树鼩呼肠孤病毒RT-nPCR检测方法的建立及初步应用

目的建立树鼩呼肠孤病毒(TRV)RT-nPCR检测方法,为树鼩的质量控制提供检测方法。方法从三批野外来源的具有感染临床症状的树鼩粪便中分离得到三株病毒,经电镜观察和聚丙烯酰胺凝胶电泳鉴定为哺乳动物呼肠孤病毒(MRV)。根据GenBank中已发表的MRV L1基因的保守区域,设计合成巢式引物,对所分离的三株树鼩呼肠孤病毒(TRV1、TRV2、TRV3)的RNA进行RT-nPCR扩增,优化反应条件,进行特异性、敏感性试验。应用RT-nPCR方法对25只野外来源的相同症状疑似病例样本进行检测。结果针对分离到的三株树鼩呼肠孤病毒的RNA进行RT-nPCR扩增,均得到513 bp的特异性目的片段,培养细胞及甲肝病毒、轮状病毒、单纯疱疹病毒阴性对照均未扩增出条带。敏感性试验结果显示可检测到的最小RNA模板浓度为0.01 pg/μL。25只树鼩粪便样本经RT-nPCR检测,有14只TRV阳性,其中死亡动物组10只,检出率为100%;存活动物组15只,检出率为27%。结论建立的TRV RT-nPCR检测方法特异、敏感、稳定,可用于TRV的常规检测。     

川牛膝MSAP反应体系的优化及引物筛选

目的：首次对川产道地药材川牛膝表观遗传多样性进行研究,建立并优化川牛膝甲基化敏感扩增多态性技术（MSAP）最佳反应体系。方法：以川牛膝苗期叶片为材料,利用正交试验设计对川牛膝MSAP反应体系中预扩增和选择性扩增中关键影响因素进行考察,建立川牛膝MSAP反应最佳体系。结果：川牛膝MSAP最佳反应体系为：酶切体系（20 μL）,300 ng DNA、1.5 μL EcoRI、1.5 μL HpaII或1.5 μL MspI（HpaII）;连接体系（25 μL）：15 μL酶切产物、1 μL EcoRI adaptor、1 μL HpaII/MspI adaptor、0.2 μL T4连接酶;预扩增体系（25 μL）：连接产物1 μL、rTaq酶1 U、引物分别1.5 μL、dNTP 2 μL;选择性扩增体系（25 μL）：预扩产物稀释50倍、rTaq酶1 U、引物1.5 μL、dNTP 3 μL,并运用优化后的体系,最终从川牛膝MSAP的256对引物中筛选出有效引物6对。结论：优化后的体系保证了稳定的图谱、清晰的条带,筛选出的6对引物组合特异性好,能够用于后续川牛膝DNA甲基化相关实验研究,同时,为其他药用植物MSAP分析提供了参考。     

HLA-DRB1基因芯片与PCR-SSP分型的比较

目的：比较基因芯片和PCR-SSP用于汉族南方人群HLA-DRB1基因分型的结果，评价分型芯片的准确性。方法：77份待分型样本，分别用等位基因特异的PCR-SSP和基因分型芯片对DRB1分型。芯片分型通过组间特异引物扩增基因组DNA，扩增用荧光标记。扩增标记后的产物与分型芯片的探针杂交，通过杂交产生的荧光信号确定样品的HLA-DRB1基因亚型，比较两种方法分型所获得的结果，不吻合的样本全部经第三方验证或测序。结果：77份样本，两种方法分型全部成功。分型结果的吻合率为93％。不吻合样本6份，其中SSP定型纯合子4份，芯片分型全部为杂合子。经第三方验证，证实芯片对纯合子和杂合子的分型全部正确。另外2份不吻合的样本经测序，证实SSP分型错误1份，芯片分型错误1份。芯片的重复率为96％。结论：基因芯片是一种理想的分型方法，具有特异性高、重复性好、操作简便、所需样本量少、一次可作多份样本的优点。     

一种新型通用引物多重PCR技术可特异性检测Y染色体序列标签位点

目的:建立通用引物多重PCR(UP-M-PCR)技术,检测Y染色体微缺失。方法:选取50例健康男性,针对Y染色体AZFa,AZFb,AZFc和AZFd区15个标签位点(STS),选择15对特异性引物和1对UP,将UP加在特异性引物对的5’端,按扩增片段大小组建4组稳定可靠的UP-M-PCR体系,分析其扩增效率和特异性。结果:UP引入M-PCR对检测体系无影响。UP-M-PCR可特异性扩增AZF区15个STS,且较M-PCR特异性更好,条带更清晰。结论:UP-M-PCR体系较M-PCR操作方便,扩增效率均衡,特异性高,是男性不育患者Y染色体微缺失筛查的新方法。     

用TaqMan探针实时定量PCR法快速检测鼠疫耶尔森菌

目的建立一种快速、准确、特异的定量检测鼠疫耶尔森菌的方法。方法针对鼠疫耶尔森菌特异的染色体标识序列设计引物和TaqMan探针，进行实时定量聚合酶链反应(polymerase chain reaction，PCR)分析，优化反应体系，检测其灵敏度、特异性和重复性。结果以EV76灭活培养物为对象，检测灵敏度可达每反应体系0．08CfU；30株非鼠疫菌株的检测结果表明，对照菌株均为阴性，检测特异性良好；对同一样品进行17次重复检测，其循环阈值的标准偏差为0．35。结论TaqMan探针法能够灵敏、特异、稳定地对鼠疫耶尔森氏菌进行定量检测，为鼠疫监控、诊断提供有力手段。     

Failure to demonstrate human papillomavirus DNA in epithelial ovarian cancer by general primer PCR

One of the recent controversies with substantial clinical interest is the role of HPV in pathogenesis of ovarian cancer. The available highly conflicting data are based on analysis of 175 ovarian carcinomas so far. As an attempt to further elucidate this issue, the first systematic study of HPV detection in ovarian cancer was carried out using a highly sensitive general primer PCR (confirmed by hybridization for low- and high-risk HPV types separately) in a series of 98 histologically and clinically well-characterized epithelial ovarian malignancies. Despite the high (fg) sensitivity and a wide HPV type coverage of the technique used, all 98 ovarian carcinomas failed to demonstrate any signs of HPV DNA whatsoever. The preexisting 12 reports comprising a total of 175 ovarian tumors analyzed for HPV were summarized, giving highly discrepant results (i.e., detection rates from 0 to 100%) with the overall HPV DNA detection rate of 25.7%. The reasons for these discrepant findings are most probably technical. Our data are consistent with those of the majority of the most recent reports failing to disclose HPV DNA in ovarian neoplasia. The present completely negative results make the authors inclined to conclude that HPV is highly unlikely to play any causal role in the pathogenesis of epithelial ovarian neoplasia.     

引物特异性HCV基因分型检测方法的建立及应用

[目的]建立引物特异性HCVRNA基因分型的方法，并应用于临床诊断。[方法]采用型特异性引物建立单管逆转录聚合酶链反应（RT-PCR）和双管引物特异性套式聚合酶链反应（NEST-PCR）检测技术，1b和3a一管内扩增，1a、2a和2b一管内扩增，分别对147例HCV抗体阳性的标本，设计HCV的C区特异性引物，采用Trizol浓缩RNA进行双管巢式PCR分型。[结果]统计结果表明我国丙型肝炎流行主要以1b（Ⅱ）和2a（Ⅲ）2种基因型为主，但有部分1a、2b、3a的HCV感染者检出，其中1a为1．36％（2／147），1b为65．99％（97／147），2a为6．12％（9／147），3a为1．36％（2／147），1b／2a型混合占22．45％（33／147），1a／2b混合型为0．68％（1／147），未分出型或其它型者分别为2．04％（3／147）。[结论]本文建立的方法具有良好的特异性和敏感性，可同时区分5种HCV基因型，我国北方地区发现有1a和3a基因型感染病例。     

Evolutionary dynamics of an expressed MHC class IIβ locus in the Ranidae (Anura) uncovered by genome walking and high-throughput amplicon sequencing

The Major Histocompatibility Complex (MHC) is a genomic region encoding immune loci that are important and frequently used markers in studies of adaptive genetic variation and disease resistance. Given the primary role of infectious diseases in contributing to global amphibian declines, we characterized the hypervariable exon 2 and flanking introns of the MHC Class IIβ chain for 17 species of frogs in the Ranidae, a speciose and cosmopolitan family facing widespread pathogen infections and declines. We find high levels of genetic variation concentrated in the Peptide Binding Region (PBR) of the exon. Ten codons are under positive selection, nine of which are located in the mammal-defined PBR. We hypothesize that the tenth codon (residue 21) is an amphibian-specific PBR site that may be important in disease resistance. Trans-species and trans-generic polymorphisms are evident from exon-based genealogies, and co-phylogenetic analyses between intron, exon and mitochondrial based reconstructions reveal incongruent topologies, likely due to different locus histories. We developed two sets of barcoded adapters that reliably amplify a single and likely functional locus in all screened species using both 454 and Illumina based sequencing methods. These primers provide a resource for multiplexing and directly sequencing hundreds of samples in a single sequencing run, avoiding the labour and chimeric sequences associated with cloning, and enabling MHC population genetic analyses. Although the primers are currently limited to the 17 species we tested, these sequences and protocols provide a useful genetic resource and can serve as a starting point for future disease, adaptation and conservation studies across a range of anuran taxa.     

蛤蚧及其伪品微型DNA条形码的引物筛选

文章旨在筛选出适合于扩增蛤蚧及伪品微型DNA条形码引物。通过引物设计软件,经过多序列比对,设计了两对引物mini-barcode F2,mini-barcode R2;mini-barcode F3,mini-barcode R3,并用这两对引物和目前已报道的微型条形码引物PCR扩增蛤蚧及伪品61个样本,结果表明mini-barcode F2,mini-barcode R2和mini-barcode F3,mini-barcode R3这两对引物的PCR扩增成功率分别为100%和90%,而目前已报道的微型条形码引物PCR扩增蛤蚧及其伪品得到了长度约500 bp非微型条形码序列片段。因此目前已报道的微型条形码引物不适用于扩增蛤蚧及伪品,该研究筛选出了适合于蛤蚧及伪品的微型条形码引物。     

The insect cytochrome oxidase I gene: evolutionary patterns and conserved primers for phylogenetic studies

Insect mitochondrial cytochrome oxidase I (COI) genes are used as a model to examine the within-gene heterogeneity of evolutionary rate and Its implications for evolutionary analyses. The complete sequence (1537 bp) of the meadow grasshopper ( Chorthippus parallelus ) COI gene has been determined, and compared with eight other Insect COI genes at both the DNA and amino acid sequence levels. This reveals that different regions evolve at different rates, and the patterns of sequence variability seems associated with functional constraints on the protein. The COOH-terminal was found to be significantly more variable than Internal loops (I), external loops (E), transmembrane helices (M) or the NH2 terminal. The central region of COI (M5-M8) has lower levels of sequence variability, which Is related to several Important functional domains In this region. Highly conserved primers which amplify regions of different variabilities have been designed to cover the entire insect COI gene. These primers have been shown to amplify COI in a wide range of species, representing all the major insect groups; some even In an arachnid. Implications of the observed evolutionary pattern for phylogenetic analysis are discussed, with particular regard to the choice of regions of suitable variability for specific phylogenetic projects.