摘要：【目的】建立蜂蜜中嗜渗酵母菌的快速检测方法,为保障蜂蜜品质提供理论基础。【方法】根据GenBank中嗜渗酵母菌26S rDNA D_(1)~D_(2)基因序列设计特异性引物,pcr扩增获得嗜渗酵母D_(1)~D_(2)基因片段,构建重组质粒pMD-18T-JM为阳性标准品,对荧光定量pcr反应条件进行优化,建立嗜渗酵母菌TaqMan探针实时荧光定量pcr方法,并对160份蜂蜜样品进行验证。【结果】建立的蜂蜜中嗜渗酵母菌TaqMan探针实时荧光定量pcr检测方法特异性好,灵敏度达0.004 pg/μL。TaqMan实时荧光定量pcr检测阳性率达98.5%,比普通pcr检测(阳性率84.1%)高14.4百分点,检测敏感性达100%。【结论】嗜渗酵母菌TaqMan探针实时荧光定量pcr方法特异、灵敏、快速、重复性好,适合于蜂蜜中嗜渗酵母菌的快速检测。

摘要：The duck circovirus （DuCV） infection in sick ducks from Fujian Province was investigated. The liver samples of 43 sick Muscovy ducks with infectious serositis were collected from 12 duck farms in Fujian Province Based on the published sequences of DuCV, two primers were designed for the detection of DuCV and four pairs of primers were designed to amplify four overlapping fragments that cover the complete genome of DuCV. The specific pcr products were amplified from positive samples. The fragments were then cloned into pMD18-T vector and sequenced, and the full length genomic sequence of the FJ0601 isolate of DuCV was obtained. pcr analysis showed that the proportion of ducks which were positive for circovirus was 79% and 10 out of the 12 farms were positive. Sequence analysis showed that the complete genome of DuCV-FJ0601 was 1988 bp and possessed features common to the family Circoviridae which included a stem-loop structure and the Rep protein motifs. Homology analysis showed that FJ0601 isolate of DuCV had 97.3%-97.5% nucleotide sequence identity to all the four Taiwan isolates （TC1/2002, TC2/2002, TC3/2002, TC4/2002）, 82.9% identity to the America （33753-52） isolate and 82.3% identity to the Germany isolate. Phylogenetic analysis with Clustal W, however, showed that FJ0601 isolate of DuCV was on a common branch with Taiwan isolates, and Germany and America isolates belonged to the other branch.

摘要：Objective:To study virulence and regulatory genes(hlyA,ctxB,tcpI) in clinical strains of Vibrio ckolerae(***),***:Three important genes,tepI,hlyA and ctxB were used for detection of toxigenic and pathogenic *** by chain reaction assay method. Results:According to the results of the pcr,the incidence of hlyA,tcpI,and ctxB genes in clinical isolates was obtained as 94.7%(72 sample),90.8%(69 sample),and 92.1%(70 sample), *** strains possessed all genes except ctxB,six strains possessed all genes except tcpI,four strains possessed all genes except hlyA,one strain possessed only hlyA and 60 strains contained a combination of three *** hlyA,ctxB and tcpI,Conclusions:Result show that this method could be reliable to detect toxigenic-pathogenic strains of *** in Iran.

摘要：将对虾白斑杆状病毒的一段特异性DNA设计成分子信标探针 ,用于该病毒的pcr检测 .温度与荧光强度之间的关系表明 ,所设计探针的发夹既可以形成也可以打开 ,符合pcr对分子信标探针的要求 .结果表明 ,在pcr同时加入分子信标探针不影响pcr扩增 ,分子信标探针只能与目的DNA杂交 ,具有较高的特异性 .随着pcr循环数的增加以及含目的DNA的质粒拷贝数的增加 。

摘要：多重pcr技术广泛应用于多个研究领域,其中引物设计及扩增条件是提高多重pcr实验效率的关键因素.为探讨优化多重pcr实验的方法,以小鼠5个看家基因为研究对象,使用实验室新近开发的MPprimer程序设计多重pcr引物,并通过改变多种反应条件来优化多重pcr实验.结果表明,MPprimer程序能够设计出理想的多重pcr引物,并且通过对退火温度及延伸时间进行优化,可显著提高多重pcr实验效率,对于提高基因表达的规模化检测能力具有积极的促进作用.

摘要：本文设计并开发出用于DNA聚合酶链式反应(pcr)和毛细管电泳(CE)分离的微流体芯片分析仪控制系统。本系统采用32位嵌入式微控制器ARM实现pcr扩增所需的3条恒温区的闭环温度控制和毛细管电泳分离功能所需的高压电场自动控制。由于对恒温区温度的精确控制是影响pcr反应的关键因素,因此温度控制系统采用模糊免疫PID算法实现对温度的精确控制,其控制性能优于常规PID控制器。本仪器还包括矩阵式键盘、小型液晶显示屏、串行通信模块和毛细管电泳分离模块。实验结果表明本系统不但完全满足设计要求,而且pcr温度控制更加精确,分析软件功能全面,系统体积小,便于室外使用。

摘要：分析了基因扩增分析(pcr)仪的工作原理及提高pcr仪温度控制精度的技术,详细讨论了温度控制系统的软硬件设计,研制了一种新颖的pcr温度控制系统。

摘要：　选用高分子量麦谷蛋白亚基(HMW-GS)在Glu-D1位点已知的 26个普通小麦品种,包括 15个 (TriticumaestivumL. )川育系列品种及其 11个亲本,根据其亚基Dx5和Dy10基因的特异序列设计引物.通过对Dx5基因的pcr扩增及对Dy10基因的AS-pcr扩增,结果表明,所设计的引物对Dx5和Dy10都能扩增出特异条带,而携有Dx5基因的材料同时也携有Dy10基因.说明用所设计的引物可快速有效的鉴定出普通小麦中是否带有优质的 5 +10亚基.图 4参

摘要：目的建立直接检测粪便样本中溶组织内阿米巴的pcr方法。方法根据溶组织内阿米巴标准株基因组中小亚基核糖体RNA (small subunit ribosome RNA, SSU rRNA)序列合成4对特异性引物(2对自主设计引物和2对参考引物),建立pcr检测方法,用该方法对221例临床腹泻患者粪便标本进行检测,同时采用病原学碘染涂片镜检法和酶联免疫吸附实验(enzyme-linked immunosorbent assay, ELISA)检测抗原,对3种方法的阳性率进行比较,并采用Kappa检验进行一致性分析,分析3种检测方法结果的准确性。结果 4对引物均扩增出溶组织内阿米巴特异的目的片段,建立了粪便样本溶组织内阿米巴检测的pcr法。选择其中2对引物(1对自主设计引物和1对参考引物)对221例粪便样本进行pcr扩增,同时用碘染涂片镜检法查病原体,用ELISA法进行抗原检测,3种方法溶组织内阿米巴阳性检出率分别为2.26%、0.90%和9.50%,差异有统计学意义(χ~2=23.34,P<0.01)。pcr法与碘染涂片镜检法比较,Kappa值为0.216,一致性微弱; pcr法与ELISA法比较一致性差,Kappa值为-0.134。pcr法的结果与临床诊断的一致性最好。结论本研究通过自行设计引物建立的粪便样本溶组织内阿米巴pcr检测法在准确性上优于镜检法和ELISA抗原检测法,为该方法用于临床诊断提供了实验基础。

摘要：本文针对鱼翅中的鲨鱼成分进行检测鉴定开发了一种快速灵敏的pcr检测方法,可检测鱼翅类食品中是否存在鲨鱼成分。根据鲨鱼线粒体的细胞色素亚基I基因序列设计了鲨鱼特异性引物,扩增长度为228 bp;为了评价方法的特异性,将设计的引物分别针对22份鱼翅样品DNA和37种其它种类DNA进行pcr检测,结果显示,只在鲨鱼鱼翅中能检测出特异的228 bp条带,其它37种物种中均无条带检出。为了评价方法的灵敏度,将鱼翅DNA中掺入了不同比例土豆DNA的样品采用本方法进行了pcr分析,显示方法可检测灵敏度为0.1%(m/m)。随机抽取45份不同类型的鱼翅样品,检测出22份鲨鱼翅均含鲨鱼成分,而21份仿鱼翅均不含鲨鱼成分而含有植物成分。该样品前处理方法、DNA提取方法以及pcr检测方法可广泛应用于食品中鲨鱼成分检测鉴定。