硅对PCR过程的抑制作用研究

探讨不同氧化程度的硅材料对PCR扩增的抑制作用及其机理。将不同氧化程度的硅纳米颗粒加入PCR反应液中,使其与Taq酶、模板等充分接触,通过离心将硅纳米颗粒沉降在管壁上,取出上清或保留硅纳米颗粒上机扩增,扩增产物采用凝胶电泳法检测。结果表明,随着硅材料表面面积与PCR反应液体积之比的增大,核酸扩增效率将明显下降,并且在所研究的范围内,氧化程度高的硅材料对PCR过程抑制作用更强;通过对抑制作用机理进行初步的实验研究,表明硅材料对PCR反应液中的Taq酶的吸附是导致抑制现象产生的主要原因,而对模板的吸附影响较小;并且,反应管内是否保留硅材料对核酸扩增影响较小,硅材料没有明显的直接化学抑制作用。     

利用Taq DNA聚合酶对PCR产物直接进行顺序分析

Taq DNA聚合酶具有反应速度快、温度作用范围广及良好的续进性等特点,可视为一种理想的DNA顺序分析酶。本文首先对非对称性PCR扩增过程中单、双链DNA产物的积累情况进行了分析,然后采用标记延伸二步法,对Taq DNA聚合酶的性质及影响因素进行分析。为进一步改进Taq DNA聚合酶测序的方法,本反应建立了“Klenow-型”的直接掺入标记同位素测序法,即在反应液中加入与标记核苷酸相应的一定浓度的冷dNTP。此法不但解决了二步法中引物后部分DNA顺序无法读出的缺点,而且简化了反应步骤,亦能得到令人满意的顺序分析结果,每次可读出至少400碱基的序列。     

大肠杆菌表达的TaqDNA聚合酶的纯化

Taq DNA聚合酶是分子生物学研究中最常用的热稳定DNA聚合酶之一,与其他热稳定DNA聚合酶具有相似的特征,其纯化策略不但有潜在的应用前景,也对同类聚合酶的分离具有指导意义。已报道的适宜大量制备Taq酶的方案所需成本较高,而文章介绍了一种利用国产阳离子交换树脂廉价制备Taq酶的方案。在本方案中,采用热变性、(NH4)SO4沉淀与724离子交换层析分离大肠杆菌表达的Taq酶,约18 g Na型树脂干粉一次可回收比活约8 131.98 U/mg、总酶活2.2×105U、近27.07 mg Taq酶。纯化的产率可达48.92%,纯化倍数约59.35。所制酶SDS-PAGE电泳只检测到94 kDa单一蛋白条带,未检测到DNA核酸酶污染,与商品酶的PCR扩增能力无区别。此纯化方法成本低,适合实验室一般性的制备和生产应用。     

线虫PCR:直接针对秀丽线虫进行PCR扩增基因组DNA的新方法

目的:直接针对秀丽线虫进行PCR反应,以便快速扩增基因组DNA,从而提高钓取目的基因和鉴定基因组是否发生突变的效率.方法:根据生物信息学分析,针对不同基因设计单重或多重PCR引物;在不含砌DNA聚合酶的PCR反应体系中加入蛋白酶K消化秀丽线虫染色体中的组蛋白,然后加入Taq酶,直接针对野生型或突变型秀丽线虫个体进行PC...     

落叶松cDNA扩增因素优化组合的研究

为了探讨落叶松cDNA扩增因素优化组合,以落叶松总RNA为模板逆转录为cDNA,然后进行PCR扩增,采用四因素三水平正交试验设计,分析比较了几种因素对PCR扩增的影响,结果显示,主要影响因素是Taq酶和Mg^2 ,随机引物和dNTP的影响较小,不同水平的影响作用是：Taq酶以高水平,即用量为2U,Mg^2 以中低水平,即用量为1.5-2.0mmol/L,可以得到多而清晰的条带;反之,则扩增效果差。     

芒AFLP反应体系的建立

以芒DNA为材料,对AFLP分子标记分析中的基因组酶切体系选择性扩增中Mg2+、dNTP和Taq酶浓度等4个因素进行了比较。结果表明20μL基因组双酶切体系中,使用1UEcoRⅠ和1UM seⅠ酶切3 h能够实现完全酶切;选择性扩增的PCR 10μL反应体系中1.4 mmol.L-1Mg2+,0.4 mmol.L-1dNTP及0.6 U Taq酶是进行芒AFLP分析的最佳反应条件,能够得到丰富稳定的带纹。该体系的构建为AFLP技术在芒相关研究中的应用奠定了基础。     

四种鲟鱼线粒体PCR-RFLP鉴定方法的研究

利用线粒体序列开发高效准确的分子鉴定方法广泛应用于水产品易混物种的鉴定中。应用PCR-RFLP技术,对鲟鱼易混种开展了分子生物学鉴定方法研究。结果表明,利用一组引物对8种鲟鱼线粒体基因进行PCR扩增,分别应用限制性内切酶TaqαI、Ava II和Eag I-HF、Nae I对扩增产物进行酶切,并用3.0%的琼脂糖凝胶检测PCR产物的酶切结果,可从8种鲟鱼易混种中分别鉴别出中华鲟、小体鲟、达氏鳇以及欧鳇。所建立的方法操作简单,在保证鱼种存活基础上只需剪取少量鳍条,便可快速准确地进行常见鲟鱼和不同鲟鱼产品的鉴别,大大增加了鉴定结果的准确性和可信度,极大地提高了工作效率。     

甜樱桃品种SSR-PCR反应体系的优化

以甜樱桃品种那翁为试材,研究了樱桃SSR技术中PCR反应体系的主要成分对SSR扩增结果的影响,并比较了采用聚丙稀酰胺凝胶及琼脂糖电泳检测扩增产物多态性的差异.结果表明:在PCR反应体系中,DNA最适浓度30～45 ng;Mg2+的最适浓度范围为1.5～3.0 mmol/L;dNTP最适浓度为0.2～0.3 mmol/L;引物的最适浓度为0.3～0.4 μmol/L;Taq聚合酶在20 μl反应体系中宜加入0.5 U.利用此反应体系,对24份樱桃代表资源进行了SSR反应,用6%的非变性聚丙稀酰胺凝胶电泳检测,扩增产物在100～250 bp之间,不同品种间DNA谱带多态性丰富.琼脂糖电泳检测的DNA多态性不如聚丙稀酰胺凝胶丰富.     

红曲菌基因组DNA提取及RAPD反应体系优化

为了建立一套适合红曲属真菌RAPD反应的优化体系,用改进的CTAB法提取红曲菌基因组DNA,采用单因素试验探讨RAPD反应体系中模板DNA、随机引物、Taq酶、Mg^2＋、dNTPs对扩增结果的影响。结果在20μL体积中,模板DNA20 ng、随机引物0、2μmol/L、Taq酶、Mg^2＋1.5mmol/L,dNTPs 1mmol/L的反应体系可得到稳定清晰的RAPD扩增图谱,为采用RAPD技术进行红曲菌种质资源遗传多样性研究奠定基础。     

穗花杉ISSR引物反应条件的优化与筛选

在研究穗花杉(Amentotaxus aragotaenia)的遗传多样性过程中,为了获得清晰、重复性好ISSR扩增结果,对影响ISSR-PCR的多个因素包括模板浓度、Taq酶的选择和用量、Mg²⁺和dNTPs浓度及退火温度等指标等进行了筛选和优化,确定了穗花杉ISSR-PCR分析的最适扩增条件： 20 μL PCR反应体系中,2 μL 10×Taq酶配套缓冲液,1.8 U Taq聚合酶（上海生工公司）,0.2 μmol·L^-1引物,0.18 mmol·L^-1 dNTP,1.5～2.5 mmol·L^-1 MgCl₂,10 ng·μL^-1模板DNA。用来自不同居群7个个体,以100个ISSR引物进行PCR扩增,筛选出扩增效果较好的10个引物。得到了92个位点,其中45个多态性位点,多态性位点比例为49%。     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

Enterovirus 71 3C proteolytically processes the histone H3 N-terminal tail during infection

Highlights
1. The N-terminal tail of histone H3 is specifically cleaved during EV71 infection.
2. Viral protease 3C is identified as a protease responsible for proteolytically processing the N-terminal H3 tail.
3. Our finding reveals a new epigenetic regulatory mechanism for Enterovirus 71 in virus-host interactions.     

Detection of EBV and HHV6 in the Brain Tissue of Patients with Rasmussen’s Encephalitis

Rasmussen’s encephalitis (RE) is a rare pediatric neurological disorder, and the exact etiology is not clear. Viral infection may be involved in the pathogenesis of RE, but conflicting results have reported. In this study, we evaluated the expression of both Epstein-Barr virus (EBV) and human herpes virus (HHV) 6 antigens in brain sections from 30 patients with RE and 16 control individuals by immunohistochemistry. In the RE group, EBV and HHV6 antigens were detected in 56.7% (17/30) and 50% (15/30) of individuals, respectively. In contrast, no detectable EBV and HHV6 antigen expression was found in brain tissues of the control group. The co-expression of EBV and HHV6 was detected in 20.0% (6/30) of individuals. In particular, a 4-year-old boy had a typical clinical course, including a medical history of viral encephalitis, intractable epilepsy, and hemispheric atrophy. The co-expression of EBV and HHV6 was detected in neurons and astrocytes in the brain tissue, accompanied by a high frequency of CD8⁺ T cells. Our results suggest that EBV and HHV6 infection and the activation of CD8⁺ T cells are involved in the pathogenesis of RE.     

Perinatal Vertical Transmission of Chikungunya Virus in Ruili,a Town on the Border between China and Myanmar

正Dear Editor,Chikungunya virus (CHIKV), an arbovirus in the family of Togaviridae, genus Alphavirus, is transmitted by the A.aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia (Silva and Dermody 2017; Suhrbier 2019). It was first reported in 1953 in Tanzania, and caused only a few outbreaks and sporadic cases in Africa and Asia in last century. However, in the epidemic in 2004, CHIKV acquired mutations that conferred enhanced transmission by the A. albopictus mosquito(Schuffenecker et al. 2006). Since then, it has successively caused outbreaks in Africa, the Indian Ocean, South East Asia, the South America, and Europe (Zeller et al. 2016).     

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.