乙型肝炎病毒SYBR GreenⅠ实时荧光定量PCR方法的建立及临床应用

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。

实时荧光定量PCR检测皱纹盘鲍Δ5脂肪酸去饱和酶基因表达方法的建立及应用

研究以皱纹盘鲍(Haliotis discus hannai Ino)体内存在的2条Δ5 Fad(Hdhfad1和Hdhfad2)为目的基因,β-肌动蛋白(β-actin)和核糖体蛋白S9(Ribosomal protein S9,RPS9)为内参基因,应用2–ΔΔCt方法建立了实时荧光定量PCR(Real-Time quantitative PCR,RT-qPCR)检测体系,并应用此体系分析了鲍鱼肌肉组织Δ5 Fad在不同饲料处理下的表达差异。实验饲料含有不同的脂肪源,分别是棕榈酸甘油酯(Tripalmitin,TP饲料)、富含二十碳四烯酸(Arachidonic acid,ARA)的油脂(AO饲料)和富含二十碳五烯酸(Eicosapentaenoic acid,EPA)的油脂(EO饲料)。结果表明:针对Hdhfad1、Hdhfad2、β-actin和RPS9所设计的引物特异性强。各引物对的PCR扩增效率(Efficiency,E)分别为1.05、0.99、0.97和0.98,满足2–ΔΔCt方法对E的要求。当退火温度为52℃,反应体积为25μL时,RT-qPCR的扩增效果最好。所建立的体系能够准确定量Δ5 Fad的基因表达。利用该方法分析Δ5 Fad在不同饲料处理下的表达结果显示,与TP对照组相比,EO和AO饲料显著降低了鲍鱼肌肉组织Δ5 Fad(Hdhfad1和Hdhfad2)的表达量。

鹿布鲁氏菌实时荧光定量PCR检测方法的建立

本研究依据布鲁氏菌的特异性基因Omp25c的部分片段作为靶基因,设计探针引物,且优化了反应体系,筛选出引物、探针的最优浓度配比。将扩增产物连接到PGEM-T载体上,制备标准品及标准曲线,建立鹿布鲁氏菌荧光定量PCR检测方法,并对其特异性、稳定性、敏感性进行评价。由标准曲线可知该方法的最低检测浓度可达到36拷贝/μL,比常规PCR灵敏度高出很多。

实时荧光定量PCR在食品致病菌及转基因成分检测中应用

与传统定性PCR技术相比,实时荧光定量PCR有更多的优点,其速度快,特异性更强、灵敏度更高、无污染性、自动化水平高等,且能对DNA模板进行定量。本文综述了实时荧光定量PCR技术原理、分类及其应用,并对其存在的问题和发展前景进行了展望。

实时荧光定量PCR检测腮腺沃辛瘤中Gadd45β表达

目的：从分子学水平检测腮腺沃辛瘤相对于瘤旁腮腺组织中生长抑制和DNA损伤修复基因45β（ Gadd45β） mRNA表达情况并探讨其在沃辛瘤的形成及发展中的意义。方法搜集30例腮腺沃辛瘤组织及瘤旁正常腮腺组织。提取总RNA，然后进行逆转录得到cDNA，再进行实时荧光定量PCR检测，测出CT值，通过2－△△CT的相对定量的方法对荧光定量PCR检测的CT值进行标准化，从而比较肿瘤组织及瘤旁正常腮腺组织中Gadd45βmRNA表达情况。结果 Gadd45β在正常腮腺组织及肿瘤组织中均有表达，分别为1．516±0．104和1．177±0．089， Gadd45β在沃辛瘤中表达明显下调（P＜0．05）。结论 Gadd45β表达下调可作为沃辛瘤发病机理一部分。

3种核酸提取方法及3种实时荧光定量PCR仪检测结果的比较

目的 探讨不同核酸提取方法以及不同实时荧光定量PCR仪检测结果之间的差异.方法 随机挑选呼吸道、肠道病毒阳性标本各25份,采用3种方法（方法A、B、C）进行核酸提取,比较不同核酸提取方法之间的差异;随机挑选呼吸道、肠道病毒阳性核酸各25份,在3种实时荧光定量PCR仪（仪器A、B、C）上进行实时荧光定量PCR,比较不同实时荧光定量PCR仪之间的差异.检测结果采用定量资料的随机区组方法分析.结果 3种核酸提取方法检测结果存在差异（x2=42.9162,P〈0.001）,其中方法A、B,方法B、C之间的差异具有统计学意义（Z=7.025,P〈0.001;Z=7.9,P〈0.001）,方法A、C之间的差异无统计学意义（Z=0.837,P=0.3816〉0.05）.3种实时荧光定量PCR仪的检测结果也存在差异（x2=23.773,P〈0.001）,其中仪器A、B,仪器A、C之间的差异有统计学意义（Z=5.70,P〈0.001;Z=6.45,P〈0.001）,仪器B、C之间的差异无统计学意义（Z=0.75,P=0.4533〉0.05）.结论 在相同条件下,用不同的核酸提取方法,以及用不同的实时荧光定量PCR仪检测的结果均有差异,在实际工作中要综合考虑评价检测结果.

From Phenotypes to Molecules: Revolutionizing Gut Microbiota Identification Methods

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.

Application of Real-time Fluorescent Quantitative PCR in Plant

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.

Development of a real-time PCR assay(SYBR Green I) for rapid identification and quantification of scyphomedusae Aurelia sp.1 planulae

The complicated life cycle ofAurelia spp., comprising benthic asexually-reproducing polyps and sexually-reproducing medusae, makes it hard for researchers to identify and track them, especially for early stage individuals, such as planulae. To solve this problem, we developed a real-time PCR assay （SYBR Green I） to identify planulae in both cultured and natural seawater samples. Species-specific primers targeting Aurelia sp.1 mitochondrial 16S rDNA （mr 16S rDNA） regions were designed. Using a calibration curve constructed with plasmids containing the Aurelia sp. 1 mt 16S rDNA fragment and a standard curve for planulae, the absolute number of mt 16S rDNA copies per planula was determined and from that the total number ofplanulae per sample was estimated. For the field samples, a 100-fold dilution of the sample DNA combined with a final concentration of 0.2 μg/μL BSA in the PCR reaction mixture was used to remove real- time PCR inhibitors. Samples collected in Jiaozhou Bay from July to September 2012 were subsequently analyzed using this assay. Peak Aurelia sp.1 planula abundance occurred in July 2012 at stations near Hongdao Island and Qingdao offshore; abundances were very low in August and September. The real-time PCR assay （SYBR Green I） developed here negates the need for traditional microscopic identification, which is laborious and time-consuming, and can detect and quantify jellyfish planulae in field plankton samples rapidly and specifically.

A real-time PCR targeted to the upstream regions of HlyB for specific detection of Edwardsiella tarda

Edwardsiella tarda has become one of the most important emerging pathogens in aquaculture industry. Therefore, a rapid, reproducible, and sensitive method for detection and quantification of this pathogen is needed urgently. To achieve this purpose, we developed a TaqMan-based real-time PCR assay for detection and quantification orE. tarda. The assay targets the hemolysin activator HlyB domain protein of E. tarda. Our optimized TaqMan assay is capable of detecting as little as 40 fg of genomic DNA per reaction. A standard curve was generated from the threshold cycle values （y） against log10 （E. tarda genomic DNA concentration） as x. The intra- and inter-assay coefficient of variation （CV） values were less than 2.06% and 1.05% respectively, indicating that the assay had good reproducibility. This method is highly specific to E. tarda strains, as it shows no cross-reactivity to Edwardsiella ictaluri, a member of the same genus, or to nine other fish-pathogenic bacteria species belonging to three other genera. This sensitive and specific real-time PCR assay provides a valuable tool for diagnostic quantitation of E. tarda in clinical samples.

Detection of Syndecan-1 and Heparanase-1 Genes in Esophageal Carcinoma by Quantitative RT-PCR

OBJECTIVE To quantitatively explore the expression of Syndecan-1 and heparanase-1 in esophageal cancer tissue as well as their relationship with the clinicopathological factors, in order to evaluate their roles in tumor invasion and metastasis.METHODS Real-time fluorescence quantitative PCR （Q-PCR） was used to analyze the expression levels of Syndecan-1 and heparanase-1 genes￡？participants included 67 cases with esophageal cancers and 32 healthy volunteers.RESULTS The expression of Heparanase-1 gene in esophageal cancers was higher than that in normal esophageal tissue （P 〈 0.001）, and the expression of Syndecan-1 gene in the normal esophageal tissue was higher compared with esophageal cancers （P 〈 0.001）. The positive rates of Syndecan-1 and Heparanase-1 gene in esophageal cancer were 13.4% （9/67） and 85.1% （57/67）.The expression of Syndecan-1 and Heparanase-1 genes was signifi cantly related to di. erentiation, depth of infi ltration, lymph node metastasis, vessel metastasis, and TNM stages of disease （P 〈 0.05）. In an attempt to measure the association between the 2 agents, this study found that the expression of Syndecan-1 mRNA had a significantly negative correlation with the expression of Heparanase-1 mRNA by using Spearman rank correlation test （OR = -0.572, P 〈 0.001）.CONCLUSION Syndecan-1 and Heparanase-1 play important roles in the invasion and metastasis of esophageal cancer. The reduction of Syndecan-1 and/or the increase of Heparanase-1 may influence the invasion and metastasis of malignant tumors.Thus the combination assay of Syndecan-1 and Heparanase-1 may contribute to the diagnosis and treatment of malignant tumors.

Detection of Survivin mRNA in nasopharyngeal carcinoma by real-time fluorescence quantitative RT-PCR

Objective： To establish the method of real time fluorescence quantitative RT-PCR for detecting the expression of Survivin mRNA in nasopharyngeat carcinoma （NPC） tissues. Methods： The total RNA was extracted from NPC cell line CNE-2 and tissues with Trizol and then been transcribed reversely to cDNA, a method of real time fluorescence quantitative RT-PCR for detecting the expression of Survivin mRNA in NPC tissues had been established, in which chronic nasopharyn-gitis patients＇ nasopharynx tissues treated as control group. Results： The expression of Survivin mRNA all could be detected either in CNE-2 cells, NPC tissues or in chronic nasopharyngitis patients＇ nasopharynx tissues, and there was higher the expression level of Survivin mRNA in NPC tissues than which in chronic nasopharyngitis patients＇ nasopharynx tissues, the difference was significant （P 〈 0.01）. The expression of Survivin mRNA could be detected both in stage Ⅰ ＋ Ⅱ and stage Ⅲ ＋ Ⅳ NPC, and there was no significant difference in relative quantifications of gene expression between these two groups （P 〉 0.05）. There was no relationship between Survivin mRNA expression and age and sex of NPC patients （P 〉 0.05）. Conclusion： Real time fluorescence quantitative RT-PCR is a rapid, effective and high sensitive method for detecting the expression of Survivin mRNA in NPC tissues. The overexpression of Survivin mRNA may play some roles in pathogenesis of NPC.

Detection of PRL-2 gene expression in hepatocellular carcinoma by real-time fluorescence quantitative PCR

Objective:To quantitatively detect the expression level of PRL-2 in primary hepatocellular carcinoma using real-time fluorescence quantitative PCR.Methods:Total RNA isolated from human HCC and liver tissue adjacent to the tumor was reversely transcribed into cDNA.Real-time fluorescence quantitative PCR(Q-PCR) method was used to analyze the expres-sion level of PRL-2 gene.Results:The Q-PCR method was performed successfully to precisely detect RNA level.PRL-2 was expressed in all portal vein tumor thrombosis(PVTT) and HCC,but only in some paratumor tissue.The highest expression level of PRL-2 gene was recorded in PVTT;meanwhile expression level of PRL-2 was higher than that in paratumor liver tis-sues and in HCC(P < 0.01),and it was higher in HCC than that in paratumor liver tissues.Conclusion:The Q-PCR may be the most precise method to quantitatively detect RNA level and can be used in gene expression changes.The PRL-2 gene has higher expression in PVTT than that in HCC and in paratumor liver tissue cells,indicating that it plays an important role in the development and metastasis of the HCC.

Selection of Reference Genes in Saccharopolyspora Spinosa for Real-Time PCR

Reverse transcription quantitative PCR （RT-qPCR） combined with the published genome information of Saccharopolyspora spinosa can allow sophisticated studies about S. spinosa, including Studying the regulation of spinosyn biosynthesis, finding new target genes for engineering, and discovering and exploiting other macrolide secondary metabolites. Studies have demonstrated that appropriate internal control is needed to normalize target genes at transcription levels. However, many studies have shown that no single reference gene is universal for all strains under all experimental conditions. Thus, eight candidate reference genes of three different S. spinosa strains in two different cultures were studied to find suitable reference gene（sl. The number of amplification cycles of these candidate genes was calculated by BestKeeper, NormFinder and geNorm. The results indicated that the most suitable reference genes for normalization during the fermentation of S. spinosa were 16S rRNA and rbL13.

Developmental Changes of the FAS and HSL mRNA Expression and Their Effects on the Content of Intramuscular Fat in Kazak and Xinjiang Sheep

Twenty-four male Kazak sheep and 30 Xinjiang fine wool sheep at different ages were selected to investigate the development-dependent expression levels of fatty acid synthase （FAS） gene and hormone-sensitive lipase （HSL） gene in muscle and their effects on the contents of intramuscular fat （IMF）. Longissimus dorsal muscle was sampled to measure IMF and total RNA was extracted to determine FAS and HSL mRNA expression levels by real-time PCR. The results showed that： l） The IMF content increased continuously with growth and showed significant differences （P 〈 0.05） between different age groups in male Kazak sheep, but in Xinjiang fine wool sheep there was no such difference observed. Furthermore, the IMF contents in Kazak were much higher （P 〈 0.01） than that of the other breed from day 30 to 90. 2） FAS mRNA expression level was the highest （P 〈 0.05） on day 0 in Kazak sheep and then declined with growth, in the other breed the gene showed a d‘ecline-rise-decline-rise＇ expression manner as the animals grew. HSL mRNA expression level had a similar model in two breeds, in Kazak sheep it was the highest on day 0 （P 〈 0.05） and in Xinjiang fine wool sheep on day 30 （P 〈 0.01）, then both decreased after this term. 3） In male Kazak sheep, FAS and HSL mRNA expression level were both negatively related to IMF content （r= -0.485 （P = 0.02）, r= -0.423 （P = 0.05））, and the ratio of FAS/HSL expression exhibited significantly negatively related IMF contents. In male Xinjiang sheep, there were no obvious relationship between FAS and HSL expression and IMF content （P 〉 0.05）.

贵州地方猪骨骼肌中3种microRNAs的表达差异

为探明miR-1、miR-133、miR-206基因在不同品种猪肌肉组织中的表达及其与屠宰性能间的关系,以6月龄大白猪、糯谷猪、关岭猪及黔南黑猪的背最长肌为材料,采用实时荧光定量PCR方法,研究了3种microRNAs(miRNAs)在贵州地方猪种和大白猪背最长肌中的表达差异。结果表明:成年期miR-133的表达量高于另外2种miRNA分子。miR-133在糯谷猪中的表达量是大白猪的1.58倍(P<0.05),与其眼肌面积、花油重高度负相关,与屠宰率高度正相关。miR-206在糯谷猪中的表达量是大白猪的2.23倍(P<0.05),与其瘦肉率高度负相关,与板油重中度正相关。在关岭猪背最长肌中miR-133的表达量最高,是大白猪的2.67倍(P<0.05),miR-133和miR-206的表达量与眼肌面积、胴体重高度正相关,与花油重、板油重和脂肪比负相关;但miR-206的表达量明显低于其他猪种。不同品种猪中miR-133和miR-206存在差异表达,对地方猪品种的骨骼肌生长和脂肪沉积有一定的调节作用。

Analysis of Seed-specificity of Silencing fad_2 Gene Expression in Transgenic Rapeseed Line W-4(Brassica napus L.)

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.

Quantitative Study on Expression of MSTN Gene in Different Tissues of Tibetan Sheep at Different Ages

[Objective] The aim was to investigate the difference in MSTN gene expression in different tissues of Tibetan sheep at different ages.[Method] According to the sequence（NM_001009428.1）published in GenBank,a pair of specific primers was designed to amplify part of cDNA sequence of MSTN by using QRT-PCR technique.The relative expression level of MSTN gene in rennet stomach,rumen,leg muscle and cardiac muscle of Tibetan sheep at different ages were analyzed.[Result] After normalization with β-actin gene,the relative expression level of MSTN gene in the 6-month-old Tibetan sheep was the highest and it was 2.52 times than that in 12-month-old Tibetan sheep（P0.05）,the relative expression level of MSTN gene in leg muscle was the highest among all tissues and it was 3 984.78 times than that in rumen（P0.01）.[Conclusion] The results established theoretical foundation for the correct use of MSTN antibody.

大鳍鳠IgL2型基因cDNA的克隆及表达分析

应用RT和RACE-PCR方法获得大鳍鳠(Mystus macropterus Bleeker)免疫球蛋白轻链(IgL)2型基因cDNA序列,分析了该基因在组织中的表达。该基因cDNA全长为929 bp,包含5'非编码区43 bp,3'非编码区159 bp,开放阅读框720 bp,编码239个氨基酸。编码的氨基酸序列分为可变区(VL)和恒定区(CL)。系统进化树分析显示,大鳍鳠IgL蛋白质序列与斑点叉尾IgL 2型(G型)合为一支,与其它硬骨鱼类IgL2型聚为一簇。组织分布研究表明,大鳍鳠IgL2基因表达量在脾脏最高,其次是鳃和头肾,这明显与大鳍鳠IgL3型基因表达方式不同。注射嗜水气单胞菌后,大鳍鳠IgL2基因转录表达量在脾脏、鳃和头肾都随时间发生变化。IgL2基因转录表达量在脾脏4 d就到达高峰,在鳃1 d就达到峰值。这些应答规律与IgL3基因差异显著,推测,IgL2的表达主要参与鳃、肠和皮肤主导的粘膜免疫。

SOCS-3在大鼠慢性肝损伤中的表达研究

目的通过研究细胞因子信号转导抑制因子-3(Suppressor of cytokine signaling-3,SOCS-3)在慢性肝损伤中的表达,为阐明其在慢性肝损伤中的作用提供实验基础。方法采用CCL4法构建大鼠慢性肝损伤模型,通过实时荧光定量PCR、免疫组化检测大鼠肝脏组织SOCS-3的表达。结果慢性肝损伤组SOCS-3 mRNA相对表达量及SOCS-3蛋白高于对照组,差异有统计学意义(P<0.05)。结论在慢性肝损伤中,随着肝脏损伤加重,因炎症因子持续刺激,SOCS-3表达逐渐增加,提示SOCS-3在慢性肝损伤发生发展过程中起重要作用。