Development and Application of a GeXP-multiplex PCR Assay for Detection of Seven Foodborne Pathogenic Bacterias

This experiment was developed to simultaneously detect the seven common foodborne pathogenic bacterias include E. coli O157:H7, Salmonella, V. cholera, L. monocytogenes, C. jejuni, V. Parahemolyticus and S. aureus. Seven pairs of specific primers were designed according to the conserved sequences of the genes from each pathogen available in the GenBank database. Single and mixed pathogen DNA templates were used to evaluate the specificity of the GeXP-multiplex assay. Control group was set up, recombinant plasmids were constructed, and samples of different DNA concentrations were randomly combined to verify the sensitivity, specificity, accuracy and anti-interference of the established GeXP method. To certify the accuracy and reliability of the GeXP assay, it was evaluated using 120 clinical specimens that were compared with the single PCR. The obtained results showed that the corresponding specific fragments of genes were amplified by the single and the multiplex GeXP PCR assay. The detection limit of GeXP was 10³ copies·μL^-1 when all of seven bacterial pathogens were detected. The results of the interference assay showed the presence of specific amplification peaks when different templates. The detection rate of the GeXP multiplex PCR method was 2.50% (3/120)-15.83% (19/120) while the conventional PCR was 2.50% (3/120)-15.00% (18/120), and GeXP multiple PCR detected 8 more positive cases, which means that, the GeXP was more sensitive and accurate in the detection of the clinical samples. In conclusion, this GeXP-based multiplex PCR is a high-throughput, specific and sensitive test to detect seven common foodborne pathogenic bacterias. This assay provides a method in rapid molecular diagnosis for mix clinical seven common foodborne pathogenic bacterias.     

基因组测序技术解析耐除草剂转基因水稻G2-7的分子特征

Molecular characterization, such as copy number and flanking sequence of foreign DNA fragment insertion site, is the important identity information, provided during safety assessment of genetic modified crop. In this study, the T-DNA insertion site, copy number and flanking sequences were identified in transgenic glyphosate-tolerant rice G2-7 based on whole genome sequencing in combination bioinformatics analysis method. 47.13 Gb clean sequence data for G2-7 was generated on Illumina NovaSeq 6000 platform. The junction reads mapped to boundaries of T-DNA and flanking sequences in G2-7 were identified by comparing with sequence of transformation vector and rice reference genome. The results showed that exogenous T-DNA fragments was integrated in the position of Chr. 1 36,189,491-36,189,507 with a single copy, 16 bp rice genome sequence was deleted at the insertion site and no insertion of vector backbone. 375 bp and 353 bp flanking host DNA sequence of 5′-end and 3′-end of the insertion DNA fragment were also obtained, respectively. The putative insertion location and flanking sequences were further confirmed by PCR amplification and Sanger sequencing. The results not only provided data support for safety assessment and event specific detection, but also demonstrated that WGS was an effective technique for identifying molecular characterization in rice.     

毛果杨全基因组磷酸根转运蛋白家族成员序列分析

磷酸根转运蛋白是一类主动转运磷酸根的重要载体蛋白.主要探讨毛果杨Populus trichocarpa全基因组中磷酸根转运蛋白(phosphate transporter)家族成员的系统发生和部分成员的理化性质、结构特点及亚细胞定位.以从毛果杨全基因组数据库中搜索并筛选得到的目标蛋白序列为基础,利用相关生物信息学软件对其进行系统发育分析,并利用一系列在线服务器对部分成员的理化参数、亲/疏水性、跨膜域、二级结构、三级结构和亚细胞定位进行了重点分析.结果表明,在毛果杨全基因组中共发现了31个磷酸根转运蛋白家族成员,根据其序列相似性,可分为4个亚家族,即PtrPHT1,PtrPHT2,PtrPHO1和PtrPHO2;PHT亚家族内的序列相似性高于PHO,并且高度保守;它们均为疏水性的全α型蛋白质,PHTs一般具有12个跨膜域,多于PHOs;不同亚家族成员的亚细胞定位不尽相同.因此,毛果杨磷酸根转运蛋白不同亚家族间分开较早,它们在杨树磷素代谢中可能起着不同的作用,共同维持并调控杨树体内的磷素平衡.     

利用AFLP技术分析植物基因组实验课程教学存在的问题及改革措施

《利用AFLP技术分析植物基因组》实验课在教学过程中存在技术原理理解难、实验操作难度大、优秀学生求知欲难满足等突出问题,通过编写教材、制作视频课件、实施开放性实验3个方面的尝试,收到了较好的教学效果。     

核果类果树中microRNAs的生物信息学预测及验证

microRNA(miRNAs)是一类非编码小分子RNA,在植物生长发育、新陈代谢以及逆境生理中起重要作用。基于生物信息学的基因搜索和同源搜索,从NCBI数据库中登录的核果类果树的EST和GSS中寻找miRNAs。从核果类果树的107982条ESTs和48273条GSSs中搜寻到了11条miRNAs前体序列,编码11条成熟体序列,并预测到19个靶基因,分别编码与生长发育、新陈代谢和转录调节等相关的蛋白。利用miRbase数据库进一步分析表明:11条miRNAs属于8个microRNA家族,分别来自桃和梅,其大小为20～23bp,其中6条为21bp,而且ppe-miR162a和ppe-miR164f保守性最高。采用poly(A)RT-PCR方法随机对预测的ppe-miR156h,pmu-miR482,ppe-miR399a,ppe-miR171a进行组织表达验证,发现ppe-miR156h在果梅花芽中表达,pmu-miR482,ppe-miR399a,ppe-miR171a在桃叶片中表达。     

上海市猪圆环病毒2型全基因组的克隆与序列分析

参照国外发表的猪圆环病毒2型(Porcine circovirus type 2,PCV-2)全基因组序列,设计合成了1对特异性引物,从上海市临床病料中提取PCV-2基因组DNA,进行PCR全基因扩增。回收PCR产物,将其插入pMD18-T载体,并对筛选出的阳性质粒进行测序。应用DNAStar序列分析软件,对所测PCV-2序列与GenBank中登录的国内外PCV-2毒株进行同源性比较。结果显示,PCV-2上海株与国内外毒株的核苷酸同源性高达95.0%～100%。进化树分析结果显示,其中9株PCV-2病毒属于PCV-2b亚型,3株PCV-2病毒属于PCV-2a亚型,且9株PCV-2b亚型毒株部分核苷酸位点显示其属于强毒力毒株。研究表明,上海市PCV-2感染以PCV-2b亚型为主,且氨基酸位点表明其具有较高的毒力。     

A组轮状病毒的基因组及其蛋白研究进展

轮状病毒基因组由11个片段组成,编码6种结构蛋白(VP1~VP4,VP6和VP7)和6种非结构蛋白(NSP1~NSP6)。文本对近年来A组轮状病毒基因组及其编码的结构蛋白和非结构蛋白的研究进展进行了综述。     

白菜型油菜和菜薹的InDel 标记开发及其RILs群体遗传连锁图谱的构建

 通过白菜型油菜‘R-O-18’和菜薹‘L58’的基因组重测序数据与白菜基因组的参考序列（‘Chiifu-401-42’的基因组序列）的比对,在全基因组范围内检测到了18 479 个短插入缺失变异位点（≤5 bp）。从中挑选了500 个插入/缺失片段为4 ~ 5 bp 的InDel 变异位点,将其设计成InDel 分子标记并进行试验验证,结果有452 个标记通过PCR 扩增出单一条带,但仅有106 个在‘R-O-18’和‘L58’间表现出多态性,346 个没有多态性,48 个在PCR 中没有扩增。亲本间具有多态性的106 个InDel 标记可用来检测以‘R-O-18’和‘L58’为亲本构建的RILs 基因型,并构建了一张包含99 个标记的遗传连锁图谱。     

Practical capability of a DNA pool‐based genome‐wide association study using BovineSNP50 array in a cattle population

Genome‐wide association mapping for complex traits in cattle populations is a powerful, but expensive, selection tool. The DNA pooling technique can potentially reduce the cost of genome‐wide association studies. However, in DNA pooling design, the additional variance generated by pooling‐specific errors must be taken into account. Therefore, this study aimed to investigate factors such as: (i) the accuracy of allele frequency estimation; (ii) the magnitude of errors in pooling construction and in the array; and (iii) the effect of the number of replicate arrays on P‐values estimated by a genome‐wide association study. Results showed that the Illumina correction method is the most effective method to correct the allele frequency estimation; pooling errors, especially array variance, should be taken into account in DNA pooling design; and the risk of a type I error can be reduced by using at least two replicate arrays. These results indicate the practical capability and cost‐effectiveness of pool‐based genome‐wide association studies using the BovineSNP50 array in a cattle population.     

狂犬病病毒的分子生物学研究进展

狂犬病是由狂犬病病毒引起的一种急性致死性人兽共患病,危害极大,因此,对狂犬病病毒的研究已成为一大热点。目前各国研究的重点主要集中在其病毒的分子生物学方面,以便更有效地防制本病。文章从病毒基因组及其编码的蛋白、致病机理等方面系统综述了狂犬病病毒分子生物学方面的研究进展,为该病的早期诊断、有效预防和控制该病提供理论基础。