实时荧光定量PCR检测三七SS基因表达的初步实践

运用含有SYBR Green I的Real Time RT-PCR法分析SS基因在一年生三七根、茎、芦头3个部位中转录水平的相对表达差异。统计分析表明SS基因在根中的表达量最高。本研究取得了特异性高、重复性好的结果,标准曲线斜率均在-3.33～-4范围内,扩增效率均在95%～100%之间,熔解曲线分析显示产物特异性的单一峰,为Real Time RT-PCR技术用于三七植物基因的差异表达分析建立了相应的技术平台。     

SYBR Green实时荧光PCR高通量检测转基因大豆油

采用SYBR Green实时荧光PCR技术,建立了食用大豆油转基因成分的检测方法.根据转基因大豆中内源参照基因lectin和外源基因35S启动子、NoS终止子和ep4 epsps基因,设计特异性引物,在Roche荧光PCR仪上进行实时荧光PCR扩增.荧光曲线表明,SYBR Green实时荧光PCR可特异性地检测大豆油中的转基因成分,方法准确、快速,并运用熔解曲线进行产物分析,验证了试验结果的特异性和准确性,检测方法灵敏度高.     

食品中阪崎肠杆菌快速检测鉴定方法的研究

通过设计特异性的引物,采用SYBR Green I实时荧光PCR,经引物的优化筛选、特异性和重现性试验,以及模拟污染样品检验,建立了食品中阪崎肠杆菌的快速检测和鉴定方法。     

一种基于免费在线工具的定量PCR引物设计方法

定量PCR(real-time polymerase chain reaction)技术在分子生物学领域被广泛应用,其中SYBR Green染料法因其简便、经济,深受分子生物学家和检测机构青睐。本研究介绍一种针对SYBR Green染料法的定量PCR引物设计方法,该方法基于免费在线工具的组合设计定量PCR引物,通过Primer Quest、Primer-BLAST、UNAFold以及Beacon Designer TM Free Edition获得最佳引物。     

卡他莫拉菌实时荧光PCR检测方法的建立

[目的]建立基于SYBR GreenⅠ染料法的卡他莫拉菌实时荧光PCR检测方法。[方法]选取卡他莫拉菌两个的基因(uspA1和copB)设计特异性引物;提取卡他莫拉菌、嗜肺军团菌等11种呼吸道病原体的DNA,通过常规PCR和实时荧光PCR对引物的特异性进行验证;以卡他莫拉菌DNA为模板进行实时荧光PCR,获取扩增曲线、标准曲线、熔解曲线和熔解峰图,并判断检测方法的灵敏度;进行重复性试验,评估检测方法的组内和组间重复性;通过模拟临床样本,对检测方法的灵敏度进行验证。[结果]共设计出3对引物,对嗜肺军团菌等10种呼吸道病原体具有特异性;对卡他莫拉菌的最低检出浓度为1.0×10^(3)cfu/mL;组内和组间最大变异系数分别为1.78%、1.89%;模拟的临床试验结果与预期相符。[结论]成功建立了卡他莫拉菌的实时荧光PCR检测方法,与10种常见的呼吸道病原体无交叉反应,且重复性变异系数小于2%,模拟临床试验灵敏度结果达到1.0×10^(3)cfu/mL。     

实时荧光定量PCR方法检测小RNA

<正>Real-time QuantitativePCR Detecting System,即实时定量核酸扩增检测系统,也称为实时定量基因扩增检测系统,简称定量PCR(qPCR)。荧光定量PCR一般分为非特异性荧光定量PCR和特异性荧光定量PCR。本文主要介绍一种应用非特异性荧光定量PCR(以SYBR Green I为例)方法对小RNA进行定量分析。SYBRGreen I qPCR分析小RNA的优点为:实验设计简单,一般需要一条特异性反转录引物,一条特异性正向PCR引物,反向PCR引物可通用于所有小RNA分析,无需象TaqMan方法那样专门设计探针;实验成本相对较低;可通过溶解曲线分析来检测扩增反应的特异性。这些特点有利于初学者掌握该技术,而对于一般的分析也可以完全达到实验目的。操作方法如下:     

腐皮镰刀菌SYBR Green实时荧光定量PCR快速检测方法的建立

目的建立一种能够快速、灵敏、特异的鉴定腐皮镰刀菌的SYBR Green实时荧光定量PCR。方法运用SYBR Green实时荧光定量PCR反应体系检测腐皮镰刀菌,并对此方法的特异性、灵敏度和稳定性进行评价。结果通过对45例样品的检测,结果显示SYBR Green实时荧光定量PCR特异性好,其检出率高于普通PCR;灵敏度高,对重组质粒标准品的检测灵敏度为1.0×10~2copies/μL;稳定性好,对质粒为1.0×10~7copies/μL、1.0×10~5copies/μL、1.0×10~3copies/μL的标准品重复检测10次,结果显示扩增反应Ct值的变异系数为0.96%~1.68%。结论SYBR Green实时荧光定量PCR检测腐皮镰刀菌,不仅特异性好,灵敏度高,稳定性好,而且简便、快速、易操作。     

大鼠尿激酶SYBR Green I real time RT-PCR引物的设计

设计用于SYBR Green I法实时定量逆转录多聚酶链反应(QRT-PCR)检测大鼠尿激酶型纤溶酶原激活因子(uPA)mRNA的引物。从基因库获取靶基因及相关序列,充分收集争分析相关生物信息学数据,应用Oligo 6.22设计出一对长度为21bp的引物,其GG含量为52.4%;上下游引物3’最稳定二聚体和及发夹结构的能量分别为-1.5、-0.40 kcal/mol和-3.5、-O.90 kcal/mol,引物间最稳定二聚体为-3.1 kcal/mol。5’端和中间△G值较高,高于3’端△G;引发效率分别455和403。实验证明,该引物能够高效、特异地实现对靶序列的检测,适用于SYBR Green I法实时定量检测(uPA)mRNA。     

关于用定量PCR方法测定基因的转录水平的几个问题

<正>定量PCR的方法已经广泛应用于生命科学各学科的研究。作者发现有些用SYBR Green I的定量PCR的工作不甚规范,影响了实验结果的准确性,这里结合实验从原理说明SYBR Green I的定量PCR的应用方法。此外,生物学研究中大量采用了相对测定,然后对结果归一化的策略。这里我们分析相对测定策略存在的不足,并介绍每拷贝基因的转录水平的概念和双外参跟踪标定法。     

鸭冠状病毒实时荧光定量PCR检测方法的建立及应用

为建立一种鸭冠状病毒（Duck coronaviruses,DuCoV）的临床快速诊断方法，根据鸭冠状病毒1b基因保守区域设计特异性引物，成功建立了用于检测鸭冠状病毒的特异性SYBR GreenⅠ荧光定量PCR方法。该方法特异性强、敏感性高、重复性好，对鸭冠状病毒有特异性扩增，最低检测限为8.04×100拷贝/μL，比普通PCR方法敏感10倍，批内变异系数与批间变异系数分别为0.28%～0.34%、0.25%～0.36%，均小于1%。对临床可疑鸭泄殖腔拭子进行检测，本方法与常规PCR方法的检测结果阳性符合率为100%，阴性符合率为96.43%，样本总符合率为97.62%。本研究建立的鸭冠状病毒SYBR GreenⅠ实时荧光定量PCR方法，可用于鸭冠状病毒的临床快速诊断及流行病学监测。     

EasyExonPrimer

EasyExonPrimer is a web-based software that automates the design of PCR primers to amplify exon sequences from genomic DNA. EasyExonPrimer is written in Perl and uses Primer3 to design PCR primers based on the genome builds and annotation databases available at the University of California, Santa Cruz (UCSC) Genome Browser database (http://genome.ucsc.edu/). It masks repeats and known single nucleotide polymorphism (SNP) sites in the genome and designs standardised primers using optimised conditions. Users can input genes by RefSeq mRNA ID, gene name or keyword. The primer design is optimised for large-scale resequencing of exons. For exons larger than 1 kb, the user has the option of breaking the exon sequence down into overlapping smaller fragments. All primer pairs are then verified using the In-Silico PCR software to test for uniqueness in the genome. We have designed >1000 pairs of primers for 90 genes; 95% of the primer pairs successfully amplified exon sequences under standard PCR conditions without requiring further optimisation. AVAILABILITY: EasyExonPrimer is available from http://129.43.22.27/~primer/. The source code is also available upon request. CONTACT: Xiaolin Wu (forestwu@mail.nih.gov).     

粪便中肠球菌SYBR GreenI荧光定量PCR检测方法的建立

目的利用SYBR GreenI荧光定量PCR方法,建立肠球菌实时荧光PCR检测方法,并初步应用于粪便中肠球菌的检测。方法根据GenBank发表的肠球菌23S rRNA基因序列的保守区域设计合成特异性的引物;利用构建的质粒标准品绘制两种标准曲线,构建基因拷贝数、细菌数为分析指标的定量分析模型并初步应用于粪便标本的检测分析。结果所建立的SYBR GreenI荧光定量PCR方法检测灵敏度可达7个拷贝数/reaction。粪便样本根据实时荧光定量PCR方法所得的理论数值与培养菌值之间差异无显著性(P>0.05)。非炎性腹泻标本中菌数与健康成人标本中菌数差异无显著性(P>0.05)。灵敏度曲线所得的数值大于菌数标准曲线,可能由于DNA提取过程中存在部分的损失。检测粪便标本结果显示SYBR GreenI荧光定量PCR方法较平板计数法敏感、快捷、简便。结论本研究建立了一种灵敏、特异、简便易行的肠球菌定量检测方法。     

Comparison between two standardized cultural methods and 24 hour duplex SYBR green real-time PCR assay for Salmonella detectionin meat samples

Food-borne diseases caused by Salmonella represent a worldwide public health problem. Salmonella must be absent in an established amount depending on the kind of the product and usually cultural methods have to be applied to evaluate the compliance of the products. ISO 6579:2002 in Europe and FSIS MLG 4.04.:2008 in the USA have usually been employed to detect Salmonella in meat, poultry and egg products. A Real Time PCR method using probes has recently been validated against the NMKL (Nordic Committee on Food Analysis) standard method. This method has been modified using the less expensive Sybr Green Real Time PCR approach and applied directly in the 18 hours preenrichment broth for the purpose of detecting Salmonella in meat products in less than 24 hours. The purpose of this study was to: - compare the effectiveness of ISO and FSIS cultural methods; - develop a new 24 hour duplex Sybr Green Real Time PCR-melting curve analysis; - evaluate the performance of Salmonella, Standard Method, Rapid Method, SYBR Green Real Time PCR. The equivalence between ISO and FSIS methods was demonstrated and the use of SYBR Green Real Time PCR as a screening tool for negative results seems appealing especially to evaluate compliance with the HACCP systems.     

Characterization of Fe-hydrogenase genes diversity and hydrogen-producing population in an acidophilic sludge

According to the DNA sequences of six Fe-hydrogenase genes (FHG) of Clostridium species retrieved from the GenBank, a set of primers specific for Fe-hydrogenase genes were identified from their common conserved regions. The length of DNA fragments amplified using these two primers averaged 313 bps. This primer set was then used to investigate the FHG diversity in an acidophilic rice-degrading sludge by methods based on polymerase chain reaction (PCR). Eight new Fe-hydrogenase gene fragments were identified from the sludge, as a result. Similarity based on amino acids among the 14 hydrogenase genes (8 newly found plus 6 known ones) was 39-97%, which is comparable to the similarity of 41-82% among the 6 known hydrogenase genes alone. The low similarity indicates a great diversity on Fe-hydrogenase among the Clostridium species. The primer set was then used to monitor the change of hydrogen-producing microbial population in a batch reaction using the technique of quantitative real-time polymerase chain reaction (qRT-PCR) with SYBR Green I as the fluorescent reagent. Results showed that the hydrogen producers had an average generation time of 4.2h, and a production rate of 7.0 x 10(16) H2-molecule cell(-1)h(-1).     

ELXR: a resource for rapid exon-directed sequence analysis

ELXR (Exon Locator and Extractor for Resequencing) streamlines the process of determining exon/intron boundaries and designing PCR and sequencing primers for high-throughput resequencing of exons. We have pre-computed ELXR primer sets for all exons identified from the human, mouse, and rat mRNA reference sequence (RefSeq) public databases curated by the National Center for Biotechnology Information. The resulting exon-flanking PCR primer pairs have been compiled into a system called ELXRdb, which may be searched by keyword, gene name or RefSeq accession number.     

RTPrimerDB: the real-time PCR primer and probe database

The real-time polymerase chain reaction (PCR) methodology has become increasingly popular for nucleic acids detection and/or quantification. As primer/probe design and experimental evaluation is time-consuming, we developed a public database application for the storage and retrieval of validated real-time PCR primer and probe sequence records. The integrity and accuracy of the data are maintained by linking to and querying other reference databases. RTPrimerDB provides free public access through the Web to perform queries and submit user based information. Primer/probe records can be searched for by official gene symbol, nucleotide sequence, type of application, detection chemistry, LocusLink or Single Nucleotide Polymorphism (SNP) identifier, and submitter's name. Each record is directly linked to LocusLink, dbSNP and/or PubMed to retrieve additional information on the gene/SNP for which the primers/probes are designed. Currently, the database contains primer/probe records for human, mouse, rat, fruit fly and zebrafish, and all current detection chemistries such as intercalating dyes (SYBR Green I), hydrolysis probes (Taqman), adjacent hybridizations probes and molecular beacons. Real-time PCR primer/probe records are available at http://www.realtimeprimerdatabase.ht.st.     

Rapid duplex PCR assay for the detection of pathogenic Yersinia enterocolitica strains

For the detection of pathogenic Yersinia enterocolitica strains, a duplex PCR has been developed based on differences observed between the fingerprint profiles of pathogenic and non-pathogenic strains. The profiles were obtained by using a primer derived from the Enterobacterial Repetitive Intergenic Consensus (ERIC) sequences. From the sequence of one pathogen-specific amplified fragment, a discriminative primer has been designed bridging the sequence of the highly conserved core region and 3' end of the ERIC element. In combination with three other primers, all located within the detected open reading frame that resembled the sequence of the bipA gene, this primer was applied in a duplex PCR assay to simultaneously detect Y. enterocolitica and to discriminate between pathogenic and non-pathogenic strains. The same primer combinations were used in an on line rapid cycling real-time PCR assay. The used SYBR Green I format allowed for the easy translation of the PCR conditions and confirmation of the resulting amplicons. The time of analysis was reduced to approximately 60 min.