平行等位基因特异序列检测技术对低比率乙型肝炎病毒耐药突变位点的检测

目的评价应用平行等位基因特异性序列检测技术(parallel allele-specific sequencing,PASS),对乙型肝炎病毒(HBV)进行耐药突变位点检测效果。方法应用PASS方法对17例未经抗病毒治疗和50例经抗病毒治疗失败的乙肝患者血浆进行耐药突变检测,其中50例乙肝抗病毒治疗失败的患者血浆同时采用测序法进行耐药分析。结果 17例未接受抗病毒治疗的患者样本中有8例检测到低比率的耐药突变位点(0.04%～16.89%),9例未发现有耐药突变。50例抗病毒治疗失败患者标本有7例PASS法和测序都未检测到耐药突变,16例PASS法检测到低比率耐药突变(0.03%～5.95%),测序法未检测到任何突变位点,27例PASS法和测序法都检测到了耐药突变(36%～100%)。应用PASS法进行连锁分析表明当多个耐药突变位点以高比率在同一患者体内检测到时,它们多以连锁的关系存在于同一病毒基因组。结论 PASS方法对检测HBV感染个体中耐药病毒群具有高度的灵敏性和特异性,并能对多个耐药突变位点进行连锁性分析。

HBV前S基因芯片检测研究

新发明的芯片法检测前S基因缺失突变发现一个乙肝携带者体内往往存在多种突变，传统分析方法耗时耗力。经研究发现，单个PCR反应产物足够对前s基因缺失分型。个体基因克隆后的产物用于前S芯片法杂交检测，比传统DNA测序法省时经济。前S芯片为0．7cm^2大小的尼龙膜，对于没有测序仪器的临床机构来讲更方便经济。同时，芯片自动检测多种前S克隆，更适合于对慢性乙肝病毒携带者大范围的突变扫描。双盲法对比芯片法和传统测序法，二者对前S突变的检出率相近。

基于16S rDNA与多拷贝基因hyvⅠ/hyvⅡ的柑橘黄龙病菌PCR检测体系比较

柑橘黄龙病是由α变形菌纲细菌黄龙病菌耐热性亚洲种(Candidatus Liberibacter asiaticus,CLas)引起的柑橘毁灭性病害,世界柑橘产量居前三的中国、巴西、美国近年受灾严重。目前对CLas的分子检测主要靶点基因序列有16S rDNA、β-操纵子和外膜蛋白基因(Bove,2015),但检出率及灵敏度相对较低,而利用多拷贝基因序列检测黄龙病的研究较少。

Detection and Sequence Analysis of Escherichia coli Virulence Genes

[Objective] This study aimed to explore the pathogenic mechanism of E., cherichia coll. [Method] An E. coil strain isolated from raccoon dogs in vivo was studied, which had been identified, PCR was used to detect the gene of irp2 （301 bp） and fyuA （953 bp） related to E. coil virulence and PCR products were s, quenced. [Result] The genes of irp2 and fyuA were successfully amplified in boi strains isolated from raccoon dogs. Compared with the GenBank, the identity of tTr irp2 gene sequence and the fyuA gene sequence of the strain reached 98.5% 99.2% and 98.9%-100% respectively. Compared with each other, the identity of tt- two gene sequences of irp2 was 99.3%, and that between the two fyuA gene se quences was 98.9%. [Conclusion] This study provided scientific experimental data fi E. coil pathogenicity, prevention, diagnosis and treatment.

连续入侵性信号扩大反映(SISAR)原理及应用简介

连续侵入性信号扩大反应(the serial invasive signal amplification reac-tion,SISAR)不同于以前的 Third Wave Technologies 公司推出的侵入信号扩大反应,它应用两个连续的侵入反应在恒温系统中将靶位点的信号放大,并通过激发荧光来显示信号。具有较高的灵敏性和严紧度,是不需要热循环 PCR 反应和限制性酶切反应的一种另类序列检测手段。

重组生命

微电子技术与生命科学是本世纪后期发展最为迅速的两大领域,目前,凝结这两大学科研究成果的产品层出不穷,其代表就是DNA生物芯片,也可称之为基因芯片。 美国加州的Affymetrix公司于1996年首次在市场上推出商业化的基因芯片和芯片阅读器,最初的产品只用于分析爱滋病毒两种酶的基因变异。今天,美国已有6家生产基因芯片的公司在市场上推出了20余种基因芯片。虽然当前生产基因芯片的都是一些小公司,而且都还没有盈利,但投资者的热情却丝毫未减。因为他们相信,基因芯片将会象计算机芯片一样迅速普及到生物科学的各个领域并发展成为一门新兴工业。 这一新兴工业的创始人名叫Zafaroni。

Analysis of the Flanking Sequence and EventSpecific Detection of Transgenic Line W-4 of Brassica napus

The genetically modified high-oleic rapeseed （Brassica napus L.） line W-4 was obtained by transforming a binary vector which harbored an inverted repeat expression cassette of fad2 gene into the rapeseed cultivar Westar.The transformation was mediated by Agrobacterium.The flanking sequences to both the left and right borders of T-DNA insertion site were amplified by thermal asymmetric interlaced PCR （TAIL-PCR） from the genomic DNA of the transgenic rapeseed line W-4.The flanking sequences to the right border was 290 bp in length and the nucleotide composition was 31.27％ for G＋C content while 68.73％ for A＋T content.The flanking sequence to the left border was 365 bp in length and the G＋C content was 32.6％ and the A＋T content was 67.4％,indicating that the T-DNA was integrated in the A/T-rich region.Further more,sequence alignment analysis showed a deletion of 62 bp including the right border of pCNFIRnos and the integration of the whole left border except a change of G to A.That was to say,the integration of the T-DNA in the transgenic line W-4 not involved in the vector sequences.Based on both flanking sequences as well as the left and right borders of the T-DNA sequences,two pairs of specific primers TLF/TLR and TRF/TRR were designed.Using the primers the event-specific PCR detection method for transgenic rapeseed line W-4 was established.By the PCR,two fragments of 485 and 405 bp were amplified from the W-4 genomic DNA as expected,while no products were amplified from the genomic DNA of other transgenic rapeseed lines and non-transgenic rapeseed line.And by the PCR it is possible to detect the W-4 genomic DNA from a mixed sample of genomic DNA.The limit of the detection for the qualitative PCR assay was 0.1％.The method developed in this work is highly specific,sensitive and suitable for event-specific detection of the transgenic rapeseed line W-4.

Molecular cloning and mRNA expression analysis of myosin heavy chain(MyHC)from fast skeletal muscle of grass carp,Ctenopharyngodon idella

The myosin heavy chain(MyHC)is one of the major structural and contracting proteins of muscle.We have isolated the cDNA clone encoding MyHC of the grass carp,Ctenopharyngodon idella. The sequence comprises 5 934 bp,including a 5 814 bp open reading frame encoding an amino acid sequence of 1 937 residues.The deduced amino acid sequence showed 69%homology to rabbit fast skeletal MyHC and 73%–76%homology to the MyHCs from the mandarin fish,walleye pollack,white croaker,chum salmon,and carp.The putative sequences of subfragment-1 and the light meromyosin region showed 61.4%–80%homology to the corresponding regions of other fish MyHCs.The tissue-specific and developmental stage-specific expressions of the MyHC gene were analyzed by quantitative real-time PCR.The MyHC gene showed the highest expression in the muscles compared with the kidney,spleen and intestine.Developmentally,there was a gradual increase in MyHC mRNA expression from the neural formation stage to the tail bud stage.The highest expression was detected in hatching larva.Our work on the MyHC gene from the grass carp has provided useful information for fish molecular biology and fish genomics.

Detection and Protein Modelling for Consensus Region of hsp70 Gene Family in Egyptian Arundo donax

Degenerate primers are particularly useful in amplifying homologous genes from different organisms. This paper describes a method for designing degenerate primers for a given multiple alignment of DNA sequences of hsp70 gene family using ClustalW algorithm and detect the consensus region in gene family of hsp70 in a plant species which have not any recorded information about hsp70 gene family in the Genbank of National Centre of Biotechnology Information （NCBI） like Arando donax .The sequenced consensus sequence ofArando donax is considered a gene marker in building the genome map sequence. The BLASTn program is used to find a homology between more than one accession numbers of DNA sequences, （X67711.2） was for Oryza sativa （hsp70）, （AY372071. l） was for Nicotiana tabacum （hsp70） and （L41253.2） was for Lycopersicon esculentum （Hsc70）. In silco PCR module was performed to detect the melting temperatures （Tm） and predicted the PCR product size （783 bp）.The result of designed degenerate primers showed that there was a homology founded among the designed primers and the DNA templates of the recorded sequences （AY372071.1, X67711.2 and L41253.2） with at least 80% identity. The designed degenerate primers were used to isolate a consensus region ofhsp70 gene family ofArando donax at the expected molecular weight （783 bp）. The isolated PCR product, （783 bp） ofArando donax was sequenced and submitted to the Database of Japan （DDBJ） with accession number AB819871. The ORF finder tool translated the accession number AB819871 and gave a selected frame which used to build 3D structure model. In conclusion, this study focused on the importance of designing the degenerate primers to isolate the gene family and predict the 3D structure of gene family depending on the ORF finder tool of Genebank.