随机扩增多态性DNA在细菌鉴定分型中的影响因素

为探讨各种试剂的浓度及不同DNA扩增仪对随机扩增多态性DNA(randomlyamplifiedpolymorphicDNA ;RAPD)的影响因素。用不同浓度引物、4×dNTP等试剂及 3种不同型号DNA扩增仪对 11株致病弧菌进行RAPD扩增。结果 11株致病弧菌分成 4型 ,重复性好。不同引物、4×dNTP对RAPD构成一定影响 ,3种DNA扩增仪产生的DNA指纹图谱基本一致。提示用于RAPD的试剂可以标准化 ,不同实验室间存在可比性     

随机扩增多态性DNA最佳模板提取方法的探讨

目的　获得最佳模板提取方法用于随机扩增多态性DNA(RAPD)。方法　对 4种方法 (挑丝法、沉淀法、水清法和裂清法 )提取的模板DNA进行琼脂糖电泳、紫外线扫描及随机扩增多态性DNA ,比较其片段大小、纯度及RAPD指纹图差异。结果　挑丝法和沉淀法均可提取出大于 2 3kb且纯度较高的模板DNA ,二者指纹图完全一致且均有 2～ 4kb的较大片段。水清法和裂清法提取的模板有弥散、碎裂的DNA并纯度较差 ,指纹图上仅有 6 0 0～ 2 0 0 0bp的较小片段。 结论　用挑丝法或沉淀法提取的模板DNA最适于进行RAPD。     

酶谱法分型检测人疱疹病毒DNA方法的建立及应用

单纯疱疹病毒Ⅰ型、Ⅱ型(HSV-Ⅰ,HSV—Ⅱ)、Epstein-Barr病毒(EBV)和人巨细胞病毒(CMV)是引起中枢神经系统、生殖道感染及肿瘤等多种疾病的共同病原体。以往的实验室诊断方法,包括已建立的各种疱疹病毒的聚合酶链反应法(PCR)技术,一般只能检测一种病毒,易造成漏诊。笔者在上述4种病毒的高度同源序列DNA聚合酶基因中设计一对引物,能对4种病毒DNA进行扩增,继而用凝胶电泳和限制性内切酶BamHI或SmaⅠ酶切分析扩增产物进行鉴别,建立了能一次性分型检测上述4种病毒的PCR——酶谱法。敏感性和特异性试验表明,灵敏度可测到1 fg DNA,相当于6个病毒颗粒,且引物仅对4种疱疹病毒扩增,与临床常见的其它病原体无交叉反应。对影响PCR效果的因素如Mg~(2+)浓度、引物浓度及循环参数等进行了优化选择。用此法检测10例疱疹病毒性脑炎患者脑脊液和18例慢性宫颈炎患者宫颈分泌物中的疱疹病毒,结果分别有8例和15例出现DNA扩增区带,并经电泳和酶切能区分各型,本方法的建立为疱疹毒感染的临床早期诊断、药物疗效考核和流行病学研究提供了有效的手段。     

单引物耐热链替代扩增HBV X区方法的初步探讨

目的 :以HBVX区为模型 ,对单引物耐热链替代扩增 (SDA)反应的影响因素进行初步探讨。SDA是一种等温的体外核酸扩增技术 ,其主要依据限制性内切酶识别并切割半硫酸磷酸化DNA未修饰链 (即打缺口 )以及 5′→ 3′外切酶缺陷的DNA聚合酶在切口处聚合延伸并替代下游链的特性 ,这种“打缺口—聚合 替代”不断循环往复 ,达到靶序列的扩增。方法 :以HBV为模型 ,设计一条典型的SDA引物 (含 5′悬端、BsoBⅠ酶切位点及 3′端靶序列互补区 ) ,采用耐热BsoBⅠ BstDNA聚合酶 5 3℃恒温反应体系 ,微孔板杂交检测最终产物。结果与结论 :对SDA反应的重要的影响因素进行了初步探讨 ,并实现了SDA对模板的线性扩增     

聚合酶链反应—酶谱法分型检测人疱疹病毒DNA方法的建立及应用研究

在疱疹病毒高度同源序列DNA聚合酶基因中设计一对引物，能对HSV－Ⅰ、HSV－Ⅱ、EBV和CMV4种疱疹病毒DNA进行扩增，继而用凝胶电泳和限制性内切酶BarnHⅠ或SmaⅠ酶切分析扩增产物进行鉴别，建立了能一次性分型检测上述4种病毒的POR－酶谱法。敏感性和特异性试验表明，灵敏度可测到1fgDNA，相当于6个病毒颗粒，且引物仅对4种疱疹病毒扩增。对影响PCR效果的有关因素如Mg2＋浓度、引物浓度、循环参数等进行了优化选择。用建立的PCR－酶谱法检测病毒性脑炎患者脑脊液和慢性宫颈炎患者宫颈分泌物中的疱疹病毒，取得较好结果。本方法的建立为疱疹病毒感染的临床早期准确诊断、药物疗效考核和流行病学研究等提供了有效的手段。     

应用原位逆转录PCR 检测石蜡切片中HCV RNA

目的：检测石蜡包埋组织中的HCV RNA。方法：原位PCR是一种能够检测单拷贝、低拷贝（20bp）DNA或RNA序列的新技术，本研究应用原位逆转录PCR检测石蜡包埋人肝外胆管癌组织中的HCV RNA。结论和结论：（1）基因组DNA去除一定要彻底。（2）选择合适的逆转录酶。（3）用原位PCR仪专用的封片装置封好，以防扩增液流出。（4）无水乙醇后固定10min防止扩增产物扩散。（5）对于不同组织来源的DNA或RNA进行原位PCR扩增的次数不同，不是循环次数越多越好，次数增多有可能使扩增产物从细胞内溢出，增加非特异性。     

脂血和溶血对HBV DNA实时荧光定量PCR检测的影响

目的 探讨溶血和脂血两种因素对HBV DNA实时荧光定量PCR方法 的影响.方法 (1) 收集临床HBV DNA标本,按HBV DNA浓度分为Ⅰ水平、Ⅱ水平;收集HBV DNA阴性的重度乳糜状脂血标本和正常人标本.将以上标本按不同比例混合,制作成极高TG浓度不同HBV DNA水平:AⅠ、AⅡ组;高TG浓度不同HBV DNA水平:BⅠ、BⅡ组;正常TG浓度不同HBV DNA水平:CⅠ、CⅡ组.实时荧光定量PCR扩增.(2)收集临床HBV DNA标本,每人采集3份血标本,其中2份标本进行-20 ℃冷冻不同时间,制成重度溶血DⅠ、DⅡ组;中度溶血EⅠ、EⅡ组;无溶血对照组FⅠ、FⅡ组.实时荧光定量PCR扩增.(3)利用方差分析各检测结果 之间是否有统计学差异.结果 不同浓度脂血和溶血,对两种水平HBV DNA荧光定量PCR检测结果 均无统计学差异.结论 溶血和脂血对实时荧光定量PCR检测HBV DNA结果 无影响.     

两种全血基因组DNA提取方法的比较

目的比较两种不同的DNA提取方法提取人全血基因组DNA,对DNA提取率、纯度以及PCR扩增的效果影响.方法分别用传统的酚、氯抽提法和改良法两种不同的方法,提取人全血基因组DNA.结果两种方法提取的DNA纯度用光吸收比值为指标检测A280/260值为:1.45和1.82,提取DNA总量分别为187μg/ml和260 μg/ml.用0.8%琼脂糖凝胶电泳鉴定改良法制备的DNA效果较好,PCR扩增效果有差异.结论用改良法提取的DNA总量明显较高,提取效率高,PGR扩增的效果好,是全血基因组DNA提取中首选考虑的方法.     

正交设计优化CYP2C19基因多态性PCR体系

目的 建立通用的CYP2C19*2和CYP2C19*3基因PCR反应体系,筛选出各反应因素的最佳水平,并确定最佳退火温度.方法 采用正交设计L16(45)在4个水平5个因素(Taq酶、引物、Mg2+、dNTP和模板DNA)对CYP2C19*2和CYP2C19*3基因PCR反应体系进行实验,两次结果分别用统计软件MINI...     

木村病与穿孔素基因突变的研究

目的 探讨木村病与穿孔素基因突变的关系,企图证实其发病机制是否与病毒感染有关.方法 从1例19岁男性木村病患者的外周血细胞提取DNA,将穿孔素主要功能区的外显子2、3和连接部位的内含子2的基因分成3段设计引物,应用PCR扩增然后进行测序分析.结果 患者穿孔素第2外显子扩增后测序发现R4C（rs12161733）、R22H杂合基因突变.结论 木村病是一种临床综合征,病毒感染导致穿孔素基因突变可能是本病发生机制之一.     

正交试验法优选小鼠四氯化碳矸损伤模型

目的;采用正交试验法优选小鼠四氯化碳肝损伤最佳模型制作条件,方法：选择对小鼠血清转氨酶（ALT）有影响的四个因素（1）四氯化碳浓度;（2）肝毒剂中毒剂量;（3）中毒次数;（4）中毒途径,生个因素取3个水平,按L9(3^4)正交试验表安排是试验,以阳性药联苯双酯双小鼠四氯化碳肝损伤改善率为考察指标,评定四氯化碳中毒方案的优劣,结果：采用0．1%(v/v)的四氯化碳,按每10g小鼠0.2ml剂量腹腔一次注射的给药方案为佳,结论 以正交试验法优选出的小鼠四氯化碳肝损伤模型制作方案是可行的。     

Discrepancies in determination of abdominal aortic aneurysms maximum diameter and growth rate,using axial and orhtogonal computed tomography measurements

Purpose

Maximum diameter and growth rate of abdominal aortic aneurysms (AAAs) which are currently used as the only variables to set the indication for elective repair are recorded through computed tomography (CT) measurements on an axial plane or on an orthogonal plane that is perpendicular to vessel centerline, interchangeably. We will attempt to record possible discrepancies between the two methods, identify whether such differences could influence therapeutic decisions and determine in which cases this should be expected.

Materials and methods

We retrospectively reviewed sixty CT-scans performed in thirty-nine patients. Three-dimensional reconstruction of AAAs has been performed and differences in maximum diameter measured on axial and orthogonal planes were recorded. A measure for asymmetry was introduced termed ShapeIndex defined as the value of section minor over major axis and was related with differences in maximum diameter recordings. Growth rates were also determined using both axial and orthogonal measurements.

Results

Axial measurements overestimate maximum diameter by 2 ± 2.7 mm (P < 0.001) with a range of 0–12.3 mm. Overall, 20% of the CTs had an axial maximum diameter >5.5 cm indicating the need for intervention whereas, orthogonal diameter was below that threshold. Asymmetry of the axial sections with ShapeIndex ≤ 0.8 was found to be related to an overestimation of maximum diameter by >5 mm. There were no significant differences in growth rates when determined using orthogonal or axial measurements in both examinations (median growth rate: 2.3 mm and 3.3 mm respectively P = 0.2). However there were significant differences when orthogonal measurements were used at initial and axial measurements used at follow-up examination or vice versa (median growth rate: 4.9 mm and 0.9 mm respectively P < 0.001).

Conclusions

Although the mean difference between measurements is low there is a wide range among cases, mainly observed in asymmetrical AAAs. ShapeIndex may identify those which are more likely to be misestimated. CT measurements performed to establish AAA growth rates should consistently use either the axial or orthogonal technique to avoid inaccuracies from occurring.     

黄金菊提取工艺的正交设计试验

目的通过四因素三水平的正交试验，结合紫外分光光度法，优选黄金菊中黄酮的最佳提取条件。方法以乙醇浓度、溶剂倍量、回流时间、提取次数为因素，每个因素3个水平，以测定的总黄酮含量作为评价指标进行正交试验。结果得到最佳提取条件为：乙醇浓度85％，溶剂倍量20倍，回流时间1h，提取次数3次。工艺验证试验表明：平均提取得率高达6．902％，平均回收率为98．41％，RSD为2．67％（n=5）。结论本提取方法为提取黄金菊中总黄酮提供了依据。     

泛素连接酶CUL4A-DDB1复合体参与DNA损伤修复的研究进展

泛素化修饰在DNA损伤信号中发挥重要功能,包括细胞周期监控、DNA修复、细胞衰老和程序性死亡的调控。CUL4A-DDB1泛素连接酶通过DCAFs靶向调控特异性的底物,启动DNA切除修复机制对受损DNA进行修复。近期的研究表明CUL4A-DDB1泛素连接酶协助DNA修复因子与受损DNA的识别,来维持基因组的稳定性和正确性。     

Comparison of volume and diameter measurement in assessing small abdominal aortic aneurysm expansion examined using computed tomographic angiography

Aim

First we aimed to assess the reproducibility of a computer tomography angiography (CTA) based technique for measuring infra-renal aortic volume and diameter. Second we sought to investigate whether changes in aortic volume and diameter were similar during follow-up.

Materials and methods

A prospective series of 57 patients, with aortic diameter initially measuring between 25 and 55 mm, were assessed with 2 CTAs a median of 14 months apart. Aortic volume and maximum diameter (both axial and orthogonal) were measured by a semi-automated workstation protocol based on previously defined techniques. Intra- and inter-observer reproducibility were assessed by repeat assessment of the initial CTA images of the first 33 patients included in the study, in order to estimate the 95% limits of agreements. Changes in aortic dimensions between the first and follow-up CTA, were defined for volume and diameter separately as changes greater than their respective 95% limits of agreement.

Results

Reproducibility of aortic volume and diameter was excellent with an average coefficient of variation <4%. The median (inter-quartile range) increases in total volume, orthogonal and axial diameters were 4.9 cm³ (0.01-14.18), 1.2 mm (0.40-3.50) and 1.4 mm (−0.15 to 3.55) respectively. Forty-two percent of patients who had increased aortic volume above the 95% limit of agreement did not display corresponding axial or orthogonal diameter changes.

Conclusions

Infra-renal total aortic volume, axial and orthogonal diameter can all be measured reproducibly from CTA. Aortic volume changes are not always reflected by similar changes in diameter and therefore provide complementary information when assessing AAA expansion over time.     

Application of three-dimensional orthogonal neural network to craniomaxillary reconstruction

The purpose of this investigation is to establish a practical method to predict and create surface a profile of bone defects by a well-trained 3-D orthogonal neural network. First, the coordinates of the skeletal positions around the boundary of bone defects are input into the 3-D orthogonal neural network to train it to learn the scattering characteristic. The 3-D orthogonal neural network avoids local minima and converges rapidly. After the neural network has been well trained, the mathematic model of the bone defect surface is generated, and the pixel positions are derived. Herein, to verify its performance the proposed method is applied on a patient with a craniofacial defect.     

The effects of microgravity on ligase activity in the repair of DNA double-strand breaks

PURPOSE: In recent years, contradictory data have been reported about the effects of microgravity on radiation-induced biological responses in space experiments. The aim of the present study was to clarify whether enzymatic repair of DNA double-strand breaks is affected by microgravity using an in vitro enzymatic reaction system. MATERIALS AND METHODS: The DNA repair activity of T4 DNA ligase (EC 6.5.1.1) was measured in vitro for a DNA substrate damaged by restriction enzyme digestion during a US Space Shuttle mission (Discovery; STS-91). After the flight, the amount of ligated DNA molecules was measured using an electrophoresis method. RESULTS: Ligated products (closed circular DNA, open circular DNA and multimeric ligated products) were produced by T4 DNA ligase treatment of linear DNA containing double-strand breaks, and they increased with increasing T4 DNA ligase concentration (0-3 units per microg of plasmid DNA). Almost no difference in T4 DNA ligase activity was detected between the space experiments and the control ground experiments. CONCLUSIONS: No significant effect of microgravity on ligation of damaged DNA was found during space flight. Therefore, other mechanisms must account for the synergism between radiation and microgravity, if it exists.     

一种细菌DNA提取方法的正交设计优化

目的通过正交实验优化异硫氰酸胍-硅胶提取细菌DNA的方法,以缩短其提取时间,进而缩短细菌特别是炭疽芽孢杆菌等恐怖细菌的核酸分子检测时间。方法以基因芯片的杂交信号值为指标,以异硫氰酸胍-硅胶提取DNA过程中的煮沸时间、室温放置时间、乙醇洗涤次数为考察因素通过正交实验法优化异硫氰酸胍-硅胶提取细菌DNA的条件。结果优化后的因素水平为煮沸10 min,室温放置15 min,乙醇洗涤2次。结论实验证明,优化后的提取时间比原来缩短了25 min,且信号值比原方法更高。     

Spiral CT optimization for measurement of bronchial lumen diameter using an experimental model

The aim of the study was to determine optimal parameters for demonstrating sublobar bronchi on spiral CT. Measurements were obtained from five parallel polyethylene tubes embedded in foam matrix with similar radiographic characteristics to segmental and subsegmental bronchi and to lung parenchyma, respectively. Collimation widths of 1.5, 2, 3 and 4 mm were used, with a pitch of 1 or 1.5 and a reconstruction interval of 1 mm or 2 mm. Various slice planes were used. Images acquired orthogonally were viewed normally. Images acquired in planes oblique or parallel to the long axes of the tubes were reformatted into a plane orthogonal to the long axes of the tubes to be comparable with the directly acquired orthogonal images. Tube diameters were measured at lung window settings (L, -400; W, 1300) and compared with known true inner and outer tube diameters. Measurements from images acquired orthogonal to the tube long axes were accurate regardless of slice thickness. Images acquired obliquely or parallel only produced accurate measurements at the lowest slice thickness (1.5 mm). Pitch and reconstruction interval had no effect on measurement error in any scan plane. It is concluded that a slice thickness of 1.5 mm or less, with a pitch of 1.5, should be used when acquiring images at angles other than orthogonal to the long axes of experimental tubes equivalent to the segmental and subsegmental bronchi. It is suggested that similar parameters should be used in vivo and that the examination should be targeted to the area of the bronchial tree in question to reduce patient dose and length of breath-hold.     

Lumpectomy Specimen Radiography: Does Orientation or 3-Dimensional Tomosynthesis Improve Margin Assessment?

PurposeOur purpose was twofold. First, we sought to determine whether 2 orthogonal oriented views of excised breast cancer specimens could improve surgical margin assessment compared to a single unoriented view. Second, we sought to determine whether 3D tomosynthesis could improve surgical margin assessment compared to 2D mammography alone.Materials and MethodsForty-one consecutive specimens were prospectively imaged using 4 protocols: single view unoriented 2D image acquired on a specimen unit (1VSU), 2 orthogonal oriented 2D images acquired on the specimen unit (2VSU), 2 orthogonal oriented 2D images acquired on a mammogram unit (2V2DMU), and 2 orthogonal oriented 3D images acquired on the mammogram unit (2V3DMU). Three breast imagers randomly assessed surgical margin of the 41 specimens with each protocol. Surgical margin per histopathology was considered the gold standard.ResultsThe average area under the curve (AUC) was 0.60 for 1VSU, 0.66 for 2VSU, 0.68 for 2V2DMU, and 0.60 for 2V3DMU. Comparing AUCs for 2VSU vs 1VSU by reader showed improved diagnostic accuracy using 2VSU; however, this difference was only statistically significant for reader 3 (0.73 vs 0.63, P = .0455). Comparing AUCs for 2V3DMU vs 2V2DMU by reader showed mixed results, with reader 1 demonstrating increased accuracy (0.72 vs 0.68, P = .5984), while readers 2 and 3 demonstrated decreased accuracy (0.50 vs 0.62, P = .1089 and 0.58 vs 0.75, P = .0269).Conclusions2VSU showed improved accuracy in surgical margin prediction compared to 1VSU, although this was not statistically significant for all readers. 3D tomosynthesis did not improve surgical margin assessment.