西伯利亚蝗基因组DNA提取及RAPD分析条件的优化

以西伯利亚蝗Gomphocerus sibiricus(L.)为研究材料,利用改良的SDS法提取高质量的DNA,分别测试了dNTP浓度、镁离子浓度、TaqDNA聚合酶用量、模板DNA的量等因素对反应结果的影响。通过各因子的组合比较,建立了西伯利亚蝗RAPD优化体系:25μLPCR反应体系,10×buffer2·5μL;dNTP0·24mmol/L;MgCl22·0mmol/L;Taq DNA聚合酶1U;DNA模板45ng;引物30ng。扩增程序为:94℃预变性1min45s、94℃变性30s、35℃退火1min30s、72℃延伸2min,45个循环、72℃延伸10min。结果表明,利用优化的反应条件进行西伯利亚蝗基因组DNA分析,实验有着良好的重复性和稳定性。     

广东野百合DNA提取和RAPD条件的优化

以野百合(Lilium brownii)新鲜叶片、硅胶干燥叶片及鳞片为材料,研究了DNA的提取方法,并对影响随机扩增多态DNA(RAPD)反应的各因素进行了优化。建立了野百合RAPD的优化反应体系及程序,即在20μl反应体系中,含20 ng模板DNA,2.0 mmol/L Mg2 、0.2 mmol/L dNTPs、1.5 U Taq DNA聚合酶、0.3μmol/L随机引物S1519;扩增程序为:94℃预变性5 min,然后94℃30 s,38℃50 s,72℃1 min,35个循环,最后72℃延伸10 min,4℃保存。     

金龟甲基因组DNA RAPD反应条件的优化

以华北大黑鳃金龟[Holotrichia oblita (Faldermann)]和矮臀鳃金龟(H.ernesti Reitter)为供试材料,对DNA模板、Mg2+、dNTPs浓度、淬火和循环内延伸时间及循环数进行优化.提出了金龟甲RAPD分析的最佳反应体系,即25 μL体系中含DNA模板1.2 ng·μL-1、Mg2+ 2. 5 mmol·L-1、dNTPs 0. 25 mmol·L-1;并确定最适淬火时间为40 s,循环内延伸时间为2 min;最佳循环数为40.     

番茄随机扩增DNA多态性体系的条件优化

利用改进的SDS法提取代号为03748的栽培番茄叶片基因组DNA。对影响番茄随机扩增DNA多态性（RAPD）扩增结果的因素进行了分析,确定了模板、Mg^2＋、dNTPs、引物和Tap DNA聚合酶的适宜浓度及反应的最佳循环次数。实验结果表明,在以下条件下,番茄的RAPD扩增效果较好：20μL反应体系中使用20-40ng的模板、1．5-2．0mmol／L的Mg^2＋、0．15＋0．20μmol／L的dNTPs、0．15-2．0μmol／L的引物、1．0U的Taq DNA聚合酶;94℃预变性5min,然后经94℃变性1min、360℃ 1min、720℃ 1．5min,进行35个循环,最后在72℃时再延伸10min。     

正交试验优化八角ISSR-PCR反应体系

利用正交试验设计研究Taq DNA聚合酶、DNA模板、引物(UBC 886)、dNTP、Mg2+浓度5个因素对云南八角ISSR-PCR反应的影响,建立其最佳反应体系。结果表明:25μL的反应体系中5个因子的最佳水平为:Mg2+3 mmol/L、Taq DNA聚合酶0.5 U、DNA模板0.016 ng、引物0.8μmol/L、dNTPs 0.2 mmol/L。PCR最佳反应程序为:94℃预变性5 min;94℃变性30 s,46℃退火45 s,72℃延伸1 min,40次循环;72℃最后延伸7 min,4℃保存。     

红锥基因组RAPD反应体系的建立和优化

以红锥(Castanopsis hystrix A.DC.)嫩叶为材料,应用改良的CTAB方法成功提取了红锥基因组DNA,并对影响随机扩增多态DNA(RAPD)反应的各因素进行了优化,建立了红锥RAPD的优化反应体系及程序.在25μl反应体系中,模板DNA 0.8 ng μl-1,10×Buffer 2.5 μl,Mg2 2.0 mmol/L,Taq DNA聚合酶0.8 U,dNTPs 0.35 mmol/L,随机引物S42 0.28 μmol/L.PCR循环程序为:94℃预变性3 min,然后94℃ 30 s,39℃ 1 min,72℃ 2 min,35个循环,最后72℃延伸10 min,4℃保存.     

小麦随机扩增多态性DNA的反应体系探索

随机扩增多态性DNA(RAPD)是近年来发展起来的一种DNA多态性检测技术。通过以小麦基因组DNA为模板 ,对RAPD反应程序中的一些重要参数进行摸索和优化试验 ,建立了随机扩增多态性DNA分析应用反应体系。即反应体积为 2 5μ1,Mg2 浓度为 2 0mM ,dNTP浓度为 150 μM ,模板DNA量 5ng～2 5ng ,随机引物量 30ng～ 4 5ng ,Tap酶 1单位 ;反应过程为 90℃变性 30s ,38℃结合 1min ,72℃延伸 2min(最后一个循环 10min) ,共 4 0个循环。该体系可有效地应用在小麦遗传育种研究中 ,重复性好 ,可靠性高。     

药用植物草珊瑚RAPD扩增条件优化

采用CTAB-DNA提取方法,从草珊瑚植物的嫩叶中提取总DNA。以此DNA为模板,优化了草珊瑚RAPD-PCR的反应条件。结果表明,PCR扩增体系最适宜的条件为:反应体积25μL,内含2.5mmol/L Mg2+、1.0UDNA聚合酶、0.4μmol/L引物、60ng模板DNA和0.16mmol/L dNTP。扩增程序为:94℃预变性2min;94℃变性30s,37℃复性30s,72℃延伸80s,40个循环;72℃延伸10min;4℃保存10min。     

土壤微生物RAPD分析体系的优化研究

采用正交实验设计,对影响土壤微生物RAPD扩增体系的Mg2+、dNTP浓度及引物浓度进行了研究,同时对退火温度、延伸时间及循环次数进行摸索。结果表明,适宜土壤微生物PCR扩增反应在25μl体积中进行,包括7ng土壤微生物DNA样品、20pm随机引物l、.5uTaq酶、3.0mmol.L-1Mg-CL2和0.2mmol.L-1dNTP。PCR扩增反应进程如下:94℃3min,使土壤DNA变性;然后再进行39个循环,每个循环包括94℃1min,37℃40s,72℃90s,结束后72℃延伸7min。     

长心卡帕藻RAPD-PCR反应体系的正交优化研究

采用SDS(十二烷基硫酸钠,20%)法提取了大型海藻长心卡帕藻(Kappaphycus alvarezii)基因组DNA.通过单因子梯度试验,确定了影响随机扩增DNA多态性(RAPD)扩增结果的模板、Mg2 、dNTPs、S22引物、Taq的适宜浓度、退火温度和反应的最佳循环次数;利用正交试验优化了模板、Mg2 、dNTPs、S22引物、Taq的配比浓度.结果表明,在进行长心卡帕藻的RAPD扩增时,在总体积为25μl的反应体系中,模板、Mg2 、dNTPs、S22引物、Taq的最佳浓度分别为15ng、2.0mmol/L、0.2mmol/L、0.25μmol/L、2.5U;退火温度为37℃.反应程序为94℃预变性5min,然后经94℃变性30s、37℃退火1min.72℃延伸2min,进行30次循环,最后在72℃再延伸10min.     

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.