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| 1 | 实时荧光定量PCR技术及其应用显示文摘实时荧光定量PCR技术是一种多色荧光检测核酸定量技术 ,该文简要介绍实时荧光定量PCR技术的原理及其应用。 | 欧阳松应 杨冬 欧阳红生 马鹤雯 | 2004 | 生命的化学2004，24，1： | 156 |
| 2 | 柑桔黄龙病的常规PCR及荧光定量PCR检测显示文摘目的为柑桔黄龙病的早期诊断和寄主体内病原菌的动态监测提供一种稳定、可靠的检验检疫技术。方法利用亚洲韧皮杆菌核糖体蛋白基因rplJ/rplL设计了2对PCR引物CQULA03F/CQULA03R、CQULA04F/CQULA04R和1条TaqMan探针CQULAP1,以此为基础建立了常规PCR和TaqMan探针法、SYBRGreenI荧光染料法两种荧光定量PCR(共3种)反应体系;确定了3种体系各自的检测灵敏度、特异性和准确性,据此对3种体系进行了比较;从2004年7月到2005年5月还利用常规PCR和TaqMan探针荧光定量PCR体系完成了对柑桔黄龙病病原菌在寄主体内的周年变化的动态监测。结果两种荧光定量PCR方法的灵敏度比常规PCR高出至少2~3个数量级,而TaqMan探针法由于使用了杂交探针,其特异性尤其可靠,另外两种定量PCR较小的产物片段使得它们具有更好的稳定性,加上荧光定量PCR方法本身受污染可能性小、操作简便等固有优势,使得前者更适合于柑桔黄龙病的检测。结论本研究建立的2种荧光定量PCR方法可以为柑桔黄龙病的病害早期诊断以及柑桔黄龙病病原菌近缘种的甄别提供准确、灵敏、快速的检验检疫技术。 | 胡浩 殷幼平 张利平 赵云 夏玉先 王中康 覃健 | 2006 | 中国农业科学2006，39，12： | 57 |
| 3 | 实时荧光定量PCR及其在微生物生态学中的应用显示文摘定量描述微生物群落的组成,在微生物生态学的许多研究领域都是非常重要的。然而由于可培养技术的局限性,定量描述微生物群落成为比较困难的事情。最近包括PCR技术在内的分子生物学技术为人们提供了有力的工具,使对微生物群落的分布、丰度等有了进一步的了解。实时荧光定量PCR技术作为核酸定量检测技术,自从发明以来在微生物生态学研究中逐渐得到了广泛的应用。从微生物生态学角度,综述了实时荧光定量PCR技术的原理、发展、优缺点及其在微生物生态学研究中的应用与研究进展,并探讨了实时荧光定量PCR技术的发展和应用前景。 | 张晶 张惠文 张成刚 | 2005 | 生态学报2005，25，6： | 43 |
| 4 | 利用实时荧光定量PCR法检测转基因水稻外源基因拷贝数的研究显示文摘转基因植物中外源基因拷贝数是影响目的基因表达水平和遗传稳定性的重要因素,因此外源基因拷贝数的检测成为转基因研究的关键.利用高通量、快速、灵敏的SYBR GreenⅠ荧光定量实时PCR法,检测了转大麦烟酰胺合成酶基因(NAS1)水稻中外源基因拷贝数.以蔗糖磷酸合成酶基因(SPS)作为水稻的内源参照基因,通过梯度稀释法,分别获得了NAS1和SPS基因的Ct值与起始模板数的相关性标准曲线,相关系数分别为0.99976和0.99571,相关性高.通过目的基因NAS1和水稻内源参照基因SPS起始模板数的比较,获得了目的基因在转基因水稻中的拷贝数,在8株转基因株系中,1株为假阳性,1株拷贝数为1,3株拷贝数为2,其余3株拷贝数分别为3、4和7,而阴性对照拷贝数为0.这种方法快速、简便、准确,可以满足转基因育种工作中对后代优良株系的选择. | 王育花 赵森 陈芬 肖国樱 | 2007 | 生命科学研究2007，11，4： | 48 |
| 5 | 虾肝肠胞虫(Enterocytozoon hepatopenaei)实时荧光定量PCR检测方法的建立及对虾样品的检测显示文摘根据Gen Bank中公布的虾肝肠胞虫(Enterocytozoon hepatopenaei)(EHP)SSU r DNA序列设计1对特异性引物,建立并优化了EHP的SYBR Green I实时荧光定量PCR(q PCR)检测方法。结果显示,该方法在60℃的退火温度时扩增效果最好,产物的熔解曲线为1个单峰,构建的方法对8.3×101–8.3×108 copies/μl的EHP SSU r DNA片段的检测响应具有良好的线性关系,扩增产物阈值循环数(Ct)与模板起始量的对数[log(Sq)]的关系为Ct=–3.369 log(Sq)+39.364(R2=0.992),扩增效率为98.1%,检测灵敏度下限为8.3×101 copies/μl,在线性范围内具有良好的组内和组间重复性。对实际样品的检测表明该方法比已报道的套式PCR的检测灵敏度约高4倍。利用本方法对采集自江苏、海南和山东的3批凡纳滨对虾样品的肝胰腺组织DNA(Hp DNA)中的EHP SSU r DNA进行了q PCR检测,结果显示,EHP的载量指数与对虾生长速率呈负相关关系,肝胰腺中EHP载量在103 copies/(ng Hp DNA)时代表了较高的风险水平。本研究建立的q PCR方法具有特异、灵敏、快速、定量的优点,所建立的方法及检测数据可为EHP的防控提供技术参考。 | 刘珍 张庆利 万晓媛 马芳 黄倢 | 2016 | 渔业科学进展2016，37，2： | 45 |
| 6 | 四步法消除SYBR GreenⅠ实时定量RT-PCR中引物二聚体的影响显示文摘为建立一种新的SYBRgreenⅠ实时定量RT PCR方法 ,使之能够有效消除引物二聚体 (PDs)对实时定量结果的影响 .对RT PCR特异性扩增产物和PDs分别进行了凝胶电泳检测和熔解曲线分析 .依据PDs和扩增产物的熔解温度 (Tm)特点 ,在通用的三步法的延伸步骤之后 ,增加一个短暂的 (5s)恒温和荧光检测步骤 ,使这个步骤的温度高于PDs的Tm,但低于扩增产物的Tm,简称该法为四步法 .PDs的Tm 通常高于 72℃ ,但低于扩增产物的Tm .将四步法第四步的温度设置在高于PDs的Tm ,但低于扩增产物的Tm 时 ,四步法能够有效地消除PDs的影响 .对三步法和四步法SYBRgreenⅠ实时定量RT PCR进行了比较 ,发现三步法根本不能用于RNA的实时定量 ,而四步法能够实现包括低丰度RNA在内的RNA的定量 .选择Tm 值尽可能小的引物 ,使PDs与扩增产物Tm 值之间有足够的差距 ,将更有利于四步法的应用 。 | 张驰宇 张高红 杨敏 贲昆龙 | 2004 | 中国生物化学与分子生物学报2004，20，3： | 47 |
| 7 | Indocyanine green clearance test and model for end-stage liver disease score of patients with liver cirrhosis显示文摘BACKGROUND:The indocyanine green(ICG)clearance test(clearance rate(K)and retention rate at 15 minutes (R15))is a sensitive indicator to evaluate liver function. The model for end-stage liver disease(MELD)score has emerged as a useful tool for estimating the mortality of patients awaiting liver transplantation and has recently been validated on patients with liver diseases of various etiologies and severity.In this study,we investigated the correlation between the ICG clearance test and MELD score of patients with liver cirrhosis. METHODS:From June 2007 to March 2008,52 patients with liver cirrhosis admitted to our center were classified into Child-Pugh class A(8 patients),B(14)and C(30).The ICG clearance test(K value and R15)was performed by ICG pulse spectrophotometry(DDG-3300K),and the MELD scores of patients were calculated. RESULTS:As the Child-Pugh classification of liver function gradually deteriorated,the K value decreased, while R15 and MELD score increased.There were significant statistical differences in K value,R15 and MELD score in patients with different Child-Pugh classifications. Significant correlations were found between the parameters of the ICG clearance test(K value and R15)and MELD score.A negative correlation was observed between K value and MELD score(r=-0.892,P<0.05),while a positive correlation was observed between R15 and MELD score(r=0.804,P<0.05).CONCLUSIONS:The ICG clearance test and MELD score are good parameters for evaluating liver function.Moreover, K value and R15 have significant correlations with MELD score,especially the K value,which may be a convenient and appropriate indicator to evaluate liver function of patients with liver cirrhosis. | Sheng, Qin-Song Lang, Ren He, Qiang Yang, Yong-Jiu Zhao, De-Fang Chen, Da-Zhi | 2009 | Hepatobiliary & Pancreatic Diseases International2009，8，1： | 37 |
| 8 | 实时荧光定量PCR技术在转基因检测中的应用显示文摘实时荧光定量PCR技术是一种新型的核酸定性、定量检测、分析技术。随着转基因产品大规模商业化,转基因生物的环境安全和食用安全性问题引起了全球范围内广泛的争论和关注。实时监测、定量准确、灵敏度高、反应速度快、重复性好及PCR反应后不需电泳检测等优点,使RQ-PCR逐步成为转基因检测的重要工具。 | 敖金霞 高学军 仇有文 郭士成 | 2009 | 东北农业大学学报2009，40，6： | 29 |
| 9 | Green chemistry of chromate cleaner production显示文摘Research topics and methods of green chemistry in chromate production are introduced in this paper . New original green chemical process of the heterogeneous reaction and separation system of liquid phase oxidation of chromite in molten salt of sodium hydroxide-one way separation in high concentration medium-metastable phase separation-carbonate recycle conversion has been developed. The green commercial process for comprehensive utilization of mineral resources-reactant recycle inside the process-zero emission was established . | 张懿 李佐虎 齐涛 王志宽 郑诗礼 | 1999 | Chinese Journal of Chemistry1999，17，3： | 27 |
| 10 | 抗草甘膦转基因大豆PCR定量检测研究显示文摘试验建立大豆中转基因成分的定量测定方法。采用双链DNASYBRGreenI结合染料实时荧光定量PCR技术,扩增编码大豆凝集素的内参照基因lec和抗草甘膦转基因大豆中的外源花椰菜花叶病毒CaMV35s基因,绘制2种基因扩增的循环数拷贝数标准曲线图,根据标准曲线方程计算样品中的转基因含量;并作重现性和熔解曲线分析。结果表明,lec和CaMV35s基因标准曲线方程线性好,R2值分别达到0.9997和0.9992。不同转基因含量的标准及模拟大豆样品定量显示,实测值与实际值接近,相对偏差5%~11%。SYBRGreenI实时定量PCR方法可用于定量测定大豆中转基因品种的含量。 | 朱元招 尹靖东 李德发 王凤来 | 2005 | 中国农业大学学报2005，10，3： | 27 |
| 11 | 基因宏阵列和荧光定量PCR方法对西瓜枯萎病害土壤中尖孢镰刀菌的快速检测和定量显示文摘采用基因宏阵列(Macroarray)和荧光定量PCR(Real-time PCR)的方法,对引起西瓜枯萎病害的病原菌尖孢镰刀菌(Fusarium oxysporum)进行了快速监测和快速绝对定量,并且对实验条件进行了优化和摸索。结果显示,在西瓜枯萎病害发病较为严重的处理N-+P-和N-+P+的根际土壤中,Macroarray检测到强烈的阳性信号,表明尖孢镰刀菌的大量存在,同时荧光定量PCR的结果也表明在这两个处理的根际土壤中尖孢镰刀菌数量最多,分别为每g土壤8.89×105和2.24×105个拷贝数;而在未发生枯萎病害的处理N++P-和N++P+中,Macroarray未检测到阳性信号,根际土壤中尖孢镰刀菌数量分别为每g土壤6.23×103和3.28×103个拷贝数;而四个处理的土体土壤中尖孢镰刀菌的数量均维持在每g土壤102~103个拷贝数,Macroarray未检测到阳性信号。与传统检测方法如病原菌的分离培养、平板稀释计数等相比,上述分子生物学方法对病原真菌的检测和定量更为准确快速、省时省力。 | 赵爽 罗佳 凌宁 徐大兵 朗娇娇 胡江 沈其荣 | 2010 | 土壤学报2010，47，4： | 26 |
| 12 | A new scoring system for assessment of liver function after successful hepatectomy in patients with hepatocellular carcinoma显示文摘BACKGROUND:Whether a major liver resection is safe has been judged mainly from the patient's hepatic reserve.However,a safe limit for liver resection does not exist yet.This study aimed to construct a new scoring system as a guide to determine a safe limit for liver resection and avoid liver dysfunction after hepatectomy.METHODS:Eighty-six patients with hepatocellular carcinoma who had undergone hepatectomy in West China Hospital from March 2007 to June 2010 were reviewed.The patients were classified according to the levels of total bilirubin after hepatectomy and the parameters in the perioperative period were compared.Receiver operating characteristic (ROC) analysis was made to assess the liver function compensatory (LFC) value to predict liver dysfunction of the patients after hepatectomy.LFC value is defined as the preoperative KICG value×22.487+standard remnant liver volume (SRLV)×0.020.RESULTS:Patients were classified into group Ⅰ (normal group,n=69) and group Ⅱ (with total bilirubin >85.5 μmol/L for 7 days after hepatectomy,n=17) based on the levels of total bilirubin after hepatectomy.Group II was further divided into two subgroups:recovered subgroup (n=14) and fatal subgroup (n=3).There were no significant differences in preoperative data or intraoperative findings except the indocyanine green test parameters (KICG and ICG R15) and SRLV.ROC analysis showed that the sensitivity and specificity of an LFC value ≤13.01 were 94.1% and 82.6% respectively for predicting liver dysfunction of the patients after hepatectomy.CONCLUSIONS:The LFC value appears to be a good predictor of postoperative liver dysfunction in patients who undergo hepatectomy for HCC.An expected LFC value of 13.01 seems to be a safe limit for liver resection. | Zheng-Gui Du,Bo Li,Yong-Gang Wei,Jie Yin,Xi Feng and Xi Chen Department of Liver and Vascular Surgery,West China Hospital,Sichuan University,Chengdu 610041,China | 2011 | Hepatobiliary & Pancreatic Diseases International2011，10，3： | 23 |
| 13 | 荧光定量RT-PCR法检测运输应激猪热休克蛋白mRNA转录水平显示文摘目的探讨应激时热休克蛋白mRNA转录水平的变化。方法根据HSP90、HSP70和GAPDHmRNA序列,设计并合成引物,将PCR扩增的基因片段克隆到pGEM-T载体,重组质粒经筛选、鉴定,体外转录出RNA,梯度稀释作为阳性模板,用于标准曲线的制定和样品检测,建立了一步法实时荧光定量RT-PCR技术平台,并用于检测长途运输猪心脏和肝脏中HSP70及HSP90mRNA转录水平的改变。结果经1、2、4、6和10h的运输应激后,组织中HSP70mRNA的转录水平随着运输应激时间的延长一直呈现出上升的趋势,运输应激到10h时HSP70mRNA的转录水平升到最高,肝脏和心脏组织中HSP70mRNA的转录水平分别为对照组的2.5和4.1倍;HSP90mRNA的转录水平则随着运输应激时间的延长呈下降趋势。结论运输应激可以影响HSPmRNA转录水平的变化,并且对不同家族的HSPmRNA转录水平的影响不同;HSP70mRNA的转录水平有望成为猪运输应激的判定指标之一。 | 李玉保 鲍恩东 王志亮 赵茹茜 | 2006 | 中国农业科学2006，39，1： | 23 |
| 14 | 实时荧光定量PCR方法检测大肠杆菌O157:H7显示文摘针对大肠杆菌(Escherichiacoli)O157:H7的致病基因eae设计了特异性引物,并用荧光染料SYBRGreenⅠ进行了实时定量聚合酶链式反应(Real-timePCR)。熔解曲线显示产物特异性较强,无非目的条带和二聚体产生。并对反应程序进行了优化,确定了最佳的反应程序,最终在合适的模板浓度内得出了标准曲线。结果显示Real-timePCR比普通PCR灵敏1000倍。 | 陈思 黄昆仑 许文涛 李媛 罗云波 | 2006 | 农业生物技术学报2006，14，5： | 23 |
| 15 | Fundamental Theories and Key Technologies for Smart andOptimal Manufacturing in the Process Industry显示文摘Given the significant requirements for transforming and promoting the process industry, we present themajor limitations of current petrochemical enterprises, including limitations in decision-making, produc-tion operation, efficiency and security, information integration, and so forth. To promote a vision of theprocess industry with efficient, green, and smart production, modern information technology should beutilized throughout the entire optimization process for production, management, and marketing. To focuson smart equipment in manufacturing processes, as well as on the adaptive intelligent optimization of themanufacturing process, operating mode, and supply chain management, we put forward several key scien-tific problems in engineering in a demand-driven and application-oriented manner, namely:intelligentsensing and integration of all process information, including production and management information; collaborative decision-making in the supply chain, industry chain, and value chain, driven by knowledge; cooperative control and optimization of plant-wide production processes via human-cyber-physical in-teraction; and Q life-cycle assessments for safety and environmental footprint monitoring, in addition totracing analysis and risk control. In order to solve these limitations and core scientific problems, we furtherpresent fundamental theories and key technologies for smart and optimal manufacturing in the processindustry. Although this paper discusses the process industry in China, the conclusions in this paper can beextended to the larocess industry around the world. | Feng Qian Weimin Zhong Wenli Du | 2017 | Engineering2017，3，2： | 23 |
| 16 | Indocyanine green kinetics to assess liver function: Ready for a clinical dynamic assessment in major liver surgery?显示文摘Indocyanine green(ICG) kinetics(PDR/R15) used to quantitatively assess hepatic function in the perioperative period of major resective surgery and liver transplantation have been the object of an extensive, updated and critical review. New, non invasive bedside monitors(pulse dye densitometry technology) make this opportunity widely available in clinical practice. After having reviewed basic concepts of hepatic clearance, we analysed the most common indications ICG kinetic parameters have nowadays in clinical practice, focusing in particular on the diagnostic and prognostic role of PDR and R15 in the perioperative period of major liver surgery and liver transplantation. As recently pointed out, even if of extreme interest, ICG clearance parameters have still some limitations, to be considered when using these tests. | Andrea De Gasperi Ernestina Mazza Manlio Prosperi | 2016 | World Journal of Hepatology2016，8，7： | 22 |
| 17 | Ⅰ群禽腺病毒SYBR GreenⅠ荧光PCR检测方法的建立显示文摘根据GenBank中Ⅰ群禽腺病毒六邻体基因的保守序列设计了1对引物,建立了检测Ⅰ群禽腺病毒12个血清型的SYBR GreenⅠ荧光PCR方法。用Ⅰ群禽腺病毒阳性样品进行了特异性、敏感性和重复性试验。结果显示。该方法可检测到每个反应相当于1×10^2个拷贝的标准品DNA,与鸡减蛋综合征病毒、禽呼肠孤病毒、传染性腔上囊炎病毒和传染性支气管炎病毒等非Ⅰ群禽腺病毒不发生交叉反应,具有良好的重复性。对保存的3份Ⅰ群禽腺病毒的细胞培养物进行荧光定量PCR检测,结果都为阳性,检测的病毒含量为1×10^6~1×10^8拷贝/μL。表明.建立的实时荧光定量PCR具有特异、敏感、快速、定量、重复性好等优点,可用于临床上Ⅰ群禽腺病毒感染的检测。 | 文艳玲 谢芝勋 王莹 谢丽基 | 2008 | 中国兽医科学2008，38，9： | 21 |
| 18 | Effects of long-term green manure application on the content and structure of dissolved organic matter in red paddy soil显示文摘Dissolved organic matter(DOM) plays important roles in soil biogeochemistry activity and nutrients transportation in soils, but studies regarding the long-term effects of green manures on the content and structure of DOM in red paddy soil have not been reported yet. A long-term green manure experiment established in 1982 was utilized to test the DOM contents in different treatments, and the spectral characteristics of DOM were investigated by using ultraviolet-visible(UV-Vis) spectrometry and Fourier transform infrared(FTIR) spectrometry. The experiment included four cropping systems: ricerice-milk vetch(RRV), rice-rice-rape(RRP), rice-rice-ryegrass(RRG) and rice-rice-winter fallow(RRF), among them, milk vetch, rape, and ryegrass are popular winter green manure species in southern China. The results showed that the content of dissolved organic carbon(DOC), which is widely used to estimate the concentration of DOM, was significantly promoted after the incorporation of green manures compared with the other sampling stages. The contents of aromatic groups and the degree of humification of DOM increased in RRV and RRP, suggesting more complex compositions of the soil DOM after long-term application of milk vetch and rape. The contents of phenol, alcohol and carboxylic acid group at the mature stage of early rice were significantly higher than those at the stage of after green manures turned over, especially for the RRV treatment. The absorption ratio of FTIR indicated that winter plantation of rape increased the aromatic-C/aliphatic-C ratio, and ryegrass increased the aromatic-C/carboxyl-C ratio. In conclusion, long-term planting of milk vetch and rape as green manures increased the degree of aromaticity, humification and average molecular weight of DOM, and made the DOM more stable in red paddy soil. | GAO Song-juan GAO Ju-sheng CAO Wei-dong ZOU Chun-qin HUANG Jing BAI Jinshun DOU Fu-gen | 2018 | Journal of Integrative Agriculture2018，17，8： | 21 |
| 19 | SYBR Green I实时荧光定量PCR检测小麦纹枯病菌体系的建立和应用显示文摘【目的】纹枯病是小麦生产上重要的土传病害之一,早期准确定量检测病原是病害预测预报及实现有效防控的基础。传统组织分离鉴定方法费时、程序复杂,而且无法做到准确定量。为实现小麦纹枯病早期快速准确定量检测,研究旨在建立小麦纹枯病菌(Rhizoctonia cerealis)的SYBR Green I实时荧光定量PCR(real-time PCR)检测方法。【方法】根据小麦纹枯病菌的β-tubilin序列,设计1对特异性引物,建立并优化SYBR Green I real-time PCR反应体系,利用小麦纹枯病菌、立枯丝核菌(R.solani)、双核丝核菌AG-A和AG-F菌株、小麦全蚀病菌(Gaeumannomyces graminis var.tritici)、小麦根腐病菌(Bipolaris sorokiniana)、小麦茎基腐病菌假禾谷镰孢(Fusarium pseudograminearum)和禾谷镰孢(F.graminearum)等8种小麦根部或土壤病原物的菌丝DNA进行普通PCR和real-time PCR特异性检测,并对灵敏度、可重复性进行评价。利用该体系分别检测接种后5、10、60 d不同接种浓度的盆栽小麦病株。【结果】研究设计的引物特异性强,普通PCR检测结果仅小麦纹枯病菌DNA有扩增条带。Real-time检测结果表明该对引物对小麦纹枯病菌只有唯一的产物吸收峰,对其余供试菌株均未检测到荧光信号。普通PCR检测的灵敏度为6.5×103copies/μL,real-time PCR的灵敏度可达到6.5×102copies/μL。以携带目的基因片段的重组质粒为标准品,构建的real-time PCR标准曲线循环阈值与模板浓度呈良好的线性关系,熔解曲线的吸收峰单一,相关系数为0.997,扩增效率为0.91。对人工接种的盆栽小麦病株进行real-time PCR检测,结果表明与病情指数和接种量分别呈显著正相关。【结论】研究建立的基于real-time PCR技术的小麦纹枯病菌快速检测方法速度快、灵敏度高、特异性强、重复性好。可以用于小麦纹枯病菌检测、指导病害预测和防治。 | 孙炳剑 陈清清 袁虹霞 施艳 李洪连 | 2015 | 中国农业科学2015，48，1： | 21 |
| 20 | 对孔雀石绿的禁用及其思考显示文摘 | 杨先乐 喻文娟 王民权 郑宗林 | 2005 | 水产科技情报2005，32，5： | 20 |
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