三株新城疫广西分离株全基因组序列的测定与分析

根据GenBank上所公布的新城疫病毒(NDV)的全基因组序列,设计了8对引物,运用RT-PCR 方法获取了3株广西地方强毒株GX7/02、GX9/03和GX11/03的全基因组序列,并对其进行了比较分析.此3株病毒的全基因组序列均由15192个碱基组成,与GenBank公布的ZJ1、U.S/Largo/71、Italy/2736/00等7个毒株的全基因组序列长度相同,比LaSota、Clone-30和B1的全基因组序列多出6个核苷酸,此6个核苷酸位于np基因的非编码区内,相对与NDV毒株LaSota、Clone-30和B1序列的1647～1648nt 位.通过序列的比较分析,发现GX7/02、GX9/03和GX11/03与ZJ1毒株的同源性较高,而与LaSota、Clone-30和B1等毒株的同源性较低.     

三株新城疫广西分离株全基因组序列的测定与分析

根据GenBank上所公布的新城疫病毒(NDV)的全基因组序列,设计了8对引物,运用RT-PCR方法获取了3株广西地方强毒株GX7/02、GX9/03和GX11/03的全基因组序列,并对其进行了比较分析。此3株病毒的全基因组序列均由15192个碱基组成,与GenBank公布的ZJ1、U.S/Largo/71、Italy/2736/00等7个毒株的全基因组序列长度相同,比LaSota、Clone-30和B1的全基因组序列多出6个核苷酸,此6个核苷酸位于np基因的非编码区内,相对与NDV毒株LaSota、Clone-30和B1序列的1647~1648nt位。通过序列的比较分析,发现GX7/02、GX9/03和GX11/03与ZJ1毒株的同源性较高,而与LaSota、Clone-30和B1等毒株的同源性较低。     

牛血液中一株新型环状病毒的分离与全基因组序列分析

我们从广西壮族自治区哨兵动物牛上采集的血液样本中分离到一株病毒,暂将其命名为广西环状病毒(毒株号:V172/GX/2015)。病毒接种C6/36细胞后可产生明显的细胞病变,表现为细胞聚集、皱缩与脱落。高分辨率琼脂糖凝胶电泳显示,病毒基因组由10节段的双链RNA组成,在凝胶上呈现"3-4-3"的带型特征。通过全长cDNA扩增与高通量测序方法获取V172/GX/2015毒株的全基因组序列。病毒基因组大小为19 889bp,基因节段的大小在3 996bp(Seg-1)至829bp(Seg-10)之间,可编码VP1至VP7等7种结构蛋白以及NS1至NS3等3种非结构蛋白。对环状病毒属保守的VP1、VP3(T2)与VP7(T13)蛋白氨基酸序列分析与系统发育树分析显示,V172/GX/2015与蚊传播环状病毒Yunnan orbivirus、Peruvian horse sickness virus以及Mobuck virus具有较近的亲缘关系,氨基酸序列相似度在31.4%至69.9%之间;V172/GX/2015在系统发育树上形成了一个独立于其它环状病毒的进化分支。本研究首次报道了一种新型环状病毒在牛上的分离与全基因组序列,研究结果将进一步丰富我们对环状病毒属病毒的认知,为开展新型环状病毒的流行病学调查与致病性研究提供基础。     

沙门氏菌泛耐药基因组特征分析

【目的】分析沙门氏菌的泛耐药基因组特征。【方法】以EnteroBase数据库中16365株沙门氏菌为对象,利用课题组自主研发的泛耐药基因组分析软件(PRAP),进行泛耐药基因组结构的鉴定,通过曼-惠特尼秩和检验和皮尔逊检验,来分析耐药基因与血清型、序列型(sequence type,ST)及分离株样本来源信息间的相关性。【结果】沙门氏菌共有104种耐药基因,其中核心耐药基因18种,附属耐药基因86种,且沙门氏菌拥有一个开放型的泛耐药基因组;相同的血清型(或ST型)有相似的耐药基因谱,不同血清型(或ST型)间耐药基因的分布差异显著(P0.05),耐药基因与样本来源、分离国家及年份间也存在一定的相关性;在测试的23种获得性耐药基因中,43.48%(10/23)的占比逐年升高,73.91%(17/23)以单一亚型为优势。【结论】利用PRAP软件分析获得的这些结果揭示了近年来沙门氏菌耐药基因的时空分布规律,为沙门氏菌等食源性致病菌耐药性的研究提供了新思路。     

全基因组测序技术对沙门氏菌血清型和耐药性的预测能力分析

【目的】评价全基因组测序技术在沙门氏菌血清型和耐药性检测方面的应用能力。【方法】对我国1950–2015年分离的290株鸡源沙门氏菌用常规检测方法进行了血清分型和药敏试验;提取全基因组进行测序,应用SeqSero和ResFinder数据库分析沙门氏菌的血清型和耐药性;对用常规检测方法和全基因组测序分析方法得到的血清型和耐药性结果进行比较,分析两种方法所得结果的符合性情况。【结果】沙门氏菌的主要血清型为肠炎和鸡白痢(≥84.5%),常规检测方法和全基因组测序分析方法在沙门氏菌血清分型方面的总体符合率为97.6%。对11种抗菌药物的最小抑菌浓度(MIC)检测结果显示,沙门氏菌对磺胺异噁唑(39.3%)、氨苄西林(39.0%)和粘菌素(39.0%)的耐药率较高,对其他抗菌药物的耐药率较低。全基因组测序分析能够100%预测美罗培南、氟苯尼考、阿奇霉素和阿莫西林/克拉维酸的耐药性,而且对恩诺沙星、四环素、复方新诺明、氨苄西林、头孢噻呋、磺胺异噁唑的预测符合率均超过95.0%。【结论】本研究结果表明,全基因组测序技术对沙门氏菌的血清分型和耐药性的预测具有较高的准确性和敏感性,是分析沙门氏菌血清型和...     

一株鸡源性沙门氏菌的基因组分析

为研究生鲜鸡肉中沙门氏菌的基因组信息,了解其耐药机制与致病机制,从新鲜鸡肉中分离出一株沙门氏菌FC10727,采用Illumina HiSeq 4000和PacBio对其进行全基因组测序与生物信息学分析.结果表明,FC10727基因组大小为4 638 420 bp,预测有4 016个基因,GC含量为54.15％.通过注...     

水稻白叶枯病菌GX1329基因组文库的构建及含编码TAL效应物基因的克隆的分离

由革兰氏阴性细菌水稻白叶枯病菌引起的水稻白叶枯病是亚洲、北美以及非洲部分地区最严重的水稻病害之一,水稻白叶枯病可使水稻减产高达50%以上.研究表明水稻白叶枯病菌的毒力主要依靠三型分泌系统所分泌的效应物.为了解水稻白叶枯病菌广西菌株GX1329中含有avrBs3/pthA家族基因的情况,本研究应用AluⅠ部分酶切其基因组DNA,构建了含有736个克隆的菌株GX1329的基因组文库.BamHⅠ酶切分析随机挑取的15个文库克隆表明,克隆的外源DNA随机性良好,克隆的最小片段为27.7 kb,最大为58.5 kb,平均大小为39.9 kb,文库克隆容量约为2.8×103 Mb,该文库中包含基因组中任一个基因的概率为99.4%.利用来自水稻白叶枯病菌菲律宾菌株PXO86的无毒基因avrXa10的第252位~第486位核苷酸序列作为探针,通过菌落原位杂交从GX1329基因组文库中筛选到37个含avrBs3/pthA家族基因的克隆.再通过Southern杂交分析,得到了17个独立克隆.这17个克隆中至少含有13个不同的avrBs3/pthA家族基因.这些基因在GX1329基因组中有的单独存在,有的两个或两个以上串联存在.本工作基本上明确了菌株GX1329基因组中avrBs3/pthA家族基因的数量,为进一步研究菌株GX1329中avrBs3/pthA家族基因的功能奠定了基础.     

人基因组的STS图谱分析

人基因组分析是现代生物学研究的前沿之一,其目标是分析人基因组3×10~9bp的全核苷酸序列,识别所有基因的位置和功能,基因组分析的第一步是要进行物理图谱分析,然后才能分段进行序列分析,最后将全部序列连成人基因组的全序列。由于人基因组之巨大,传统的图谱分析如cosmid,由于其克隆容量只有40Kb;而经典的分析方法如遗传连锁(geneticlinkage)分析的分辩力太低(1cM,相当于1Mb,megabase,百万碱基对),不宜于作为人基因组分析的第一克隆工具。1987年,美国华盛顿大学的M.Olson小组首次在成功地将长达数百Kb的人染色体片段以YAC(Yeast Artificial Chromosome)形式克隆到酵母细胞中,     

中国东北地区人乳头瘤病毒16型全基因组克隆及HPV16.HaCaT重组细胞模型的构建及初步鉴定

为构建含东北地区人乳头瘤病毒16型(HPV16)全基因组的HPV16.HaCaT细胞模型,收集中国东北地区HPV16单一感染患者宫颈脱落细胞,提取DNA,将HPV16全基因组分成4个区段,通过4对特异性引物对HPV16全基因组进行分段扩增,测序后进行序列拼接及核酸序列分析,克隆HPV16全基因组序列;通过细胞转染,构建含HPV16全基因组的HPV16.HaCaT重组细胞模型;利用聚合酶链式反应(PCR)和细胞免疫荧光法检测重组细胞内HPV16早期基因的表达.成功克隆出中国东北地区HPV16全基因组序列(GenBank登录号:MW320358);构建了东北地区HPV16全基因组的重组质粒及HPV16.HaCaT重组细胞模型;证明了 HPV16早期基因E1-E4、E5、E6和E7在重组细胞模型内均有表达,从而获得中国东北地区HPV16全基因组序列及含有HPV16全基因组的HPV16.HaCaT重组细胞模型.     

霍乱弧菌噬菌体VP2基因组序列的测定与分析

测定并分析了霍乱弧菌噬菌体VP2基因组序列,为VP2生物学特性和功能研究提供分子遗传学基础。为此构建了VP2DNA随机文库,鸟枪法(shot-gun)测定其全基因组序列。测序结果用软件Phrad-Prap拼接成最小重叠群(contig),引物步移法测定contigs问的缝隙(gap)序列,拼接后获得VP2全基因组序列。利用生物信息学技术分析’VP2基因组,最后对VP2和相关噬菌体做DNA聚合酶(DNA pol)基因的进化树分析。结果：VP2属短尾噬菌体科,基因组全长39853bp,为环状双链DNA,G C含量为50．56％,较高于霍乱弧菌测序菌株N16961基因组G C含量;VP2的基因组有碱基使用偏性;预测和注释了45个开放读码框(ORF),分析了DNA复制基因、衣壳蛋白和DNA包装基因、侵染相关基因。DNA pol进化树比较结果,VP2与链球菌噬菌体Cp-1和芽孢杆菌噬菌体GA-1分为一群。根据对VP2基因组序列的测定和分析预测了VP2的ORF,并分析了其中的功能基因,推测VP2在进化关系上属于噬菌体phi29样噬菌体。     

Molecular fingerprinting of Salmonella enterica subsp. enterica Typhimurium and Salmonella enterica subsp. enterica derby isolated from tropical seafood in South India

Salmonella enterica subsp. enterica Typhimurium and Salmonella enterica subsp. enterica Derby strains isolated from different seafood were genotyped by PCR-ribotyping and ERIC-PCR assays. This study has ascertained the genetic relatedness among serovars prevalent in tropical seafood. PCR-ribotyping exhibited genetic variation in both Salmonella serovars, and ribotype profile (II) was most predominant, which was observed in 10/18 of Salmonella enterica subsp. enterica Typhimurium and 7/17 Salmonella enterica subsp. enterica Derby isolates. Cluster analysis of ERIC-PCR for Salmonella enterica subsp. enterica Typhimurium strains exhibited nine different banding patterns and four strains showed >95% genetic homology within the cluster pairs. ERIC-PCR produced more genetic variations in Salmonella enterica subsp. enterica Typhimurium; nevertheless, both methods were found to be comparable for Salmonella enterica subsp. enterica Derby isolates. Discrimination index of PCR-ribotyping for Salmonella enterica subsp. enterica Typhimurium isolates was obtained at 0.674 and index value 0.714 was observed for Salmonella enterica subsp. enterica Derby strains. Molecular fingerprinting investigation highlighted the hypothesis of diverse routes of Salmonella contamination in seafood as multiple clones of Salmonella enterica subsp. enterica Typhimurium and Salmonella enterica subsp. enterica Derby were detected in same or different seafood throughout the study period.     

Genome-Wide Methylation Patterns in Salmonella enterica Subsp. enterica Serovars

The methylation of DNA bases plays an important role in numerous biological processes including development, gene expression, and DNA replication. Salmonella is an important foodborne pathogen, and methylation in Salmonella is implicated in virulence. Using single molecule real-time (SMRT) DNA-sequencing, we sequenced and assembled the complete genomes of eleven Salmonella enterica isolates from nine different serovars, and analysed the whole-genome methylation patterns of each genome. We describe 16 distinct N⁶-methyladenine (m6A) methylated motifs, one N⁴-methylcytosine (m4C) motif, and one combined m6A-m4C motif. Eight of these motifs are novel, i.e., they have not been previously described. We also identified the methyltransferases (MTases) associated with 13 of the motifs. Some motifs are conserved across all Salmonella serovars tested, while others were found only in a subset of serovars. Eight of the nine serovars contained a unique methylated motif that was not found in any other serovar (most of these motifs were part of Type I restriction modification systems), indicating the high diversity of methylation patterns present in Salmonella.     

Novel haemolysins of Salmonella enterica spp. enterica serovar Gallinarum

Haemolysins of Salmonella are important due to their probable role in pathogenesis of systemic salmonellosis and use in sub-serovar level typing. The present study was undertaken to determine haemolytic potential of Salmonella Gallinarum strains through phenotypic and genotypic methods. Amplification of haemolysin gene (clyA) and cytolysin gene (slyA) was attempted in order to determine their role in haemolysin production. Study on 94 strains of S. Gallinarum revealed the production of two types of haemolysis viz., beneath the colony haemolysis (BCH) or contact haemolysis and clear zone haemolysis (CZH). Haemolysis was observed on blood agar prepared with blood of cattle, buffalo, sheep, goat, horse, rabbit, guinea pig, fowl, and human blood group A, B, AB and O. Although, haemolysis was also observed on blood agar prepared with whole blood, clarity of zone was more evident on blood agar made from washed erythrocytes. Clear zone haemolysis was best observed on blood agar prepared with washed erythrocytes of goat and a total of 12% (11 of 94) S. Gallinarum strains under study produced CZH on it. The clyA gene could not be detected in any of the 94 strains under study, while slyA gene could be amplified uniformly irrespective of haemolytic potential (CZH) and haemolytic pattern (BCH) of the strains. The study suggested that the two types of haemolysis (CZH and BCH) observed among S. Gallinarum strains may not be due to either slyA or clyA gene products and thus there may be some other gene responsible for haemolytic trait in Gallinarum serovar. Different haemolytic patterns of strains under study indicated multiplicity of haemolysins in S. Gallinarum.     

Substructure within Salmonella enterica subsp. enterica isolates from Australian wildlife

Multilocus sequence typing of 56 Salmonella enterica subsp. enterica strains isolated from Australian wildlife hosts was performed. The results of population assignment algorithms revealed that the 56 strains could be subdivided into two distinct clades. Strains belonging to the two clades were further distinguished phenotypically, genotypically, and with respect to host distribution.     

Detection of hilA gene sequences in serovars of Salmonella enterica subspecies enterica

hilA gene promoter, component of the Salmonella Pathogenicity Island 1, has been found in Salmonella serovar Typhimurium, being important for the regulation of type III secretion apparatus genes. We detected hilA gene sequences in Salmonella serovars Typhi, Enteritidis, Choleraesuis, Paratyphi A and B, and Pullorum, by polymerase chain reaction (PCR) and hybridization techniques. The primers to carry out PCR were designed according to hilA sequence. A low stringency hybridization with the probe pVV441 (hilA open-reading-frame plasmid) was carried out. To find hilA gene sequences in other Salmonella sp. suggest that these serovars could have similar sequences of this kind of virulence genes.     

The Genomic Blueprint of Salmonella enterica subspecies enterica serovar Typhi P-stx-12

Salmonella enterica subspecies enterica serovar Typhi is a rod-shaped, Gram-negative, facultatively anaerobic bacterium. It belongs to the family Enterobacteriaceae in the class Gammaproteobacteria, and has the capability of residing in the human gallbladder by forming a biofilm and hence causing the person to become a typhoid carrier. Here we present the complete genome of Salmonella enterica subspecies enterica serotype Typhi strain P-stx-12, which was isolated from a chronic carrier in Varanasi, India. The complete genome comprises a 4,768,352 bp chromosome with a total of 98 RNA genes, 4,691 protein-coding genes and a 181,431 bp plasmid. Genome analysis revealed that the organism is closely related to Salmonella enterica serovar Typhi strain Ty2 and Salmonella enterica serovar Typhi strain CT18, although their genome structure is slightly different.     

Complete genome sequence of Salmonella enterica subsp. enterica serovar Typhi P-stx-12

We report here the complete genome sequence of Salmonella enterica subsp. enterica serovar Typhi P-stx-12, a clinical isolate obtained from a typhoid carrier in India.     

Methodologies for Salmonella enterica subsp. enterica subtyping: gold standards and alternatives

For more than 80 years, subtyping of Salmonella enterica has been routinely performed by serotyping, a method in which surface antigens are identified based on agglutination reactions with specific antibodies. The serotyping scheme, which is continuously updated as new serovars are discovered, has generated over time a data set of the utmost significance, allowing long-term epidemiological surveillance of Salmonella in the food chain and in public health control. Conceptually, serotyping provides no information regarding the phyletic relationships inside the different Salmonella enterica subspecies. In epidemiological investigations, identification and tracking of salmonellosis outbreaks require the use of methods that can fingerprint the causative strains at a taxonomic level far more specific than the one achieved by serotyping. During the last 2 decades, alternative methods that could successfully identify the serovar of a given strain by probing its DNA have emerged, and molecular biology-based methods have been made available to address phylogeny and fingerprinting issues. At the same time, accredited diagnostics have become increasingly generalized, imposing stringent methodological requirements in terms of traceability and measurability. In these new contexts, the hand-crafted character of classical serotyping is being challenged, although it is widely accepted that classification into serovars should be maintained. This review summarizes and discusses modern typing methods, with a particular focus on those having potential as alternatives for classical serotyping or for subtyping Salmonella strains at a deeper level.