转基因棉花中棉41多重PCR体系的建立

利用多重PCR建立转基因棉花中棉41外源基因的检测体系.多重PCR技术的关键在于多重PCR反应条件的优化,本文从多重引物组合、PCR反应体系、PCR反应条件这三个方面对转基因棉花多重PCR反应进行优化,建立了扩增效率较高的中棉41的六重PCR反应体系,优化后的体系为HotstarTaq DNA聚合酶用量为1.25 U/50 μL,镁离子的终浓度为3.5 mM,dNTP的终浓度为400 μM,BSA(10mg/mL)的加入量为2μL,选取58℃为反应扩增的退火温度.利用优化的多重PCR体系特异性地检测到转基因棉花中棉41中的外源基因启动子CaMV 35S、终止子TNOS、报告基因NPTII和抗虫基因CrylAc.     

大黄鱼基因组DNA的提取及其RAPD分析条件的探索

通过实验筛选出一种快速、简便提取大黄鱼基因组 DNA的方法 .以该法提取的大黄鱼基因组 DNA为模板 ,对大黄鱼 RAPD分析中的 DNA模板浓度、镁离子浓度、d NTPs浓度、引物浓度和 Taq酶浓度进行了研究 ,初步确定了适于大黄鱼 RAPD分析的最佳反应体系 :2 5μL反应体系中 ,含模板 DNA2 5 ng,1 0× PCR缓冲液 2 .5μL,d NTPs1 0 0μmol/L,Mg Cl2 2 .5 mmol/L,引物 0 .2μmol/L,Taq DNA聚合酶 1 .0 μmol/min.     

铊膜DC-SQUID磁强计研制成功

高温超导体铊膜DC-SQUID(直流-超导量子干涉仪)磁强计已于1992年12月8日通过专家鉴定。整机磁通灵敏度(稳定值)达到9.3×10~4φ_0/Hz~(1/2),最佳值为3.8×10~(-4)φ_0/Hz~(1/2),磁场分辨率为9pT/Hz~(1/2),为目前同类仪器的国际先进水平。除具有极高灵敏度和广泛应用前景外,因为可以在液氮温度工作,高温超导磁强计比低温超导磁强计使用起来更方便。     

金属氧化物纳米点薄膜的模板法合成

报道了一种简便的金属氧化物纳米点薄膜的合成方法.首先制备了具有有序纳米凹坑阵列的多孔阳极氧化铝模板,然后在模板表面真空蒸镀金属薄膜,对所制备的金属薄膜进行氧化处理,得到了具有有序纳米点阵列的金属氧化物纳米点薄膜.纳米点的直径约为 1 0 0nm,高度约为 45nm,以六边形有序排列,密度约为 2× 1 0 1 3个/m2 .     

青、老年人扩张型心肌病的临床研究

探讨老年人扩张型心肌病 ( DCM)的临床特征并与青年人 DCM进行比较 .通过临床观察对 80例老年人和 140例青年 DCM的临床资料进行对比分析 .结果发现 :( 1)老年组醛固酮值( 30 4 .8± 69.1)较青年组 ( 2 13.3± 54.5,pmol/ L)明显增高 ( P<0 .0 5) ,老年组 T3和 FT3值 ( 0 .78± 0 .2 1,2 .87± 0 .73)较青年组 ( 1.2 6± 0 .33nmol/ L ,3.55± 0 .64pmol/ L )明显降低 (均为 P<0 .0 1) ;( 2 )老年组室性心律失常发生率 ( 61.3% )较青年组 ( 92 .1% )低 ( P<0 .0 1) ;( 3)老年组低血钾、低血镁发生率高 (分别为 51.3%和 2 7.5% ) ,对洋地黄敏感性增加 ,易发生洋地黄中毒( 2 8.8% ) ;( 4 )老年组的病程 [( 11.0± 4 .7)年 ]和平均生存期 [( 6.9± 4 .2 )年 ]均较青年人 [( 5.2±2 .5)和 ( 3.4± 2 .7)年 ]长 (均为 P<0 .0 5) ;( 5)老年组的主要死因是充血性心力衰竭 ( 78.9% ) ,青年组则为恶性心律失常 ( 61.9% ) .结论为老年人 DCM的预后比青年人相对要好 ;老年人 DCM常伴低 T3综合征 ;心力衰竭、电解质失衡及交感神经兴奋是老年人 DCM室性心律失常的主要原因     

五种重组人源IL-18突变子表达质粒的构建及其在BxPC-3细胞中的表达

目的:设计获得5段hIL-18异构体,定向插入pEGFP-C1质粒形成融合基因,转染胰腺癌Bx-PC-3细胞并表达,为进一步研究改建的hIL-18蛋白功能奠定实验基础.方法:设计引物从pGEM-T-hIL-18质粒中PCR扩增得到5段不同的hIL-18片段,分别连接入pEGFP-C1载体构建不同的突变子(Mu0、Mu1、Mu2、Mu3和Mu4),转染原位胰腺癌BxPC-3细胞,以荧光显微镜观察绿色荧光蛋白的表达,RT-PCR法检测转染细胞中hIL-18表达水平.结果:(1)五种重组质粒经酶切与测序证实构建成功;(2)BxPC-3细胞转染重组质粒后可观察到绿色荧光蛋白的表达.结论:(1)成功构建了hIL-18五种异构体的绿色荧光蛋白表达载体;(2)改建的hIL-18蛋白可与绿色荧光蛋白在BxPC-3细胞融合表达.     

HBV标志阳性患者血清HBV-DNA定量测定的探讨

近年来 ,HBV- DNA定量分析的价值受到越来越多研究者的重视。我们对部分 HBV标志阳性患者进行了血清 HBV- DNA定量测定 ,探讨患者血清携带 HBV- DNA检测结果的关系。1　材料与方法1 .1　标本来源375例 HBV标志阳性标本为我院门诊患者。1 .2　试剂和仪器HBV- DNA定量试剂盒购于深圳市达尔安生物有限公司 ,HBV核酸扩增 ( PCR)荧光检测试剂盒、操作严格按说明书进行。仪器为美国 PE公司产5 70 0基因扩增仪。2　结果1 0 2例血清 HBs Ag、抗 HBc及 HBc Ag阳性的患者 ,HBV- DNA定量测定结果 99例大于正常参考值 ( 4 .0× 1 0…     

应重视节水生物技术研究

1　我国及华北地区水资源简况我国每年农业用水占用水总量的 73.4 % (发达国家多在 50 %以下 ) ,当前我国灌溉用水的利用系数为 0 .3— 0 .4 (发达国家为 0 .7— 0 .9)。从ＧＤＰ用水效益上来看 ,我国 1995年用水效益为 10 .7元 /ｍ3 ,是美国 1990年的 1/ 8,日本 1989年的 1/ 2 5(按 1995年汇率计算 )。这些数据说明我国节水潜力很大。北方地区 ,特别是华北的黄淮海地区 ,是我国水资源供需矛盾最突出的地区。据 1997年统计资料 ,黄淮海地区以仅占全国 7.7%的水资源养育着全国34.3%的人口 ,支撑着全国 4 0 %的灌溉面积 ,生产出全国 39.2 %的…     

马传染性贫血病毒gp90蛋白的表达及其在诊断中的潜在应用

以质粒pVRCSV1.0-syn-gp90,利用PCR扩增马传染性贫血病毒外膜蛋白基因(gp90),构建了原核表达质粒pET-28a(+)-gp90,然后转化大肠杆菌Rosetta(DE3),诱导表达并纯化该重组蛋白,ELISA和Westem blot证明马的阳性血清可以识别该重组蛋白.以纯化的融合蛋白免疫新西兰大白兔制备多克隆抗体,测定的效价为1:13,000.     

Apocynaceae系细胞尿苷二磷酸葡萄糖焦磷酸化酶的纯化和表征

经 4步分离纯化的Apocynaceae植物培养细胞尿苷二磷酸葡萄糖焦磷酸化酶(UGPase)的比活为 2 0 4 9U mg ,与细胞粗提液相比 ,活力提高 97倍 ,活力回收率为 2 1 %,可直接用于尿苷二磷酸葡萄糖 (UDPG)的合成 ,并且储存稳定性很好 .UGPase的最适pH值为 7 2 ,UGPase的最适温度为 37℃左右 .Mg2 是UGPase发挥活力所必需的 .高浓度UTP和Glc 6 P均能抑制UGPase的活力     

Accomplishment of one-step specific PCR and evaluated SELEX process by a dual-microfluidic amplified system

One of the main obstacles for systematic evolution of ligands by exponential enrichment (SELEX) failure is the generation of a non-specific product, as selection-inherent amplification procedures tend to form by-products, which prevents the enrichment of target-binding aptamers. Herein, we reported a dual-microfluidic amplified system (dual-MAS) based on the real-time polymerase chain reaction (PCR) detection chip and the large volume PCR chip for one-step specific PCR and for evaluating the SELEX process. First, it is a simple method to accomplish analytical PCR and amplification PCR in one step, and the optimal number of cycles for generating the specific PCR product is the cycles when the slope of the linear amplification period of the real-time PCR curve begins to decrease. Second, the time used by the dual-MAS for generating a specific PCR product is reduced to 30 min, and the multi-functional dual-MAS can simultaneously evaluate the SELEX process by providing important information on the amounts of enriched sequences and the library diversity in every round of SELEX. In addition, pollution contamination and fragment loss can be significantly avoided in the closed chip. Last, the specific PCR product, the amounts of enriched sequences, and the library diversity can be obtained for every single SELEX in just 30 min. Compared with current methods, this system can reduce the time for generating a specific PCR product and SELEX, and it is easier to choose the optimal number of cycles for a specific PCR product. In a word, it is a sensitive, simple, and rapid strategy to improve the specificity of the PCR product and make the process of SELEX in a controlled way.     

Multiplex PCR for rapid detection of exonal deletions in patients of duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is the most common X-linked disorder in children affecting 1 in 3500 males. Since, as of now, we have no treatment for DMD, carrier detection and prenatal diagnosis is the most important preventive strategy. Multiplex PCR helps in rapid detection of hot spot exonal deletions (positive in 65% of cases) as many exons can be identified in a single run. 10 children with characterstic clinical features of DMD and chorionic villus samples of 10 antenatal patients with positive family history were studied. We identified a deletion mutation in exon 49 of the dystrophin gene in a 4 yr old boy referred with signs and symptoms suggestive of DMD using primers for exons 45, 48, 49, 43, 44, 19, 3, 8, 13 and muscle promoter, subjected to multiplex polymerase chain reaction (PCR) and agarose/Nu-Sieve gel electrophoresis. These genetic methods aid in prenatal diagnosis of DMD as well as confirmation of diagnosis in children with signs and symptoms suggestive of the disease. Work done as WHO fellow in Deptt. of Genetics, All India Institute of Medical Sciences, New Delhi.     

PCR技术快速鉴别牛、羊、猪皮革的初步探讨

提取牛、羊、猪3种动物皮革组织中的DNA,设计了3对特异性引物,PCR扩增,初步建立了这4种动物皮革成分的鉴定方法。DNA的提取的质量及其后的PCR鉴定依赖于皮革的加工程度,如何从皮革成品中提取有效的核酸信息需进一步研究。     

不结球白菜品种自交不亲和S基因型的鉴定

为给不结球白菜的品种配置和遗传育种提供理论基础,利用特异性PCR扩增、DNA测序和生物信息学方法鉴定了7个自交系的S基因型,PCR扩增结果表明,51-14、375-124、711-114、884-214等4个自交系的基因型为Class-Ⅰ SLG和SRK,表现为自交强不亲和.523-112和544-312的基因型为Class-ⅡSLG类型.表现为自交若不亲和,和田间试验结果一致.测序结果blast分析表明844-214 Class-Ⅰ型和Brassica rapa SLG56的相似性为99%,51-14的SRK PCR与SRK56的同源性达到100%.     

聚合酶链反应检测乙型肝炎患者血清HBVDNA与ELISA检测HBeAg、抗HBe比较研究

应用聚合酶链反应（ＰＣＲ）检测了１５３例各型乙型肝炎患者的血清ＨＢＶＤＮＡ，并与ＥＬＩＳＡ法检测的血清ＨＢｅＡｇ和抗ＨＢｅ结果比较．结果ＨＢｅＡｇ阳性８９例，ＰＣＲ法ＨＢＶＤＮＡ检出率为９２．１３％；抗ＨＢｅ阳性２７例，ＰＣＲ法ＨＢＶＤＮＡ检出率为５１．８５％；ｅ系统用性３７例，ＰＣＲ法ＨＢＶＤＮＡ检出率为４０．５４％．提示抗ＨＢｅ血清学转换不能作为乙型肝炎病毒复制与非复制、病变活动与静止的唯一指标，而应该用或同时用ＨＢＶＤＮＡ作为指标才可靠．     

Amplification of SPPS150 and Salmonella typhi DNA with a high throughput oscillating flow polymerase chain reaction device

In this paper, a novel oscillating flow polymerase chain reaction (PCR) device was designed and fabricated to amplify SPPS150 and salmonella typhi. In this new design, the samples are shuttled (oscillating flow) inside a microfluidic chip to three different temperature zones required for DNA amplification. The amplification cycle time has markedly been reduced as the reagent volume used was only about 25% of that used in conventional PCRs. Bubble formation and adsorption issues commonly associated to chip based PCR were also eliminated. Based on the performance evaluated, it is demonstrated that this oscillating flow PCR has the advantages of both the stationary chamber and continuous flow PCR devices.     

使用免疫BMPs纯化细菌PCR反应模板的比较

目的:使用免疫BMPs,对含有大肠杆菌O157的粪便标本,进行目标病原菌的捕获和洗净,建立一种快速纯化细菌PCR反应模板的方法。方法:将大肠杆菌O157抗体接种于BMPs表面,制成免疫BMPs。使用免疫BMPs,对含有不同浓度大肠杆菌O157的粪便标本,进行目标病原菌的捕获、分离和洗净,再使用洗净的病原菌制备PCR反应模板。作为对照,对含有不同浓度大肠杆菌O157的粪便标本,常规进行增菌培养及鉴别培养后,制备细菌的PCR反应模板,或粪便标本直接制备PCR反应模板。结果:使用免疫BMPs处理后制备PCR反应模板,与通过常规增菌培养和鉴别培养后制备PCR反应模板,PCR检测结果一致。而粪便标本直接取样制备PCR反应模板,大肠杆菌O157低浓度时,PCR检测的阳性率较低。结论:利用免疫BMPs捕获自然标本(粪便)中的目标病原菌,通过分离和洗净,去除标本中存在的PCR反应抑制物等影响因素,可省略增菌培养及鉴别培养,快速纯化目标病原菌的PCR反应模板。     

Diagnostic Value of PCR in Genitourinary Tuberculosis

Genitourinary tuberculosis is a disease of the genitourinary system which includes the entire urinary tract and reproductive system. Genital tuberculosis is an important cause of female infertility, especially in developing nations like India. In the present study, a total of 257 clinical specimens comprising of endometrial biopsy (109), endometrial curetting (42), menstrual blood (8), semen (17), placenta (11) and urine (70) were collected from patients and subjected for PCR, Culture and AFB detection. The endometrial biopsy, endometrial curetting, menstrual blood, semen, placenta, urine showed 30.2, 45.2,12.5, 5.8, 27.2, 31.4 %, positivity rate for tuberculosis by PCR, 7.3, 9.5, 25.0, 0, 9, 8.5 % by culture and 1.8, 2.3, 0, 0, 0, 2.8 % respectively by AFB smear. Being a novel, rapid technique, PCR is the method of choice for rapid diagnosis and management of genitourinary tuberculosis shared with the other concerned tests. This study reveals that genital tuberculosis can occur in any age group, however, the majority of patients were from reproductive age (nearly 75 % of them were from 20–45 years of age) group.     

Design of Allele Specific PCR for Rapid Detection of CYP3A5 (A6986G) and Mdr-1 (C3435T) Polymorphisms

Single nucleotide polymorphisms in CYP3A5 (A6986G) and MDR-1 (C3435T) genes have been shown to be associated with the pharmacokinetics of tacrolimus in case of renal transplant recipients. Knowing these genotypes of the recipients before undergoing transplantation, is therefore essential for physicians to adjust the starting dose of tacrolimus in order to avoid drug induced nephrotoxicity. We have designed an allele specific PCR method for easier and rapid detection of these polymorphisms. 20 Indian renal transplant recipients on tacrolimus who developed nephrotoxicity within 1 month of transplantation and 58 Indian non-transplant subjects having the risk factors for kidney disease i.e. hypertension or diabetes or the family history of these, have been studied for these SNPs by allele specific PCR method. The data suggest that the heterozygosity of CYP3A5 and mutant allele frequency of MDR-1 SNP is higher in transplant patients as well as in general population.