应用种特异性PCR技术快速鉴定辣椒实蝇

【目的】辣椒实蝇 Bactrocera latifrons (Hendel)为我国重要的检疫性有害生物,其寄主范围广泛,危害严重。由于传统鉴定方法受到饲养周期、饲养条件、虫态等因素的限制,使得果蔬进出口贸易通关速度、疫情快速鉴定受到较大的影响,因此迫切需要开发关于实蝇的快速鉴定识别的技术。【方法】本研究基于mtDNA COI序列设计了一对能够准确鉴定辣椒实蝇的种特异性引物FL680和RL1057,选用辣椒实蝇作为阳性对照,选用番石榴实蝇B. correcta (Bezzi)、桔小实蝇 B. dorsalis (Hendel)和颜带实蝇 B. cilifer (Hendel)等20种实蝇作为阴性对照,进行PCR扩增并将PCR产物进行电泳检测。【结果】仅目标种辣椒实蝇能够扩增出清晰且单一的约378 bp的条带,其余实蝇种类均未出现条带。将本实验建立的种特异性PCR（SS-PCR）鉴定方法应用于实际检疫工作中并得到了验证,表明该方法具有强的种特异性。【结论】本文提出辣椒实蝇快速鉴定识别技术可应用于实蝇的疫情监测和口岸的检疫检测工作。     

特异性单引物PCR

建立特异性单引物PCR,并用于筛选基因克隆重组子。根据α－globin的一段序列设计引物,按照设计好的特性单引物PCR扩增条有α－globin的重组质粒PLNSX－aglobin,具体扩增条件如下：（1）97℃5min变性后,94℃30s,70℃2min30s,30个循环,制备单链模板;（2）94℃1min,12℃5min,16℃3min,18℃3min,20℃3min,22℃3min,25℃1min,30℃3min,37℃3min,72℃6min,低温条件下引物3′端4－5个碱基与所扩单链模板退火配对,扩增得到一系列不同起始位点但同一序列末端长短一的核酸片段;（3）以上述所得片段为模版进行如下条件的扩增：94℃30min,70℃2min,70℃2min,72℃2min,每个循环增加1s）,45个循环。结果,特异性单引物PCR能够从pLNSX－αglobin上扩增出特异带,可有效用于筛选基因克隆重组子。     

虎物种特异性鉴定的 PCR 方法研究

为了建立可对虎DNA 进行特异性检测的PCR 方法, 应用在野外调查中获得的虎疑似样品和保护执法工作中难以检查辨认的虎产品进行物种鉴定, 从Genbank 数据库下载5 个虎亚种及其它6 种猫科动物和6 种鹿科动物的mtDNA 细胞色素b 基因序列, 并用Wdnasis (V2.5) 软件对上述不同动物的该基因碱基序列进行比较。在此基础上, 综合考虑了设计引物的基本原则, 选择了虎与其它动物碱基序列上差异位点较多的两个片段, 设计出PCR 引物(引物1、2) 。用该对引物分别对从东北虎、华南虎及8 种猫科动物和6 种非猫科动物的肌肉、脏器组织、皮或毛发中提取的DNA 进行PCR (聚合酶链反应) 扩增。结果表明, 所设计的引物对虎DNA 具有特异性,从而达到了对该物种进行特异性检测鉴定的目的。     

酒酒球菌的快速特异性PCR鉴定

以Oenococcus oeni苹果酸-乳酸酶基因(mleA)为目标基因,设计了1对特异性引物PmleaL/PmleaR进行酒酒球菌的快速鉴定研究。结果表明,直接以O.oeni的菌落为模板,通过引物对PmleaL/PmleaR的PCR扩增,可得到mleA基因的特异性条带;用此特异性引物进行供试乳酸菌的PCR鉴定,所有O.oeni菌系均得到特异性条带,而供试的其它种类乳酸菌未扩增出目标带。PmleaL/PmleaR可用于O.oeni的快速PCR鉴定。     

涅斯捷连科氏菌特异性引物PCR分子鉴定

涅斯捷连科氏菌属的建立基于对Micrococcus halobius的再分类,这是一类广泛分布于高盐土壤环境的革兰氏阳性细菌,属放线菌类.在对我国西部盐湖环境放线菌的生物多样性及分类学研究中,大量类似菌株被分离.[目的]为了对其进行快速鉴定,特别是筛选出属于优势类群的涅斯捷连科氏菌,[方法]本研究根据前人以报道的方法设计了一对针对其16S rRNA基因的特异性PCR引物(Nes1/Nes2),[结果]并通过部分典型菌和野生菌株的PCR实验验证,结合16S rRNA基因的测序验证,证明了Nes1/Nes2的PCR反应有效性及其对涅斯捷连科氏菌的特异性.[结论]利用该引物可以快速准确的对涅斯捷连科氏菌进行鉴定.     

PCR引物特异性核查系统(PSC)的构建与应用

聚合酶链式反应(PCR)虽已广泛用于分子生物学研究中,然而PCR实验中的非特异性产物问题将直接影响PCR的效率,在多重PCR实验中更是如此。为了最大限度地降低非特异性产物的出现率,同时避免用户频繁使用Blast比对检查非特异性,我们开发了基于NCBI-Blast的引物评估和模板DNA特异性结合能力评估的核查系统PSC(Primer Specificity Checking,http://biocompute.bmi.ac.cn/PSC),并基于虚拟PCR实验确定了用于引物质量核查计算的多种参数,能够在线提供多个物种的引物特异性核查结果。该系统可以有效地对引物序列可能产生的所有非特异性扩增进行预测,有助于实验前引物优化或者对非特异扩增结果进行解释,最终达到提高PCR效率的目的。     

类群特异性PCR引物设计与评估

本文以真核藻类18SrDNA类群特异性PCR引物的设计与评估为例,详细介绍了如何利用Primrose等一系列程序设计类群特异性PCR引物并评估其敏感性与特异性,并对这一方法的优势与应用类群特异性PCR引物进行群落多样性分析需要注意的问题进行了讨论。     

基于种特异性COI标记的新入侵种甘蓝粉虱快速鉴定技术

【目的】针对粉虱类害虫种类多、体型微小、形态相似、难以准确快速识别的问题,以新入侵我国大陆的甘蓝粉虱 Aleyrodes proletella (L.)为靶标,以田间常见的其他10种/隐种粉虱为参照,采用基于线粒体DNA细胞色素C氧化酶亚基I (mitochondrial DNA cytochrome c oxidase subunit I, mtDNA COI) 基因的种特异性 (species-specific COI, SS-COI) PCR方法,研究其快速分子检测技术。【方法】利用mtDNA COI基因通用型引物LCO-1490/HCO-2198获得甘蓝粉虱及其他常见粉虱的COI序列,根据测序结果设计种特异性SS-COI引物1对（APZYJF/APZYJR）,其扩增片段大小为384 bp,同时对该对引物的种特异性及灵敏性进行检测。【结果】种特异性检验结果显示,该对引物仅对甘蓝粉虱的mtDNA COI基因具有扩增效果,对我国常见的其他种类的粉虱包括温室粉虱 Trialeurodes vaporariorum (Westwood)、柑橘粉虱 Dialeurodes citri (Ashmead)、螺旋粉虱 Aleurodicus disperses (Russell)、双钩巢粉虱 Paraleyrodes pseudonaranjae Martin、非洲伯粉虱 Bemisia afer (Priesner et Hosny)以及烟粉虱B. tabaci (Gennadius)5个隐种（MED, Asia I, Asia II 1, Asia II 3和China 1）等不具有交叉反应扩增能力。灵敏性检验结果显示,该对引物不仅对不同性别的成虫具有良好的扩增效能,对2-4龄若虫甚至单粒卵或单头初孵若虫亦具有同样的扩增能力,其最低检测阈值为14.00±0.37 pg/μL（相当于1/25 600头雌性成虫）。【结论】该技术体系完全可用于甘蓝粉虱的快速准确识别及检测监测,对有效阻截其进一步扩张蔓延意义重大。     

心脏和软骨细胞特异性表达Cre重组酶转基因小鼠的基因型鉴定

我们曾经报道了分别在心脏(α—MHC—Cre)和软骨细胞(Col2A1-Cre)特异性表达Cre重组酶转基因小鼠的成功研制。为了对这2种转基因小鼠进行特异性的基因型鉴定,设计了2对特异性PCR引物,其中一条引物分别位于α-肌球蛋白重链基因(α-MHC)启动子和Ⅱ型胶原(Col2Al)启动子上。以6种不同组织特异性Cre重组酶转基因小鼠基因组DNA为模板,利用设计的特异性引物以及位于Cre编码区的通用引物进行PCR反应。结果显示,2对特异性引物可以分别将心肌细胞特异性和软骨细胞特异性Cre重组酶转基因小鼠与其他组织特异性Cre重组酶转基因小鼠有效区分开来。     

不同牛分枝杆菌特异性基因PCR方法的比较

【背景】牛结核病是我国二类动物疫病,世界动物卫生组织将其列为法定报告的动物疫病。牛主要通过患病牛呼吸道分泌物和咳嗽所产生的气溶胶感染;人则主要通过食用未经高温处理的病牛的肉或奶感染。因此,经过病原学PCR检测对疑似患病牛牛奶或屠宰组织样品进行快速检验确诊,能够最大限度地减少奶牛养殖中乳品生产业的经济损失。【目的】研究并确定适宜的牛分枝杆菌PCR扩增引物及参数,为临床快速准确诊断牛结核病提供参考。【方法】对已报道的5对PCR引物,运用降落(touch down) PCR法确定适宜退火温度(Tm);运用梯度稀释的牛分枝杆菌C68001株(国内牛结核菌素生产用菌株)基因组DNA以及不同菌液含量的人工模拟临床样本(淋巴结、肺脏和牛奶),确定不同引物PCR方法的敏感性;同时以6种常见牛感染菌(牛种布鲁氏菌2308、羊种布鲁氏菌Rev.1、牛分枝杆菌C68001和AN5、禽分枝杆菌C68202、副结核分枝杆菌C68681和胞内分枝杆菌C68226)核酸样本,确定不同引物PCR方法的特异性。【结果】所有引物在53-63℃均含有目的条带,确定引物的最佳退火温度是60℃。在细菌核酸敏感性检验中,1号和3...     

Allele-specific PCR can improve the efficiency of experimental resolution of heterozygotes in resequencing studies

Resolution of the two haplotypes present in an individual that is heterozygous at a locus has been a difficult problem for nucleotide sequence-based population genetic studies. Here, we demonstrate a method in which allele-specific polymerase chain reaction (AS-PCR) and computational phasing are combined for relatively high-throughput, efficient resolution of phase in resequencing studies. Using data from multiple loci that were fully experimentally phased, we demonstrate that the popular computational tool PHASE can accurately phase heterozygous individuals with common SNPs (single nucleotide polymorphisms) and/or common haplotypes. However, we also demonstrate that experimental phasing with AS-PCR can efficiently supplement computational phasing, providing a rapid means to phase individuals with rare SNPs or haplotypes and with heterozygous insertion/deletion polymorphisms. By following simple stepwise procedures, AS-PCR can result in much more efficient and accurate experimental phasing of haplotypes than is possible with traditional methods such as cloning.     

等位基因特异PCR技术的研究与应用

生物的单核苷酸多态性(Single-nucleotide polymorphism,SNP)具有数量多、分布广、易于分型、稳定性强等优点,很适合于用做分子标记.等位基因特异PCR(Allele-specific PCR,AS-PCR)是根据SNP位点设计3'末端与SNP位点碱基互补或错配的特异PCR引物,通过凝胶电泳等方法检测PCR扩增产物的有或无,从而检测基因型中SNP的一种技术.经过不断地改进与完善,基于SNP的等位基因特异PCR标记已逐渐成为一种快速、简便、低成本、可靠、高通量的检测基因型SNP的方法.本文应用等位基因特异PCR技术,根据小麦TaDREB1基因在旱选10和鲁麦14的120(C→A)SNP成功地开发了一个SNP分子标记,证明了该方法的有效性和可行性.     

Efficient validation of single nucleotide polymorphisms in plants by allele-specific PCR,with an example from barley

Although the increasing number of expressed sequence tags (ESTs) from the public domain has facilitated the detection of single nucleotide polymorphisms (SNPs), further validation is needed before they can be used as markers. For SNP validation, we have compared 2 independent methods: (1) the primer extension method followed by capillary electrophoresis on an ABI PRISM 3100 Genetic Analyzer and (2) nested PCR followed by agarose-based visualization. We present an assessment of the efficiency and costs associated with these methods, based on a sample of barley cultivars.     

An Allele-specific PCR Assay for Detecting Azoxystrobin-resistant Alternaria Isolates from Pistachio in California

Alternaria late blight caused by Alternaria spp. in the alternata, tenuissima and arborescens species‐groups is one of the most common fungal diseases of pistachio in California. A single point mutation resulting in the replacement of a glycine by an alanine at codon 143 (G143A) in the mitochondrial cytochrome b (cyt b) has been found in all azoxystrobin‐resistant isolates of these three species from California pistachio. In this study, a pair of allele‐specific polymerase chain reaction (PCR) primers was developed to detect this point mutation. The allele‐specific PCR assay coupled with a rapid DNA extraction method could detect azoxystrobin‐resistant Alternaria isolates in a few hours. The allele‐specific PCR method was also reliable for detecting azoxystrobin‐resistant Alternaria directly in both laboratory‐inoculated and naturally infected pistachio leaves.     

利用动态等位基因特异性杂交技术进行单核苷酸多态高通量分型

动态等位基因特异性杂交(dynamic allele-specific hybridization, DASH)是新发展起来的一种单核苷酸多态(single nucleotide polymorphisms, SNP)等位基因分型技术,具有快速、经济、准确、高通量、重复性好等优点.利用DASH技术,对96份正常人外周血DNA样品成功地进行了两个SNP位点的基因分型,并摸索实验条件,对该技术进行了优化.     

Allele-specific silencing of mutant Huntington's disease gene

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by a poly-glutamine expansion in huntingtin, the protein encoded by the HD gene. PolyQ-expanded huntingtin is toxic to neurons, especially the medium spiny neurons of the striatum. At the same time, wild-type huntingtin has important – indeed essential – protective functions. Any effective molecular therapy must preserve the expression of wild-type huntingtin, while silencing the mutant allele. We hypothesized that an appropriate siRNA molecule would display the requisite specificity and efficacy. As RNA interference is incapable of distinguishing among alleles with varying numbers of CAG (glutamine) codons, another strategy is needed. We used HD fibroblasts in which the pathogenic mutation is linked to a polymorphic site: the Δ2642 deletion of one of four tandem GAG triplets. We silenced expression of the harmful Δ2642-marked polyQ-expanded huntingtin without compromising synthesis of its wild-type counterpart. Following this success in HD fibroblasts, we obtained similar results with neuroblastoma cells expressing both wild-type and mutant HD genes. As opposed to the effect of depleting wild-type huntingtin, specifically silencing the mutant species actually lowered caspase-3 activation and protected HD cells under stress conditions. These findings have therapeutic implications not only for HD, but also for other autosomal dominant diseases. This approach has great promise: it may lead to personalized genetic therapy, a holy grail in contemporary medicine.     

Development of a simple and low-cost real-time PCR method for the identification of commonly encountered mycobacteria in a high throughput laboratory

Aims:  To facilitate efficient identification of commonly encountered mycobacteria species ( Mycobacterium tuberculosis , Mycobacterium avium , Mycobacterium intracellulare , Mycobacterium fortuitum complex , Mycobacterium chelonae/abscessus , Mycobacterium kansasii , Mycobacterium gordonae ) in high throughput laboratories, a 16s rDNA sequence based real-time PCR assay was developed and evaluated.
Methods and Results:  Oligonucleotide primers and hybridization probes were designed based on sequence differences of the mycobacterial 16S rDNA gene. This assay was evaluated with 1649 suspected non-tuberculosis mycobacterial isolates. Apart from 3 out of 40  M. avium isolates that showed false signal with M. intracellulare specific probe, 100% specificity was obtained for all tested probes. Assay sensitivity varied from 88·9 to 100% depending on species. Average cost for obtaining a definite identification was only USD 1·1 with an average turn around time of less than 3 days.
Conclusions:  A rapid, simple and inexpensive real-time PCR assay was developed for the identification of common encountered mycobacteria in a high throughput laboratory setting.
Significance and Impact of the Study:  With this assay, more than 80% of the clinically isolated nontuberculous mycobacteria could be identified in a highly cost effective manner. This helped to save resources for other laboratory activities especially in high throughput mycobacterial laboratories.     

细菌基因组重复序列PCR技术及其应用

细菌中散在分布的DNA重复序列近年来不断被报道,基因外重复回文序列和肠细菌基因间共有重复序列是两个典型的原核细胞基因组散在重复序列。重复序列在染色体上的分布和拷贝数具种间特异性,用它们的互补序列作为引物,以细菌基因组DNA为模板进行PCR扩增反应,反应产物的琼脂糖电泳可以提供非常清晰的DNA指纹图谱,使用此图谱既可对各种微生物进行快速分型及鉴定,又可对它们进行DNA水平上的遗传多样性分析。细菌基因组重复序列PCR技术具有简捷、快速、结果稳定等特点,可对细菌进行分子标记,用于菌株分型、分类鉴定和亲缘关系等方面的研究。     

PCR快速鉴定鼠疫耶尔森氏菌

建立一种简便、快速、特异的PCR检测方法,用于鼠疫耶尔森氏菌的快速鉴定。针对鼠疫耶尔森氏菌特异的一段染色体序列3a设计引物,扩增-276bp片段的鼠疫标识序列。应用该PCR反应体系,对我国17个生态型及1个待定的生态型共计275株鼠疫耶尔森氏菌及48株相关菌株的PCR扩增结果表明,实验菌株均扩增出预期的276bp片段产物带,48株相关菌株均阴性,其检测灵敏度为100pg DNA。说明该方法用于鼠疫耶尔森氏菌的检测鉴定简便、快捷,具有很高的特异性和敏感性。     

GA21转基因玉米实时荧光PCR检测方法的建立

成功建立了实时荧光PCR鉴定检测转基因玉米GA21品系的方法。该方法通过GA21玉米品系的OTPmEPSPS边界的270bp和133bp靶序列,设计品系特异性检测引物和探针,同时针对Pactin1mEPSPS边界的430bp靶序列设计品系特异性检测引物,应用实时荧光PCR和PCR技术,特异性检测GA21玉米品系。结果表明,应用实时荧光PCR的TaqMan探针技术检测转基因作物边界序列,不仅可以达到品系鉴定的目的,而且该方法和常规PCR比特异性强,简便快速,同时实验采用完全闭管检测,又降低了污染机会,为转基因作物的品系鉴定检测提供了新方法。