基于核酸侵入反应的基因突变检测方法研究进展

肿瘤靶向药物治疗需要检测特异性的基因突变。尽管已开发出多种基于模板扩增的基因突变检测方法,但由于存在扩增产物交叉污 染的风险,其应用受到极大限制。基于信号扩增的核酸侵入反应,由于不存在扩增产物污染的风险,并且具有良好的单碱基识别特异性, 非常适用于基因突变的检测。因此,一系列基于核酸侵入反应的基因突变检测方法应运而生。综述若干基于核酸侵入反应的基因突变检测 方法原理与应用研究。     

Identification of vhhP2, a novel genetic marker of Vibrio harveyi, and its application in the quick detection of V. harveyi from animal specimens and environmental samples

Aims:  To investigate the species-specific prevalence of vhhP2 among Vibrio harveyi isolates and the applicability of vhhP2 in the specific detection of V. harveyi from crude samples of animal and environmental origins.
Methods and Results:  A gene ( vhhP2 ) encoding an outer membrane protein of unknown function was identified from a pathogenic V. harveyi isolate. vhhP2 is present in 24  V. harveyi strains isolated from different geographical locations but is absent in 24 strains representing 17 different non- V. harveyi species, including V. parahaemolyticus and V. alginolyticus . A simple polymerase chain reaction method for the identification of V. harveyi was developed based on the conserved sequence of vhhP2 . This method was demonstrated to be applicable to the quick detection of V. harveyi from crude animal specimens and environmental samples. The specificity of this method was tested by applying it to the examination of two strains of V. campbellii , which is most closely related to V. harveyi . One of the V. campbellii strains was falsely identified as V. harveyi .
Conclusions:  vhhP2 is ubiquitously present in the V. harveyi species and is absent in most of the non- V. harveyi species; this feature enables vhhP2 to serve as a genetic marker for the rapid identification of V. harveyi . However, this method can not distinguish some V. campbellii strains from V. harveyi .
Significance and Impact of the Study:  the significance of our study is the identification of a novel gene of V. harveyi and the development of a simple method for the relatively accurate detection of V. harveyi from animal specimens and environmental samples.     

High-throughput screening of bacteriorhodopsin mutants in whole cell pastes

A high-throughput screening method has been developed which enables functional analysis of bacteriorhodpsin in whole cell pastes. Reflectance spectra, from as little as 5 ml of Halobacterium salinarum cells, show close correspondence to that obtained from the purified purple membrane (PM), containing bacteriorhodopsin (BR) as the sole protein component. We demonstrate accurate quantification of BR accumulation by ratiometric analysis of BR (A_max 568) and a membrane-bound cytochrome (A_max 410). In addition, ground-state light- and dark-adapted (LA and DA, respectively) spectral differences were determined with high accuracy and precision. Using cells expressing the BR mutant D85N, we monitored transitions between intermediate-state homologues of the reprotonation phase of the light-activated proton pumping mechanism. We demonstrate that phenotypes of three mutants (D85N/T170C, D85N/D96N, and D85N/R82Q) previously characterized for their effect on photocycle transitions are reproduced in the whole cell samples. D85N/T170C stabilizes accumulation of the N state while D85N/D96N accumulates no N state. D85N/R82Q was found to have perturbed the pK_a of M accumulation. These studies illustrate the correspondence between pH-dependent ground-state transitions accessed by D85N and the transitions accessed by the wild-type protein following photoexcitation. We demonstrate that whole cell reflectance spectroscopy can be used to efficiently characterize the large numbers of mutants generated by engineering strategies that exploit saturation mutagenesis.     

肌肉萎缩引起肌电功率谱变化的理论和实验研究

为了通过肌电分析实现肌肉萎缩的无创检测,建立了一种数学模型研究肌肉萎缩后,其肌电信号功率谱的相应变化,并用大鼠的后肢卸载肌肉萎缩实验模型初步验证了数学模型分析的结论。该模型根据肌肉萎缩后肌纤维横截面积减小以及肌肉由于卸栽而出现持续性收缩的性质,采用中心导体模型及其电缆方程和肌电的线性系统模型建立起肌肉萎缩和生理肌电功率谱变化之间的数学关系。通过数字仿真和动物实验均发现了肌肉萎缩引起肌电幅度增加和功率谱高频成分降低的现象。结果表明数学模型和动物实验结果吻合,采用的数学模型能较好地阐述肌肉萎缩和肌电功率谱变化之间的关系。肌肉萎缩后肌电功率谱发生相应改变这一性质将为肌肉萎缩的无创检测提供一种新的方法。     

FTIR Spectroscopic Study of Biogenic Mn-Oxide Formation by Pseudomonas putida GB-1

Biomineralization in heterogeneous aqueous systems results from a complex association between pre-existing surfaces, bacterial cells, extracellular biomacromolecules, and neoformed precipitates. Fourier transform infrared (FTIR) spectroscopy was used in several complementary sample introduction modes (attenuated total reflectance [ATR], diffuse reflectance [DRIFT], and transmission) to investigate the processes of cell adhesion, biofilm growth, and biological Mn-oxidation by Pseudomonas putida strain GB-1. Distinct differences in the adhesive properties of GB-1 were observed upon Mn oxidation. No adhesion to the ZnSe crystal surface was observed for planktonic GB-1 cells coated with biogenic MnO _x , whereas cell adhesion was extensive and a GB-1 biofilm was readily grown on ZnSe, CdTe, and Ge crystals prior to Mn-oxidation. IR peak intensity ratios reveal changes in biomolecular (carbohydrate, phosphate, and protein) composition during biologically catalyzed Mn-oxidation. In situ monitoring via ATR-FTIR of an active GB-1 biofilm and DRIFT data revealed an increase in extracellular protein (amide I and II) during Mn(II) oxidation, whereas transmission mode measurements suggest an overall increase in carbohydrate and phosphate moieties. The FTIR spectrum of biogenic Mn oxide comprises Mn-O stretching vibrations characteristic of various known Mn oxides (e.g., “acid” birnessite, romanechite, todorokite), but it is not identical to known synthetic solids, possibly because of solid-phase incorporation of biomolecular constituents. The results suggest that, when biogenic MnO _x accumulates on the surfaces of planktonic cells, adhesion of the bacteria to other negatively charged surfaces is hindered via blocking of surficial proteins.     

食用调和油中花生油含量的近红外光谱分析

采用偏最小二乘法(PLS)等方法建立了食用调和油中花生油含量定量分析的近红外光谱定标模型。采集食用调和油样品在4 000 cm-1~10 000 cm-1范围内的近红外漫反射光谱,光谱经一阶导数处理后,采用偏最小二乘法建立样品中花生油含量的定标模型,并用Leave-one-out内部交叉验证法对模型进行验证。模型相关系数为0.99961,校正均方根RMSEC为0.830%。比较不同光谱预处理方法对定标模型的影响,结果表明一阶导数Corr.coeff最好。采用不同的化学计量学方法建立的定标模型中以偏最小二乘回归法最理想。     

Oriented solids as a colloid suspension in nematic liquid crystal – New tool for IR-spectroscopic and structural elucidation of inorganic compounds and glasses

Possibilities of the linear-polarized infrared (IR-LD) spectroscopy of oriented colloid suspensions in nematic liquid crystals, for structural and local structural elucidation for first time are demonstrated of inorganic compounds and glasses. The advantages of the method for tellurite and borate glasses are shown. The IR-band assignment of the typical local structural units in the glasses are proposed by a comparison with the IR-characteristics of appropriate crystalline analogues as α-TeO₂, V₂O₅, MoO₃ · H₂O and its high temperature form. The IR-spectroscopic characteristics of BO₃, BO₄ and boroxol ring are elucidated, using crystalline β-BaB₂O₄, SrB₄O₇, H₃BO₃ and B₂O₃ as model systems, where the structural moieties have been refined by single crystal X-ray diffraction.     

Oxidative addition of 3,6-di-tert-butyl-o-benzoquinone and 4,6-di-tert-butyl-N-(2,6-di-iso-propylphenyl)-o-iminobenzoquinone to SnCl2

Addition of 3,6-di-tert-butyl-o-benzoquinone (3,6-DBBQ) to SnCl₂ in THF leads to the oxidation of Sn(II) to Sn(IV) with formation of catecholate complex (3,6-DBCat)SnCl₂ · 2THF (1), where 3,6-DBCat is 3,6-di-tert-butyl-catecholate dianion. The reaction of 4,6-di-tert-butyl-N-(2,6-di-iso-propylphenyl)-o-iminobenzoquinone (IBQ-Prⁱ) also proceeds on the oxidative-addition mechanism yielding bis-iminosemiquinonato species (ISQ-Prⁱ)₂SnCl₂(2), where ISQ-Prⁱ is anion-radical 4,6-di-tert-butyl-N-(2,6-di-iso-propylphenyl)-o-iminobenzosemiquinolate. The complexes have been characterized by IR, X-band EPR, ¹H NMR (for 1) spectroscopy and magnetochemistry (for 2). X-ray analysis data show the distorted octahedral environment of tin(IV) for both complexes. Complex 1 is diamagnetic (ground state S = 0), while 2 has triplet ground state (S = 1, biradical). Catecholate complex 1 is able to be a spin trap for different organic radicals.     

CRISPR/Cas系统在抗病毒研究中的应用

近年来,CRISPR/Cas系统因其效率高、靶向性强、易操作等优势,已被广泛应用于多种病毒研究中。本文首先简单介绍了CRISPR/Cas系统的分类,并比较了Cas9和Cas12a与Cas13a的特点;其次重点介绍了CRISPR/Cas9通过靶向破坏病毒基因组,或编辑宿主关于病毒生命周期的关键因子的策略在抗病毒方面的各种应用,CRISPR/Cas13a采用靶向破坏病毒基因组方法在抗病毒中的应用,以及CRISPR/Cas12a和CRISPR/Cas13a在病毒基因检测中的应用。最后讨论了CRISPR/Cas在病毒研究中面临的挑战,并讨论了CRISPR/Cas12a作为抗病毒工具的潜在应用前景。由于CRISPR/Cas系统自身的优势,预计该系统将会给病毒相关的疾病诊断和控制带来革命性的变化。