应用 DNA 条形码技术鉴定凭祥口岸蚊种

为探索DNA条形码技术在蚊类鉴定中的应用,本研究扩增了广西凭祥地区5种蚊虫的COⅠ基因序列,进行比对分析。基于Kimura双参数模型计算23只蚊子COⅠ序列的种内种间遗传距离结果表明种间遗传距离显著大于种内遗传距离。通过NJ法构建系统发育树聚类分析,不同种类的蚊虫位于不同的分支,同属的蚊种分支较不同属的蚊种分支较短,种内分支则更短。上述结果都与形态学鉴定结果一致。这些研究结果表明, DNA条形码技术对于本研究中所涉及的蚊种具有较好的区分效果,可以作为一种有效的工具在蚊虫鉴定中进行应用。     

基于线粒体COⅠ基因序列进行库蠓亚属(双翅目:蠓科)近似种的分子鉴定

为了更快速、准确地鉴别库蠓近似种以及弥补传统形态学在种类鉴定中存在的操作繁琐、难度大等不足,本文采用DNA测序的方法获得库蠓亚属的刺螫库蠓Culicoides punctatus、灰黑库蠓C.pulicaris和新替库蠓C.newsteadi等3种库蠓近似种的部分线粒体COⅠ基因序列,并对其进行分子鉴定;基于Kimura 2-parameter模型分析遗传距离,同时应用MEGA 6.0软件构建系统发育树。结果显示,序列经过Clustal W比对及人工校对后,上述3种库蠓的COⅠ序列长度为394 bp;遗传距离在种内和种间具显著差异(P﹤0.05);系统发育树中不同库蠓种类各自构成单系(群),同种类不同地理种群聚为一支。本研究初步证实了线粒体COⅠ序列可应用于库蠓近似种的分子鉴定。     

DNA条形码研究进展

DNA条形码（ DNA Barcoding ）技术是一种新的物种识别方法,它是分子生物学和生物信息学相结合的产物。在最近几年里,该技术已成为生物分类学中引人注目的研究热点。这一概念认为,类似于商店里使用扫描仪读取条形码,对地球上每一种生物通过快速分析其DNA中的一段基因（线粒体细胞色素c氧化酶Ⅰ亚基, mt COⅠ）加以识别。理论上, DNA条形码已被证明在生物分类鉴定中具有非常重要的作用,并推动了一系列相关学科的发展,但目前不同分类学家对其持的意见也不尽相同。本文综述了DNA条形码技术的产生、发展概况、原理与操作及其在分类中的应用,并概括了DNA条形码在应用于物种分类中可能存在的问题。     

六种蚊虫DNA条形码序列分析

传统分类学鉴定需要有完整无残缺的成虫标本,对于非昆虫分类学研究人员应用传统分类学方法鉴定蚊虫较为困难;本研究旨在发掘更多适用于蚊类鉴定的DNA条形码,实现口岸一线蚊类的快速准确鉴定。本文通过提取14个蚊虫样品的基因组DNA,使用通用引物PCR扩增ITS1、ITS2、Cytb、COI基因及序列,PCR产物双脱氧法测序,结合NCBI中相关数据分析部分ITS2、Cytb、COI序列的相似性和遗传关系。结果显示:获得了14个蚊虫样品的部分ITS1、ITS2、Cytb、COI序列的非全长碱基序列共计39条;依据种内遗传距离和相似度将14个未知蚊虫分子分类鉴定为6种蚊虫:凶小库蚊、致倦库蚊、尖音库蚊、赫坎按蚊、白纹伊蚊、里海伊蚊;碱基使用率统计表明所测样品的Cytb、COI基因符合昆虫线粒体DNA碱基组成;构建基因ITS2、Cytb、COI系统进化树,符合蚊虫系统发育遗传进化理论。本研究对14个未知蚊虫样品的进行了分子鉴定,为蚊类的快速准确识别鉴定提供基础数据。     

我国部分省份(地区)汉族人群HLA-Ⅰ类基因多态性地区差异分析

目的 探讨我国部分省份(地区)汉族人群HLA-Ⅰ类经典基冈座位HLA-A、HLA-B、HLA-Cw位点的群体遗传学特点及其基因频率分布的地区差异.方法 选取1014例无关汉族拟行造血干细胞移植治疗患者及其健康家系供者的血液样本,提取基因组.DNA后,采用序列特异性引物聚合酶链式反应(PCR-SSP)分型技术进行HLA-A、HLA-B、HLA-Cw位点基因分型,分析不同地区汉族人群及不同种族间的基因频率分布特征.基于文献报道的我国不同地区汉族人群及不同种族的HLA-Ⅰ类基因频率资料,计算种群间遗传距离(D),比较不同地区汉族人群及不同种族间遗传距离差异.结果 Hard-Weinberg吻合度检验表明,本研究抽样群体适于进行遗传学统计分析.HLA-A位点共检测出14种基因型,最常见的是A*02(0.330)、A*11(0.240)、A*24(0.155)、A*33(0.075);HLA-B位点共检测出27种基因型,最常见的是B*13(0.134)、B*15(0.143)、B*40(0.133)、B*46(0.102);HLA-Cw位点共检测出13种基因型,最常见的是Cw*01(0.157)、Cw*03(0.247)、Cw*07(0.181)、Cw*08(0.106).群体汉族与其他人种间HLA-A、HLA-B基因频率差异均有统计学意义(P<0.05);除兰州汉族人群仅同南方汉族、湖南、山东、江苏、台湾汉族人群间HLA-A、HLA-B基因频率差异有统计学意义(P<0.05)外,其余各地区汉族人群间HLA-A、HLA-B基因频率差异均有统计学意义(P<0.05).各地区汉族人群间平均遗传距离D=0.164,辽宁和北方汉族人群间遗传距离(D=0.064)最小,江苏与湖南汉族人群间遗传距离(D=0.299)最大;不同地区汉族人群间遗传距离普遍小于种族间遗传距离.结论 我国不同地区汉族人群HLA-Ⅰ类基因频率分布存在显著差异,但其差异要明显小于世界不同人种间的分布差异.我国汉族人群所特有的HLA-Ⅰ类基因频率分布格局资料对区域性疾病的个性化治疗、遗传易感性及疾病防治等研究具有很好的理论及应用价值.     

三峡库区上、下游血吸虫病流行区钉螺线粒体cox1基因遗传变异研究

目的研究三峡库区上、下游血吸虫病流行区钉螺线粒体DNA细胞色素C氧化酶亚单位t（c毗1）基因的遗传变异。方法采集三峡库区上游四川、云南及下游安徽、湖北4省共7个地、市的钉螺样本,提取基因组DNA,PCR特异性扩增线粒体cox1基因并测序,用ClustalX（1．81）软件进行多序列比对,MEGA（4．0）软件Kimura2-parameter法计算遗传距离,邻接法（NJ）和最大简约法（MP）构建系统发生树。结果上游与下游不同地域株钉螺间cox1基因差异约为16％．下游地区的肋壳与光壳钉螺cox1基因差异约为3．7％,上游不同地域株钉螺碱基差异约为5．4％。遗传距离显示,上游四川与云南地域株的遗传距离为0．022～0．050,下游安徽与湖北地域株的遗传距离为0．014～0．027,而上游与下游各地区螺群间的遗传距离在0．127～0．138之间,明显大于上游或下游各地区螺群内的遗传距离。进化树结果表明,下游湖北的荆州、石首和安徽的芜湖、宁国钉螺形成一支系,上游四川的绵竹、新都钉螺同属一支系。但两种方法构建的进化树在云南大理钉螺的归属上存在差异,MP法提示大理钉螺从上游的分支中独立出来,单独形成一类。结论上游与下游不同地域株钉螺cox1基因遗传差异较显著,下游肋壳和光壳钉螺种群内遗传变异较小,而上游光壳钉螺种群内遗传变异较大。     

东方田鼠免疫抑制模型对日本血吸虫感染性的研究

目的建立东方田鼠免疫抑制模型,初步探讨淋巴细胞在东方田鼠抗血吸虫病机制中的作用。方法成年健康的东方田鼠40只,分成4组（每组10只）：免疫抑制＋血吸虫感染组、免疫抑制组、血吸虫感染组和空白对照组。免疫抑制剂每周注射3次,连续使用6周,建立东方田鼠免疫抑制的动物模型,用脾淋巴细胞转化试验和ELISA抗体检测进行免疫抑制模型的初步鉴定。在尾蚴攻击感染各组动物后42d,麻醉处死各组动物后进行解剖,灌注冲虫,收集虫体和肝脏虫卵镜检。结果免疫抑制模型淋巴细胞增殖受到显著抑制;免疫抑制组血吸虫抗体水平显著低于未抑制组和空白对照组;免疫抑制和未抑制的东方田鼠均未检出成虫和虫卵。结论成功建立东方田鼠免疫抑制模型,淋巴细胞在东方田鼠抗血吸虫病机制中可能不发挥直接作用。     

中国钩端螺旋体rRNA基因限制性内切酶酶切位点多态性分类研究

目的初步建立我国致病性钩端螺旋体ｒＲＮＡ基因限制性内切酶酶切位点多态性分类体系。方法采用ｒＲＮＡ基因限制性内切酶酶切位点多态性（ＭＲＳＰ）方法对６５个血清型７５株国内外钩端螺旋体参考株及２７株野生株进行分析。结果１６ＳｒＲＮＡ及２３ＳｒＲＮＡ基因在ＨｉｎｆⅠ、ＤｄｅⅠ的酶切位点上呈多态性,分别存在１８种和２４种不同的图谱。结合两种方法则有３４种不同的图谱。致病性钩端螺旋体间ＭＲＳＰ谱型差别相对较小,遗传距离较近,而它们与非致病性钩端螺旋体的ＭＲＳＰ谱型相差大,遗传距离远。国内主要流行型的ＭＲＳＰ只表现为ＭＲＳＰ１型和２型,非主要流行型的ＭＲ－ＳＰ型变化较多。大多数野生株与相应的参考株的ＭＲＳＰ型相同,个别具有不同ＭＲＳＰ型的野生株与相应参考株的遗传距离较近。结论ｒＲＮＡ基因限制性内切酶酶切位点多态性（ＭＲＳＰ）可用于钩体分类,而且是钩体病分子流行病学调查的一种好方法     

广西3个少数民族17个Y-STR位点的遗传多态性

目的　研究广西侗、苗、壮3个少数民族17个Y-STR基因座的多态信息及其在法医学应用价值。方法　少数民族口腔拭子用磁珠法提取DNA后用Yfiler复合扩增试剂盒扩增,3100XL型遗传分析仪进行基因分型。计算基因频率,多样性和遗传距离。结果　获得3个少数民族309名男性17个Y-STR基因座的等位基因频率分布资料,3个少数民族单倍型共观察到275种。单倍型多样性：侗族为0.9972,苗族为0.9966,壮族为0.9964;遗传距离中苗族跟侗族的遗传距离最近。结论　由17个Y-STR位点组成的单倍型多样性在种族鉴别,父权鉴定和群体遗传学等方面有很高的应用价值。     

RAPD–SCAR marker and genetic relationship analysis of three Demodex species (Acari: Demodicidae)

For a long time, classification of Demodex mites has been mainly based on their hosts and phenotype characteristics. The study was the first to conduct molecular identification and genetic relationship analysis for six isolates of three Demodex species by random amplified polymorphic DNA (RAPD) and sequence-characterized amplified region (SCAR) marker. Totally, 239 DNA fragments were amplified from six Demodex isolates with 10 random primers in RAPD, of which 165 were polymorphic. Using a single primer, at least five fragments and at most 40 in the six isolates were amplified, whereas within a single isolate, a range of 35-49 fragments were amplified. DNA fingerprints of primers CZ 1-9 revealed intra- and interspecies difference in six Demodex isolates, whereas primer CZ 10 only revealed interspecies difference. The genetic distance and dendrogram showed the intraspecific genetic distances were closer than the interspecific genetic distances. The interspecific genetic distances of Demodex folliculorum and Demodex canis (0.7931-0.8140) were shorter than that of Demodex brevis and D. canis (0.8182-0.8987). The RAPD-SCAR marker displayed primer CZ 10 could be applied to identify the three Demodex species. The 479-bp fragment was specific for D. brevis, and the 261-bp fragment was specific for D. canis. The conclusion was that the RAPD-SCAR multi-marker was effective in molecular identification of three Demodex species. The genetic relationship between D. folliculorum and D. canis was nearer than that between D. folliculorum and D. brevis.     

东方田鼠ADCC体外杀伤日本血吸虫童虫效果的初步观察

为了进一步了解东方田鼠抗日本血吸虫病的机理,我们对东方田鼠抗体依赖细胞介导细胞毒(ADCC)体外杀伤日本血吸虫童虫效果进行了观察.用来源于东方田鼠和昆明鼠腹腔细胞的巨噬细胞(M)、嗜酸性粒细胞(Eos)作为效应细胞,分别以东方田鼠阳性/阴性血清、昆明鼠阳性/阴性血清进行ADCC试验.在培养3～6h时,所有东方田鼠M和Eos样本孔中均有大量细胞粘附于童虫,而昆明鼠M和Eos样本孔中几乎没有细胞粘附;培养20～24h、44～48h的童虫死亡率显示1.东方田鼠阳性/阴性血清的杀伤力明显高于相应的昆明鼠阳性/阴性血清的杀伤力;2.东方田鼠/昆明鼠的阳性血清诱导的童虫死亡率高于相应的东方田鼠/昆明鼠的阴性血清诱导的童虫死亡率;3,4种效应细胞样本孔以及细胞空白对照孔中的童虫死亡率基本相似.上述结果提示,东方田鼠血清具有明显的杀伤日本血吸虫童虫的作用,可能是东方田鼠抗日本血吸虫病机理中起决定作用的因子;东方田鼠M和Eos对日本血吸虫童虫具有天然粘附能力.     

Growth of Clethrionomys gapperi and Microtus pennsylvanicus in captivity.

Growth of Clethrionomys gapperi and Microtus pennsylvanicus from southern Manitoba was documented from birth to 30 days. Clethrionomys gapperi were smaller and grew more slowly (g/day) than M. pennsylvanicus, although both species were weaned at the same age. There were significant differences in growth rates between sexes in M. pennsylvanicus and among litter sizes in both species. Most of the variability in growth was due to differences among litters. The patterns of development was similar to those reported for other North American species of Clethrionomys and Microtus.     

Identification of astigmatid mites using ITS2 and COI regions

Identification of astigmatid mites based on their morphological characteristics is difficult because of the similarity of their organs, especially in immature mites. The ribosomal second internal transcribed spacer (ITS2) and the mitochondrial cytochrome oxidase subunit I (COI) regions are highly conserved in the eukaryotes and are usually used as barcodes. The ITS2 and COI regions of six species of astigmatid mites (Aleuroglyphus ovatus, Blomia tropicalis, Dermatophagoides farinae, Dermatophagoides pteronyssinus, Euroglyphus maynei, Tyrophagus putrescentiae) were obtained by polymerase chain reaction and sequenced. The lengths of the ITS2 sequences varied from 316 to 488 bp, while the COI regions were 377 or 378 bp long. Considering the ITS2 genes, the intraspecific genetic distance was in the range of 0.00–0.077844, whereas the interspecific genetic distance was 0.202426–0.912959. The values were 0.000–0.029748 and 0.138403–0.279304 for intra- and interspecific genetic distances when COI genes were used. The phylogenetic trees inferred from the ITS2 and the COI regions, by using maximum parsimony and neighbor-joining methods, were identical to those based on their morphological classification. Thus, the ITS2 and COI regions can be applied as barcodes to identify different species of astigmatid mites.     

rDNA-ITS2序列作为中国按蚊分子鉴别特征的评述：Ⅱ塞蚊亚属

为评价核糖体DNA第2内转录间隔区（ rDNA-ITS2）序列作为中国按蚊属塞蚊亚属蚊种鉴别的分子特征。笔者整理和分析了数据库中注册的所有中国记录的塞蚊亚属蚊种的rDNA-ITS2序列,并应用MEGA软件计算其在种内和种间的差异性（ p距离）。截止2013年7月, GenBank中已注册ITS2序列共442条,隶属塞蚊亚属蚊27种。计算结果显示, ITS2序列在大多数种内的变异性小于1％;而注册的浅色按蚊、乌头按蚊和溪流按蚊的序列种内差异较大,无法确定其种特异序列;忽略上述3蚊种进行计算的其他种间序列差异性范围为2.80％（大劣按蚊与大劣按蚊D）～68.3％（多斑按蚊与迷走按蚊）。结果提示rDNA-ITS2序列在中国塞蚊亚属大多数蚊种中具有良好的种内保守性和种间解析度,少数种类无法区分。     

DNA barcodes can distinguish species of Indian mosquitoes (Diptera: Culicidae)

Species identification of mosquitoes (Diptera: Culicidae) based on morphological characteristics remains often difficult in field-collected mosquito specimens in vector-borne disease surveillance programs. The use of DNA barcodes has been proposed recently as a tool for identification of the species in many diverse groups of animals. However, the efficacy of this tool for mosquitoes remains unexplored. Hence, a study was undertaken to construct DNA barcodes for several species of mosquitoes prevalent in India, which included major vector species. In total, 111 specimens of mosquitoes belonging to 15 genera, morphologically identified to be 63 species, were used. This number also included multiple specimens for 22 species. DNA barcode approach based on DNA sequences of mitochondrial cytochrome oxidase gene sequences could identify 62 species among these, in confirmation with the conventional taxonomy. However, two closely related species, Ochlerotatus portonovoensis (Tiwari & Hiriyan) and Ochlerotatus wardi (Reinert) could not be identified as separate species based on DNA barcode approach, their lineages indicating negligible genetic divergence (Kimura two-parameter genetic distance = 0.0043).     

The use of COI barcodes for molecular identification of forensically important fly species in Germany

Deoxyribonucleic acid (DNA)-based insect identification has become a routine and accurate tool in forensic entomology. In the present study, we demonstrate the utility of the mitochondrial DNA cytochrome oxidase I gene "barcoding region" as a universal marker for molecular identification of forensically important Diptera. We analyzed 111 specimens belonging to 13 species originating from Frankfurt am Main, Germany (Calliphoridae: Calliphora vicina, Calliphora vomitoria, Lucilia ampullacea, Lucilia caesar, Lucilia illustris, Lucilia sericata, Lucilia silvarum, Phormia regina, Protophormia terraenovae; Piophilidae: Parapiophila vulgaris; Muscidae: Hydrotaea dentipes, Hydrotaea ignava, Hydrotaea similis). Intraspecific variation ranged from 0 to 1.17% and interspecific variation occurred between 1.17% and 15.21%. Although differences within species were generally less than among species, divergence percentages overlapped due to low interspecific nucleotide divergence of the recently separated sister species L. caesar and L. illustris. However, all species formed distinct monophyletic clades and thus the cytochrome oxidase 1 (COI) barcode has been shown suitable for clear differentiation and identification of forensically relevant Diptera in Germany.     

Differentiation of Culicoides obsoletus and Culicoides scoticus (Diptera: Ceratopogonidae) based on mitochondrial cytochrome oxidase subunit I

Culicoides obsoletus (Meigen) and Culicoides scoticus Downes & Kettle (Diptera: Ceratopogonidae) are sibling species of the Obsoletus group. This group comprises species of biting midges that are suspect vectors of bluetongue virus (family Reoviridae, genus Orbivirus, BTV) and African horse sickness virus (family Reoviridae, genus Orbivirus, AHSV). BTV and AHSV have been isolated several times from females of this group, although it has not been possible to determine the particular species harboring the virus, because of the inability to clearly identify the females of each species based on morphology. Both sexes of C. obsoletus and C. scoticus midges from Catalonia and the Balearics (Spain) were sequenced for the mitochondrial gene cytochrome oxidase subunit I (COI), and these sequences were analyzed to determine intra- and interspecific genetic variability. Species-specific primers for C. obsoletus and C. scoticus were designed and a polymerase chain reaction (PCR)-based diagnostic assay based on the COI gene and using a hemi-nested PCR technique was developed for reliably distinguishing the females of both species. The species-specific PCR diagnostic was compared with morphological discrimination of C. obsoletus and C. scoticus females. The morphologic characters were not fully reliable.     

一种分离纯化东方田鼠肝细胞的方法改进

目的建立体外培养东方田鼠鼠肝细胞的实验体系。方法采用下腔静脉插管原位灌注结合胶原酶灌注法分离纯化出肝细胞,在改良Eagle培养基（Dulbecco’sModifiedEagleMedia,DMEM）中培养,显微镜下观察细胞形态,AO．EB染色观察测定其活率来评价细胞质量。结果一只东方田鼠肝脏一般可获得1．5×10^8～3．0×10^8个细胞,活率达95％,完全符合实验要求。刚分离的东方田鼠肝细胞呈圆球形,大小均匀,培养3h后,大部分肝细胞出现贴壁,形态呈扁平状。培养24h后,伸展良好,并成片生长。结论下腔静脉插管原位灌注结合胶原酶灌注法可以分离出高纯度和活率高的东方田鼠肝细胞。     

Genetic diversities of hantaviruses among rodents in Hokkaido, Japan and Far East Russia

Seroepizootiologic surveys among wild rodents were carried out in Japan and Far East Russia in 1995 and 1996. Seropositive animals were only identified in Clethrionomys rufocanus (23/134) in Hokkaido, Japan. On the other hand, seropositives were identified in C. rufocanus (1/8), Apodemus agrarius (2/66), Apodemus spp. (2/26) and Microtus fortis (3/22) in Vladivostok, Far East Russia. Total RNA was isolated from lungs of seropositive animals and the S genome segments were amplified by PCR, cloned and sequenced. The S and M genomes of hantavirus, derived from Japanese C. rufocanus (Tobetsu genotype), were most closely related with Puumala viruses (76-79% nucleotide and 95% amino acid identities for S genome, 70-78% nucleotide and 87-92% amino acid identities for M genome). The recombinant nucleocapsid protein of Tobetsu genotype was antigenically quite similar with that of Sotkamo. These suggest that the virus endemic in Japanese C. rufocanus belongs to Puumala virus. Phylogenetic analysis indicates that the genotype forms a distinct lineage within Puumala viruses. Partial S segment (1-1251 nt), derived from seropositive M. fortis in Vladivostok, was sequenced and analyzed. The S genome segment, which was designated Vladivostok genotype, was most closely related with Khabarovsk virus (79% nucleotide and 90% amino acid identities) which was isolated from M. fortis.