双歧杆菌属特异性测序引物筛选及优化

【背景】目前双歧杆菌的益生功能被普遍认可,越来越多的研究开始关注肠道中双歧杆菌的生物多样性。然而双歧杆菌是肠道中的低丰度物种,现有技术尚难以深入研究其多样性。【目的】基于双歧杆菌16SrRNA基因序列筛选一对适用于分析粪便样品中低丰度双歧杆菌属多样性的特异性引物。【方法】依据已有引物的相对位置及其与双歧杆菌属16S rRNA基因序列的匹配率,将引物重组优化得到扩增片段800 bp的双歧杆菌属特异性引物;通过PCR扩增和琼脂糖凝胶电泳对引物进行实验筛选和特异性验证;以细菌通用引物(27f/1492r)为参照,通过单分子实时(Single-molecule real-time,SMRT)测序技术对不同引物的3份粪便样品中细菌的DNA扩增子进行测序,在种水平上分析比较不同引物的优劣。【结果】对文献中已有的9对双歧杆菌特异性引物进行重组并优化,其中2对引物的理论特异性较好且扩增产物大于800 bp,它们分别为Bif164-f/Pbi R2和Pbi F1/Pbi R2。PCR扩增和琼脂糖凝胶电泳实验发现,Bif164-f/Pbi R2的扩增条带明亮且无拖尾。此外,利用SMRT测序平台对引物27f/1492r和Bif164-f/Pbi R2的3份粪便样品中细菌的DNA扩增子进行测序并分析。27f/1492r扩增子的分析结果显示,3份样品依次分别含1、3、4个双歧杆菌种且双歧杆菌的平均相对含量为0.34%;而Bif164-f/Pbi R2扩增子的分析结果显示,3份样品依次分别含2、6和8个双歧杆菌种且双歧杆菌的平均相对含量为98.72%。上述结果表明,Bif164-f/Pbi R2可在种水平上特异地检出粪便中低丰度的双歧杆菌,进而实现样品中双歧杆菌的多样性分析。【结论】筛选出一对双歧杆菌特异性引物Bif164-f/PbiR2,可在种水平上分析粪便样品中低丰度双歧杆菌的生物多样性,同时也验证了理论结合实验进行引物筛选这种方法的可行性。     

引物应用策略：基于2对古菌16S rRNA基因通用引物对环境样品古菌多样性分析

【目的】找到适宜的16S rRNA基因通用引物应用策略,应对复杂环境微生物多样性调查,尤其目前高速发展的高通量测序技术带来的巨大挑战。【方法】用Oligocheck软件分别将两对应试的古菌16S rRNA基因通用引物与RDP(Ribosomal database project)数据库中古菌16S rRNA基因序列进行匹配比对。用两对应试引物分别构建海洋沉积物样品的古菌16S rRNA基因文库。【结果】软件匹配结果显示引物f109/r958与目的基因的匹配程度高于引物f21/r958。该结果与古菌16S rRNA基因文库RFLP分析、古菌多样性指数分析结果相吻合。数据还表明,2对引物的综合文库能更好满足该沉积物样品的古菌多样性分析。【结论】选用与数据库中目的基因匹配性高的通用引物和多个引物的联合使用,可以有效提高环境样品微生物多样性调查的分辨率。     

不同PCR引物在根系丛枝菌根真菌群落研究中的应用比较

摘要:【目的】基础PCR的各种分子技术已广泛地应用于丛枝菌根真菌(AMF)的群落研究。为探讨不同PCR引物对丛枝菌根真菌群落的特异性差异扩增。【方法】本研究选取4对AMF特异性引物(NS31-AM1,AML1-AML2,NS31-AML2和SSUmCf-LSUmBr),通过PCR、克隆及测序技术对AMF的多样性及群落结构进行了分析比较。【结果】不同引物对AMF的扩增特异性及覆盖度均有显著性差异,且不同引物得到的AMF群落结构也存在显著性差异。SSUmCf-LSUmBr的扩增特异性及覆盖度最高,NS31-AML2和NS31-AM1次之,而AML1-AML2则相对较差。【结论】NS31-AML2的扩增区段能很好地与越来越被认可的AMF VT分类数据库(http://maarjam.botany.ut.ee)相匹配,且扩增片段长度也适合于目前的高通量测序技术。基于此,本文推荐NS31-AML2为AMF群落研究中的首选引物。     

艾丁湖沉积物放线菌多样性

摘要: 【目的】为了研究新疆艾丁湖低海拔、高盐环境中的放线菌多样性。【方法】本研究应用基于16S rRNA基因序列系统发育分析的免培养方法和选择性分离培养方法相结合的方式对艾丁湖沉积物样品进行放线菌多样性分析。利用放线菌特异性引物扩增16S rRNA 基因并构建了16S rRNA 基因克隆文库,对文库中的插入序列进行RFLP( Restriction Fragment Length Polymorphism 限制性片段长度多态性) 分析。采用不同盐浓度的9 种选择性培养基分离样品中的放线菌。【结果】通过对     

双歧杆菌属特异性定量引物的设计及验证

【目的】旨在设计一对双歧杆菌属特异性引物以检测不同样品中低丰度双歧杆菌的含量。【方法】在NCBI中下载57株双歧杆菌全基因组序列,以其共有单拷贝核心基因为目的片段设计双歧杆菌属特异性引物;并对引物进行PCR初筛和特异性复筛;之后借助ddPCR(Droplet Digital PCR,微滴式数字PCR)依次对筛选出的引物进行特异性、灵敏度和实用性验证。【结果】引物Bif-D-9特异性最好,可扩增出4株双歧杆菌而不能扩增20株非双歧杆菌中的任何一株菌;同时通过ddPCR仪定量稀释后的DNA,其扩增结果呈线性下降趋势,证明其灵敏度较好;另外,Bif-D-9结合ddPCR定量出婴儿粪便中双歧杆菌的拷贝数为71 copies/μL,母亲粪便中双歧杆菌的拷贝数为2.7 copies/μL,证明了该方法的实用性。【结论】引物Bif-D-9具有双歧杆菌属特异性,且灵敏度较高、实用性较好,适用于复杂样品中双歧杆菌属定量。     

属特异性T-RFLP技术用于乳酸杆菌的群落分析

【目的】设计乳酸杆菌属特异性T-RFLP技术(末端限制性片段长度多态性分析)对14株乳酸杆菌进行分型。【方法】采用源于16S-23S rRNA基因间隔区序列的乳酸杆菌属特异性引物LAB-rev,乳酸杆菌的属特异性引物,6-FAM荧光标记后结合16S上游通用引物7f用于乳酸杆菌的PCR扩增。【结果】选取HaeⅢ和HhaⅠ进行限制性酶切,最后对酶切后的产物末端测序得到T-RFLP峰谱图,该图谱能够快速准确地对不同种的乳酸杆菌进行定性、定量的分析。【结论】实验成功搭建T-RFLP技术用于微生态环境中乳酸杆菌检测的平台,对于在功能性食品、乳酸饮料和药物对肠道微生态的影响及菌种鉴定等领域有重大意义。     

ARDRA分型测定刺参养殖环境中蛭弧菌多样性

【目的】研究刺参养殖环境中蛭弧菌类微生物的多样性及其组成特征。【方法】对刺参养殖用水进行过滤,获得微生物并提取其总DNA,分别采用两对特异性引物Per和Bac扩增蛭弧菌类微生物相应的16S r RNA基因目的片段。通过核糖体DNA扩增片段限制性内切酶分析(ARDRA)方法,使用HaeⅢ和MspⅠ,双酶切各60个目的基因的单克隆,选不同条带克隆进行测序并对测序结果的多重序列分析比对。【结果】确定两对引物分别存在8和9种碱基差异序列,均属于噬菌弧菌属,分属于3个类群中。类群Ⅰ、Ⅱ为优势类群,且均为已知类群,类群Ⅲ为潜在的新类群;Per1(KP214541)和Bac44(KP214551)代表菌株分别为优势种。【结论】刺参养殖环境中蛭弧菌具有较高的多样性,包括已知类型和未知类型的蛭弧菌;可进一步进行蛭弧菌的分离、培养,用于刺参养殖中病害防治研究。     

双退火温度PCR扩增DNA

【目的】与设置单一退火温度的常规PCR(S-T_m PCR)不同,本研究探讨双退火温度PCR(D-T_m PCR)由高到低设置2条引物各自退火温度。【方法】以PxF61和VPel为正/反向引物,用Q5 DNA聚合酶扩增4.3 kb的模式DNA pET20b-Xyn(黑曲霉木聚糖酶基因)。PCR程序为:98°C预变性3 min,30次循环{98°C变性30 s,设置双退火[T_(m1) 70°C(Px F61)退火15 s、T_(m2) 62°C(VPel)退火15 s],72°C延伸130 s}。【结果】与S-T_m PCR(61°C)相比,D-T_m PCR扩增4.3 kb的目的条带亮度更高,减少2条杂带;经25次循环目的 DNA产物量最高。D-T_m PCR用于长片段引物扩增5.3 kb重组质粒DNA条带更明显。【结论】D-T_m PCR直接扩增目的条带,避免了探讨T_m的麻烦,不要求2条引物T_m相近,从理论上更加清晰地认识引物与各自模板分步退火过程。     

基于16S rDNA基因的谷斑皮蠹PCR检测技术

【目的】谷斑皮蠹是一种重要的检疫性害虫,在新疆周边多个国家分布,口岸检疫人员多次从进境货物中截获谷斑皮蠹,该虫对新疆的农业生产极具威胁。【方法】以谷斑皮蠹的16S rDNA基因为靶序列,用昆虫通用引物对4种供试皮蠹进行PCR扩增,将扩增产物进行克隆和测序,用生物软件设计检测谷斑皮蠹的特异性引物与探针。【结果】设计的特异性引物(TG-SNP-F/TG-SNP-R)及所建立的常规PCR方法能有效检测出谷斑皮蠹,其扩增产物的片段大小为250 bp,灵敏度为3 ng·μL~(-1)。设计的特异性引物(TG-F/TG-R)和探针(TG-probe),以及所建立的实时荧光PCR方法,对谷斑皮蠹的检测特异性强,灵敏度达0.8 fg·μL~(-1)。【结论】建立的常规PCR方法和实时荧光PCR检测方法能够对谷斑皮蠹进行准确鉴定,为口岸检疫人员检测进境货物中携带的谷斑皮蠹提供技术支持。     

两种混合样品策略对揭示杜鹃花根部真菌多样性的影响

由于土壤微生物群落物种组成的高度空间异质性,混合样品(sample pooling)被广泛应用于微生物多样性与群落结构研究。在根部真菌的分子检测中,样品混合策略以及测序的克隆数或序列数均对揭示真菌群落结构的准确性有影响。【目的】为建立一套能快速准确地反映杜鹃花属植物根部真菌的物种组成与群落结构的分子检测技术平台,【方法】本研究采集锈红杜鹃和亮鳞杜鹃多份根系样品分别提取DNA,比较PCR扩增前和扩增后混合策略构建的克隆文库中真菌物种组成的差异。【结果】在2种宿主植物根系中,多份样品在PCR扩增后混合构建的克隆文库检测到的根部真菌物种丰富度、真菌群落的Shannon-Wiener多样性指数均高于扩增前混合的克隆文库。高频度的根部真菌在2种克隆文库中均检测到,但低频度的真菌物种组成在2种克隆文库中完全不同。更重要的是,当采用广泛应用的真菌通用引物ITS1f和ITS4扩增根部真菌ITS序列时,PCR扩增后混合的方法能有效地减轻杜鹃花属植物ITS序列被优先扩增的现象。真菌物种累积曲线显示,当测序的真菌ITS片段克隆数达到50个左右,即能较全面地反映2种杜鹃花根部真菌物种组成。【结论】独立扩增多份根系样品DNA,再将PCR产物混合构建克隆文库的方法能更全面地揭示杜鹃花属植物根部真菌物种丰富度与物种组成。     

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.     

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     

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.     

Entwicklungsgeschichte und Ultrastruktur von Pollenkitt und Exine bei nahe verwandten entomophilen und anemophilen Angiospermensippen derAlismataceae,Liliaceae, Juncaceae,Cyperaceae, Poaceae undAraceae

Some closely related members of the monocotyledonous familiesAlismataceae, Liliaceae, Juncaceae, Cyperaceae, Poaceae andAraceae with variable modes of pollination (insect- and wind-pollination) were studied in relation to the ultrastructure of pollenkitt and exine (amount, consistency and distribution of pollenkitt on the surface of pollen grains). The character syndromes of pollen cementing in entomophilous, anemophilous and intermediate (ambophilous or amphiphilous) monocotyledons are the same in principal as in dicotyledons. Comparing present with former results one can summarize: 1) The pollenkitt is always produced in the same manner by the anther tapetum in all angiosperm sub-classes. 2) The variable stickiness of entomophilous and anemophilous pollen always depends on the particular distribution and consistency of the pollenkitt, but not its amount on the pollen surface. 3) The mostly dry and powdery pollen of anemophilous plants always contains a variable amount of inactive pollenkitt in its exine cavities. 4) A step-by step change of the pollen cementing syndrome can be observed from entomophily towards anemophily. 5) From the omnipresence of pollenkitt in all wind-pollinated angiosperms studied one can conclude that the ancestors of anemophilous angiosperms probably have been zoophilous (i.e. entomophilous) throughout.

     

Identification and Characterization of the First Equine Parainfluenza Virus 5

正Dear Editor,Parainfluenza virus 5 (PIV5), known as canine parainfluenza virus in the veterinary field, is a negative-sense,nonsegmented, single-stranded RNA virus belonging to the Paramyxoviridae family (Chen 2018). The virus was first reported in primary monkey kidney cells in 1954 (Hsiung1972), then it has been frequently discovered in various     

Pathogenic Characterization and Full Length Genome Sequence of a Reassortant Infectious Bursal Disease Virus Newly Isolated in Pakistan

<正>Dear Editor,Infectious bursal disease (IBD) is one of the most important diseases of the poultry. The IBD virus (IBDV), a nonenveloped virus belonging to the Birnaviridae family with a genome consisting of two segments of double-stranded RNA (segments A and B), targets B lymphocytes of bursa of Fabricious leading to immunosuppression. In Pakistan,poultry farming is the second biggest industry and IBD is the second biggest disease threating the poultry sector.However, there is limited genome information of IBDV