核酸提取方法进展

核酸提取是分子生物学的基本方法,也是核酸诊断中最关键的方法,它是下游诊断、分析和制备的前提。过去的核酸提取方法繁琐费时且有限。目前,核酸提取方法已经取得了较大进步。本文综述了核酸提取方法的进展情况,包括传统的基于溶液的抽提方法、现在常用的柱提取法、正兴起的多效生物分子抽提法和自动化抽提系统等。多效生物分子抽提法、自动抽提系统的微型化和完全自动化或者它们的组合是核酸提取技术未来发展的方向。     

从脱毒棉籽粕和食用菌中提取食用核酸的工艺探讨

目的：利用核酸含量高的植物原料,探索食用核酸提取新工艺。方法：以脱毒棉籽粕粉和平菇子实体为原料,采用去离子水一次抽提-等电点与乙醇分次沉析法和NaCl溶液分次抽提-等电点与乙醇一次沉析法从中提取核酸。结果：两种原料采用两种不同工艺,提取核酸制品的A260／A280值均大于1．6,其中水提法大于1．6,盐提法大于1．8。核酸提取率在1．0％以上,其中水提法为1．07％和2．05％,盐提法为1．74％和2．48％。结论：脱毒棉籽粕粉和平菇子实体均是提取核酸的良好原料,后者优于前者。水提法和盐提法均适合于对脱毒棉籽粕粉和平菇子实体中食用核酸的提取,盐提法更为合理。     

棉阿舒囊霉细胞色素C的纯化、结晶和专一性的比较

棉阿舒囊霉(Ashbya gossypii)细胞色素C可用氯化钠抽提法或乙酸乙酯自溶法从菌体中抽出,经阳离子交换树脂Zeroli*226柱层析、硫酸铵分部沉淀及反透析后,可被纯化和结晶。乙酸乙酯自溶法的抽提得率高于氯化钠抽提法。两种方法抽提及纯化的细胞色素C在电泳中都表I见为均一,但氯化钠抽提之细胞色素C的生物活力略高于用乙酸乙酯抽提的细胞色素C。     

不同提取方法对桂花精油品质的影响

用超临界流体萃取法(SCFE)、可食用石油醚和酒精抽提蒸馏三种不同的抽提方法,提取了咸宁桂花品种——银星的花精油;气质联用(GC-MS)分析不同方法提取精油的香气成分及其相对含量。结果表明:SCFE提取的精油得率最高(0.19%),紫罗兰酮、醇类的相对含量高达36.99%;石油醚浸提结果次之(0.13%),酒精抽提法不可取(0.07%)。     

Effect of Different Extraction Methods on Osmanthus Oil Quality

用超临界流体萃取法(SCFE)、可食用石油醚和酒精抽提蒸馏三种不同的抽提方法,提取了咸宁桂花品种——银星的花精油;气质联用(GC-MS)分析不同方法提取精油的香气成分及其相对含量.结果表明:SCFE提取的精油得率最高(0.19％),紫罗兰酮、醇类的相对含量高达36.99％;石油醚浸提结果次之(0.13％),酒精抽提法不可取(0.07％).     

14型肺炎链球菌荚膜多糖纯化工艺中两种去除蛋白方法的比较

目的苯酚抽提法和脱氧胆酸钠沉淀法去除14型肺炎链球菌荚膜多糖中蛋白质的效果比较。方法将3批次14型肺炎链球菌发酵培养液经超滤、乙醇沉淀等方法初步纯化后,平分成两份,分别采用苯酚抽提法和脱氧胆酸钠沉淀法去除蛋白,通过比较多糖收获量、多糖组分检定结果、多糖分子质量、多糖抗原活性、多糖核磁共振图谱,以此评价这两种蛋白去除方法的效果。结果与苯酚抽提法相比,脱氧胆酸钠沉淀法制备的14型肺炎链球菌纯化荚膜多糖除收获量较高,蛋白和核酸杂质含量较低外,氨基己糖含量、多糖分子质量、抗原活性和多糖核磁共振图谱的检定分析结果无显著性差异(P>0.1)。结论作为14型肺炎链球菌荚膜多糖纯化工艺中的除蛋白方法,脱氧胆酸钠沉淀法优于苯酚抽提法。     

三种人全血基因组DNA提取方法的比较

目的：比较改良酚一氯仿抽提法、盐析法、试剂盒法从人全血中提取基因组DNA的效果,以期建立一种快速、经济的提取高质量基因组DNA的方法。方法：分别用上述三种方法从人全血中提取基因组DNA,通过紫外分光光度计、琼脂糖凝胶电泳、聚合酶链式反应（PCR）、限制性内切酶酶切检测提取的基因组DNA的产量、纯度和质量。结果：改良酚一氯仿抽提法与试剂盒法提取的基因组DNA相比,DNA的产量有统计学差异,DNA的纯度无统计学差异,但试剂盒法提取的基因组DNA有较明显的降解现象：盐析法与改良酚．氯仿抽提法、试剂盒法相比,基因组DNA的产量和纯度都存在统计学差异,并且基因组DNA聚合酶链式反应（PCR）扩增的稳定性也明显劣于另外两种方法;三种方法提取基因组DNA均能进行限制性内切核酸消化。结论：改良酚一氯仿抽据取法是一种经济、快速、高效、稳定提取人全血基因组DNA的方法,适用于批量临床标本处理。     

不同DNA抽提方法对普通PCR和实时荧光定量PCR方法检测家蚕微孢子虫的影响

为了优选快速、 灵敏、 特异的家蚕微孢子虫Nosema bombycis分子检测方法和DNA抽提方法, 本文通过对家蚕微孢子虫TaqMan探针荧光定量PCR检测方法和SYBR Green荧光定量PCR检测方法的建立以及反应体系优化, 并与普通PCR方法进行比较; 再采用4种不同DNA抽提方法分别对PCR和实时荧光定量PCR方法检测家蚕微孢子虫悬浮液的效果评价。结果显示: 不经过DNA抽提, 直接将家蚕微孢子虫发芽液进行PCR反应的效果优于其他方法, 检测灵敏度由高到低依次为直接法、 酚/氯仿抽提法、 动物组织DNA试剂盒抽提法和植物组织DNA试剂盒抽提法; TaqMan探针法检测家蚕微孢子虫发芽液的灵敏度和SYBR Green法相近, 达到微孢子10²个/mL, 两者均优于普通PCR方法。实验表明, 直接采用发芽液结合荧光定量PCR方法检测家蚕微孢子虫最为简便、 快速、 灵敏。该研究结果将有助于提高家蚕微粒子病监控技术和检疫能力, 对家蚕微粒子病的检疫和防治具有积极意义。     

适于小麦叶片蛋白质组分析的样品提取方法研究

以‘铭贤169'小麦苗期叶片为材料,分别采用传统的TCA/丙酮沉淀法、酚提取-甲醇/醋酸铵沉淀法以及改进的TCA/丙酮沉淀-酚/SDS联合抽提法提取叶片总蛋白,进行双向电泳分离和胶体考染,以建立适用于小麦蛋白质组分析的样品制备方法.结果表明:TCA/丙酮沉淀法较酚提取-甲醇/醋酸铵沉淀法获得的蛋白杂质较少,在二维电泳图谱中的蛋白点较酚抽提-甲醇/醋酸铵沉淀法提取的蛋白点清晰且多.相比于以上2种提取蛋白样品方法,改进的TCA/丙酮沉淀-酚/SDS联合抽提法提取的小麦叶片蛋白杂质少、二维电泳图谱上的点明显增多、分辨率较高.所选小麦的代表性蛋白点能获得成功鉴定.该方法可推广应用于水稻叶片蛋白质组分析的样品提取.     

还原型谷胱甘肽高产菌株的初筛及其提取工艺优化

目的:筛选还原型谷胱甘肽(GSH)高产菌株并优化其提取工艺.方法:从中科院沈阳应用生态研究所菌种保藏室的酵母菌库中筛选GSH高产的酵母菌,改进培养基组分,提高胞内GSH含量,并优化热水抽提和乙醇提取两种方法,提高提取液中GSH含量.结果:筛选获得一株酿酒酵母Y,其胞内GSH含量9.60 mg/g,培养基改良后,胞内GSH含量又提高了34.8%.通过单因素和正交试验确定热水抽提法最优提取条件为:料液比1∶3,pH 2.0,在90℃水浴中,抽提10min,GSH的产量可达14.27mg/g干菌体.结论:添加氨基酸和葡萄糖都有利于酵母菌体的生长和GSH合成.热水抽提法较乙醇提取法相比,提取效果好、无污染、操作简单,为后续的分离纯化工作奠定基础.     

Neuraminidase Subtyping of Avian Influenza Viruses with PrimerHunter-Designed Primers and Quadruplicate Primer Pools

We have previously developed a software package called PrimerHunter to design primers for PCR-based virus subtyping. In this study, 9 pairs of primers were designed with PrimerHunter and successfully used to differentiate the 9 neuraminidase (NA) genes of avian influenza viruses (AIVs) in multiple PCR-based assays. Furthermore, primer pools were designed and successfully used to decrease the number of reactions needed for NA subtyping from 9 to 4. The quadruplicate primer-pool method is cost-saving, and was shown to be suitable for the NA subtyping of both cultured AIVs and uncultured AIV swab samples. The primers selected for this study showed excellent sensitivity and specificity in NA subtyping by RT-PCR, SYBR green-based Real-time PCR and Real-time RT-PCR methods. AIV RNA of 2 to 200 copies (varied by NA subtypes) could be detected by these reactions. No unspecific amplification was displayed when detecting RNAs of other avian infectious viruses such as Infectious bronchitis virus, Infectious bursal disease virus and Newcastle disease virus. In summary, this study introduced several sensitive and specific PCR-based assays for NA subtyping of AIVs and also validated again the effectiveness of the PrimerHunter tool for the design of subtyping primers.     

Locking the 150-Cavity Open: In Silico Design and Verification of Influenza Neuraminidase Inhibitors

Neuraminidase (NA) of influenza is a key target for virus infection control and the recently discovered open 150-cavity in group-1 NA provides new opportunity for novel inhibitors design. In this study, we used a combination of theoretical methods including fragment docking, molecular linking and molecular dynamics simulations to design ligands that specifically target at the 150-cavity. Through in silico screening of a fragment compound library on the open 150-cavity of NA, a few best scored fragment compounds were selected to link with Zanamivir, one NA-targeting drug. The resultant new ligands may bind both the active site and the 150-cavity of NA simultaneously. Extensive molecular dynamics simulations in explicit solvent were applied to validate the binding between NA and the designed ligands. Moreover, two control systems, a positive control using Zanamivir and a negative control using a low-affinity ligand 3-(p-tolyl) allyl-Neu5Ac2en (ETT, abbreviation reported in the PDB) found in a recent experimental work, were employed to calibrate the simulation method. During the simulations, ETT was observed to detach from NA, on the contrary, both Zanamivir and our designed ligand bind NA firmly. Our study provides a prospective way to design novel inhibitors for controlling the spread of influenza virus.     

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.     

Assay of catecholamines and dihydroxyphenylethyleneglycol in human plasma and its application in orthostasis and mental stress

A high performance liquid chromatographic method involving electrochemical detection is described which permits the assay of noradrenaline (NA), adrenaline (A), dopamine (DA), and dihydroxyphenylethyleneglycol (DOPEG) in human plasma and brings about analytical recoveries of 70% and more. This method was used to assess the effects of graded orthostasis and mental stress on the plasma levels of these catechols. Orthostasis elicited increases in plasma NA and DOPEG, but did not cause any change in plasma A and DA. The increases in NA and DOPEG were dependent on the degree of orthostasis and correlated closely (rs = 0.724; n = 30, P less than 0.001). Pretreatment with desipramine abolished the DOPEG response to standing, indicating that orthostasis - induced increases in plasma DOPEG are presynaptic in origin. Mental stress evoked pronounced increases in plasma A, less pronounced increases in plasma NA and no changes in plasma DA and DOPEG. Hence, the simultaneous measurement of plasma NA and DOPEG may help to distinguish between various types of activation of the sympathetic nervous system.     

Can non-shivering thermogenesis in brown adipose tissue following NA injection be quantified by changes in overlying surface temperatures using infrared thermography?

We aimed to investigate whether infra red thermography (IRT) can be used to measure and quantify non-shivering thermogenesis (NST) in the short-tailed field vole Microtus agrestis, by directly comparing it with a standard method, i.e. metabolic response following Noradrenaline injection (NA). Mean skin surface temperature overlying Brown adipose tissue (BAT) depot was 0.82 degrees C higher than mean surface temperature that did not overly BAT. The difference in temperature increased by 1.26 degrees C after NA was administered. Mean skin surface temperature overlying BAT increased by 0.32 degrees C after NA was administered; however, surface temperature decreased by 1.32 degrees C after saline was administered. Mean skin surface temperature overlying BAT did not change significantly between warm and cold acclimated voles; in contrast metabolic peak following NA injection significantly increased in cold acclimated voles. There was no significant correlation between change in surface temperature after NA injection and metabolic peak following NA injection. The results of this study suggest that IRT is not a sensitive enough method to measure changes in NST capacity in BAT following NA injection, or to detect changes in NST capacity induced by cold acclimation. However, IRT can distinguish between skin surfaces overlying BAT and skin surfaces that do not.     

Enhanced contrast acoustic‐resolution photoacoustic microscopy using double‐stage delay‐multiply‐and‐sum beamformer for vasculature imaging

In acoustic‐resolution photoacoustic microscopy (AR‐PAM) systems, the lateral resolution in the focal zone of the ultrasound (US) transducer is determined by the numerical aperture (NA) of the transducer. To have a high lateral resolution, a large NA is used. However, the larger the NA, the smaller the depth of focus [DOF]. As a result, the lateral resolution is deteriorated at depths out of the focal region. The synthetic aperture focusing technique (SAFT) along with a beamformer can be used to improve the resolution outside the focal region. In this work, for image formation in AR‐PAM, we propose the double‐stage delay‐multiply‐and‐sum (DS_DMAS) algorithm to be combined with SAFT. The proposed method is evaluated experimentally using hair targets and in vivo vasculature imaging. It is shown that DS_DMAS provides a higher resolution and contrast compared to other methods. For the B‐mode images obtained using the hair phantom, the proposed method reduces the average noise level for all the depths by about 134%, 57% and 23%, compared to the original low‐ resolution, SAFT+DAS and SAFT+DMAS methods, respectively. All the results indicate that the proposed method can be an appropriate algorithm for image formation in AR‐PAM systems.      

Naturally occurring motoneuron cell death in rat upper respiratory tract motor nuclei: A histological,fast dil and immunocytochemical study in the nucleus ambiguus

The mammalian upper respiratory tract (URT) serves as the common modality for aspects of respiration, deglutition, and vocalization. Although these actions are dependent on coordinated and specific neuromuscular control, little is known about the development of URT control centers. As such, this study investigated the occurrence of naturally occurring motoneuron cell death (MCD) in the nucleus ambiguus (NA) of a developmental series of rats. Standard histological techniques were used to count motoneurons in the ventrolateral brainstem where the mature NA is found. In addition, the neural tracer, fast Dil, was used to determine whether motoneurons were still migrating into the region of the NA during the period that cell counts were first taken. Furthermore, to elucidate the potential effect of inadvertently counting large interneurons on the assessment of motoneuron numbers, an antibody to γ-aminobutyric acid (GABA) was used. The results of this study have, for the first time, demonstrated that MCD occurs in a URT-related motor nucleus. Approximately a 50% cell death was observed during the prenatal development of NA, with no further loss seen postnatally. The fast DiI studies showed that by embryonic day 17, NA was fully formed, suggesting that motoneuron migration from the basal plate was complete. In addition, use of the GABA antibody showed a lack of inhibitory interneurons within the NA. The finding of MCD in the NA helps define a critical period in the formation of URT neuromuscular control. As the course of MCD is modifiable by epigenetic signals, insult to the organism during this prenatal period may compromise future URT control. © 1995 John Wiley & Sons, Inc.     

Outside‐binding site mutations modify the active site's shapes in neuraminidase from influenza A H1N1

The recent occurrence of 2009 influenza A (H1N1) pandemic as well as others has raised concern of a far more dangerous outcome should this virus becomes resistant to current drug therapies. The number of clinical cases that are resistant to oseltamivir (Tamiflu®) is larger than the limited number of neuraminidase (NA) mutations (H275Y, N295S, and I223R) that have been identified at the active site and that are associated to oseltamivir resistance. In this study, we have performed a comparative analysis between a set of NAs that have the most representative mutations located outside the active site. The recently crystallized NA‐oseltamivir complex (PDB ID: 3NSS) was used as a wild‐type structure. After selecting the target NA sequences, their three‐dimensional (3D) structure was built using 3NSS as a template by homology modeling. The 3D NA models were refined by molecular dynamics (MD) simulations. The refined models were used to perform a docking study, using oseltamivir as a ligand. Furthermore, the docking results were refined by free‐energy analysis using the MM‐PBSA method. The analysis of the MD simulation results showed that the NA models reached convergence during the first 10 ns. Visual inspection and structural measures showed that the mutated NA active sites show structural variations. The docking and MM‐PBSA results from the complexes showed different binding modes and free energy values. These results suggest that distant mutations located outside the active site of NA affect its structure and could be considered to be a new source of resistance to oseltamivir, which agrees with reports in the clinical literature. © 2012 Wiley Periodicals, Inc.