarity of 83%-90% with the BMP15 nucleotide sequences in humans, mice, ovine, sheep, cows and pigs. The expression of BMP15...Partial cDNA sequence of rabbit BMP15 was cloned by RT-PCR from rabbit ovaries, showing a similarity of 83%-90% with the BMP15 nucleotide sequences in humans, mice, ovine, sheep, cows and pigs. The expression of BMP15 in rabbit cumulus-oocyte complexs during oocytes in vitro maturation （IVM） was measured by fluorescent quantitative RT-PCR method. BMP 15 was expressed at low levels in immature oocytes and increased to the highest level at 16h of IVM, which coincides with the time of cumulus cell expansion, then declined slowly under IVM cultivation. The expression pattern of BMP 15 suggested that it might be important in cumulus expansion in rabbits.展开更多
This study was to investigate the efficiency and specificity of RNAi silencing on the expression of endogenous fad2 gene in transgenic line W-4. [Method] The relative expression of fad2 gene in seeds at different deve...This study was to investigate the efficiency and specificity of RNAi silencing on the expression of endogenous fad2 gene in transgenic line W-4. [Method] The relative expression of fad2 gene in seeds at different developmental stages of 7th, 14th, 21st and 28th day after flowering （DAF） as wel as the root, stem, leaf at winter seedling stages of both the transgenic line W-4 and non-transgenic control Westar by real-time fluorescence quantitative PCR. [Results] The results showed the relative expression of fad2 gene was gradual y increasing with the days after flowering in the seeds of the control Westar, while it was found decreasing significantly since the 21st DAF in the seeds of the line W-4. The decline was up to 60% in comparison with the control Westar. However, no significant difference in the relative expression of fad2 gene in other organs like root, stem and leaf was observed between transgenic line W-4 and non-transgenic control Westar. Fatty acid composition analysis showed the oleic acid desaturation parameter（ODP） in seeds of the line W-4 was 0.07 in average, decreased by nearly 75% than control Westar which was 0.24 in average, while no significant difference in the seedling root, stem and leaf was measured between transgenic rapeseed and control. [Conclusion] The results above validated that RNA interference in transgenic rapeseed W-4 is at a seed-specific manner, not interfering with fad2 gene expression in organs such as the root, stem and leaf. The study also found that the period of fad2 gene expres-sion decline was wel coincided with the expression of napin gene, both appeared at the 21st DAF, indicating that the expression of dsRNA of fad2 gene is precisely control ed by the napin promoter.展开更多
文摘Background: Psoriasis has been shown to highly accumulate protoporphyrin IX (PpIX), but a variable distribution within plaques after fluorescence diagnosis is seen. It is unknown what causes this heterogeneity of fluorescence in psoriatic skin, despite adequate keratolytic treatment. Variations in fluorescence might explain the variable and the mostly partial clinical response of psoriasis seen after photodynamic therapy (PDT).Objectives: This study examines morphological and immunohistochemical differences in inhomogeneous PpIX-induced fluorescence in stable plaque psoriasis. Materials and methods: Fourteen patients with stable plaque psoriasis were included in this study. In each patient one psoriatic plaque was incubated with 20%5-aminolaevulinic acid (ALA) ointment for 3 h after keratolytic treatment. Fluorescence diagnosis with ALA induced porphyrins (FDAP) was performed and subsequently high-and low-fluorescent psoriatic skin samples were biopsied. Biopsies were investigated with respect to histologicalhyperkeratosis (thickness of stratum corneum), proliferation (Ki-67 antigen), keratinization (K10, filaggrin) and inflammation (CD3). Digital images acquired with FDAP were analysed using image analysis software. Results: Inhomogeneous fluorescence was seen in 12 of the 14 plaques. A significantly thicker stratum corneum was found in low-fluorescent psoriatic skin compared with highly fluorescent skin. Fluorescence intensity and thickness of the stratum corneum proved to be negatively correlated. The variable-fluorescent parts of the lesional psoriatic skin showed no differences in epidermal proliferation, keratinization or inflammation. Conclusions: Heterogeneous ALA-induced fluorescence in psoriasis plaques related to inhomogeneous distribution of PpIX in the epidermis may result from differences in penetration of ALA and/or light within a plaque caused by differences in stratum corneum thickness. The variable clinical response seen after PDT in psoriasis could be explained by this. These findings are consistent with the general assumption that optimal desquamation prior to FDAP or PDT is required for the most favourable results.
文摘The gut microbiota is a complex ecosystem composed of many bacteria and their metabolites.It plays an irreplaceable role in human digestion,nutrient absorption,energy supply,fat metabolism,immune regulation,and many other aspects.Exploring the structure and function of the gut microbiota,as well as their key genes and metabolites,will enable the early diagnosis and auxiliary diagnosis of diseases,new treatment methods,better effects of drug treatments,and better guidance in the use of antibiotics.The identification of gut microbiota plays an important role in clinical diagnosis and treatment,as well as in drug research and development.Therefore,it is necessary to conduct a comprehensive review of this rapidly evolving topic.Traditional identification methods cannot comprehensively capture the diversity of gut microbiota.Currently,with the rapid development of molecular biology,the classification and identification methods for gut microbiota have evolved from the initial phenotypic and chemical identification to identification at the molecular level.This review integrates the main methods of gut microbiota identification and evaluates their application.We pay special attention to the research progress on molecular biological methods and focus on the application of high-throughput sequencing technology in the identification of gut microbiota.This revolutionary method for intestinal flora identification heralds a new chapter in our understanding of the microbial world.
文摘Partial cDNA sequence of rabbit BMP15 was cloned by RT-PCR from rabbit ovaries, showing a similarity of 83%-90% with the BMP15 nucleotide sequences in humans, mice, ovine, sheep, cows and pigs. The expression of BMP15 in rabbit cumulus-oocyte complexs during oocytes in vitro maturation （IVM） was measured by fluorescent quantitative RT-PCR method. BMP 15 was expressed at low levels in immature oocytes and increased to the highest level at 16h of IVM, which coincides with the time of cumulus cell expansion, then declined slowly under IVM cultivation. The expression pattern of BMP 15 suggested that it might be important in cumulus expansion in rabbits.
基金Supported by Fund for National Rapeseed Research System(CARS-13)~~
文摘This study was to investigate the efficiency and specificity of RNAi silencing on the expression of endogenous fad2 gene in transgenic line W-4. [Method] The relative expression of fad2 gene in seeds at different developmental stages of 7th, 14th, 21st and 28th day after flowering （DAF） as wel as the root, stem, leaf at winter seedling stages of both the transgenic line W-4 and non-transgenic control Westar by real-time fluorescence quantitative PCR. [Results] The results showed the relative expression of fad2 gene was gradual y increasing with the days after flowering in the seeds of the control Westar, while it was found decreasing significantly since the 21st DAF in the seeds of the line W-4. The decline was up to 60% in comparison with the control Westar. However, no significant difference in the relative expression of fad2 gene in other organs like root, stem and leaf was observed between transgenic line W-4 and non-transgenic control Westar. Fatty acid composition analysis showed the oleic acid desaturation parameter（ODP） in seeds of the line W-4 was 0.07 in average, decreased by nearly 75% than control Westar which was 0.24 in average, while no significant difference in the seedling root, stem and leaf was measured between transgenic rapeseed and control. [Conclusion] The results above validated that RNA interference in transgenic rapeseed W-4 is at a seed-specific manner, not interfering with fad2 gene expression in organs such as the root, stem and leaf. The study also found that the period of fad2 gene expres-sion decline was wel coincided with the expression of napin gene, both appeared at the 21st DAF, indicating that the expression of dsRNA of fad2 gene is precisely control ed by the napin promoter.
