狐狸奇异变形杆菌的分离与鉴定

无菌采取病死狐狸内脏,用LB液体培养基和麦康凯琼脂平板培养,分离到1株细菌,通过对该菌的形态特征、培养特性、生化特性进行分析,初步鉴定为变形杆菌。用PCR方法扩增该分离菌株的16 SrRNA基因,结果获得大小为1 539 bp的DNA片段(已登录GenBank,登录号EU643833)。经BLAST分析,结果表明,该分离株的16 S rRNA基因核苷酸序列与GenBank上登录的奇异变形杆菌分离株(AY820623、EF091150)的同源性为99.7%,证实该分离菌株为狐狸奇异变形杆菌,并命名为HU/08。致病性试验证明,该分离菌株对小白鼠有高致病性;毒素测定试验证明,该分离菌培养液的无菌滤液对小白鼠无毒性作用。     

一种简便的DNA提取方法在动物毛发检验中的应用

目的建立一种简便的DNA提取方法,用于动物毛干的DNA抽提。方法利用PCR缓冲液及蛋白酶K在PCR仪上对毛发进行消化,以此DNA为模板,扩增mtDNA 12S rRNA基因的部分片段并进行序列测定,测序结果在GenBank上进行BLAST搜索,再利用DNAMAN软件进行同源性分析。结果利用这种新DNA抽提法能从无毛囊毛发中获得后续PCR扩增所需的线粒体DNA。结论该法在无毛囊毛发样本鉴定中具有较高的应用价值。     

猪源嗜麦芽窄食单胞菌16 S rRNA基因的克隆和序列分析

从临床病猪无菌采集抗凝血 ,抽提全血基因组DNA ,用能够扩增大多数真细菌 16SrRNA基因的通用引物 ,扩增出长度为 15 0 2bp的嗜麦芽窄食单胞菌的 16SrRNA基因 ;该基因与国外报道的嗜麦芽窄食单胞菌部分临床和环境分离株核苷酸序列的同源性达 99%以上。证实嗜麦芽窄食单胞菌可感染猪。     

猪三毛滴虫长春株的形态观察及其5.8 S rRNA基因分析

对从长春地区腹泻仔猪结肠中分离到的1株毛滴虫进行了形态观察,并采用PCR扩增该毛滴虫的5.8SrRNA基因,测序后与国外已报道的三毛滴虫进行核酸同源性分析,并应用DNAStar软件绘制系统发育进化树。形态观察表明分离的毛滴虫为猪三毛滴虫。猪三毛滴虫长春分离株5.8SrRNA序列与国外已报道的三毛滴虫高度同源。系统发育进化树表明该序列与已报道的猪三毛滴虫5.8SrRNA(序列号U85966.1),牛胎儿三毛滴虫5.8SrRNA(序列号U85967.1,AF339736.1,AF466749.1,AF466750.1,AF466751.1)和T.mobiliensis5.8SrRNA(序列号U86612.1)同属于一个亚群,但与另1株猪三毛滴虫5.8SrRNA(序列号AY349190.1)亲缘关系较远。形态观察和5.8SrRNA基因分析表明,从长春地区分离的猪毛滴虫为猪三毛滴虫。     

死亡人体胸肌组织中rRNA亚基的稳定性评价

目的观察尸体胸大肌组织中rRNA各亚基表达水平的死后稳定性,探讨其在死亡时间推断中应用的价值。方法选取6例（3例成人、3例婴幼儿）外界温、湿度环境基本一致、死亡时间基本一致（24h以内）的个体,提取左侧胸大肌组织,在PBS缓冲液中低温冻存,分别在尸检即刻、2、3、4、5、6、7、8、9、10d提取微量组织保存于RNA固定液中,通过实时荧光定量RT-PCR技术检测rRNA 5srRNA、5.8srRNA、18srRNA、28srRNA亚基的表达水平。并分析年龄、死因对rRNA亚基表达水平的影响。结果死后各时间点rRNA各亚基的表达随死后经过时间延长无显著性降解,Ct值变化与死后经过时间无相关性（P值均大于0.05）,其中5s rRNA在18.503±2.655~20.937±2.340之间,5.8s rRNA在17.687±3.011~20.617±2.204之间,18s rRNA在16.457±3.920~22.330±2.571之间,28s rRNA在15.077±6.051~22.207±2.685之间。结论人体胸肌组织中rRNA各亚基稳定性好,其各亚基的表达量与死因、年龄无关,适于作为晚期死亡的推断的内参基因。     

Soil sample metagenome NGS data management for forensic investigation

Soil analysis is a valuable resource in forensic investigation. Classical forensic soil analysis involves examination of its physical characteristics and chemical composition, such as soil type, colour, particle size, shape, pH, elemental, mineral and organic content. However the limited variability of these parameters is not always allowing adequate discrimination between soil samples. As soil supports extreme diversity of microorganisms and eukaryotic communities, microbiological approaches have been proposed. Several molecular approaches for microbial DNA profiling are available; however there is a lack of published data of implementation of the next generation sequencing (NGS) approaches for forensic soil analysis.The aim of the current study was elaboration of criteria for soil metagenome data management and database searching. We used our previously sequenced collection of 11 samples collected from different environments (forests, fields, grasslands, urban park) with different flora. The single sample collection includes 9 soil samples per one sampling area (30 m × 30 m) spaced by 15 m. In the current study we concentrated mainly on 18S rRNA gene V2-V3 region for fungi however SSU rRNA region for arbuscular mycorrhizal (AMF) fungi and V2-V3 hypervariable region of 16S rRNA gene for bacterial communities were taken into account. The sequencing was performed by Roche/454 platform. For data analysis OTU based approach on mothur software and NCBI BLASTN search were used. NCBI BLASTN analysis revealed altogether 2983 AMF matches and 8997 18S matches as well as 25477 OTUs (16S) were determined. Several data filtration approaches were used for data management. We found that 18S marker results could be used to create and run a filtered database that is computationally much more efficient and flexible. Our results have broad impact; however more samples have to be analysed, additional studies performed and cooperation between soil scientists and forensic scientists is required to be able to implement these novel techniques into the routine forensic practice.     

扩增12S rRNA基因鉴定生物检材种属

目的建立一种能够在同一反应条件下鉴定多个具体物种．又满足简单、快速、特异、灵敏、准确等实用要求的种属鉴定方法。方法从GenBank中获取人、鸡、鸭、鹅、猪、兔、鼠、绵羊、水牛、狗、山羊等11个物种的12SrRNA基因序列．设计一对针对上述11个物种的通用引物和分别针对人、鸡和鸭的特异引物,同时扩增各物种12SrRNA基因。以通用引物扩增片段为内部对照．以特异引物扩增片段用于人、鸡和鸭的种属鉴定,并分别对人、鸡、鸭单一检材以及人和鸡、人和鸭、鸡和鸭等二元混合DNA进行鉴定。结果通用引物扩增,各物种均有400bp左右的扩增片段：特异引物只对各自目标种属有扩增产物,片段大小：人163bp、鸡286bp、鸭374bp;测序结果与GenBank既有序列比对,Identities分值：人100％、鸡99％、鸭100％;通用引物扩增人、鸡和鸭检材的灵敏度为2．5Pg;特异引物扩增灵敏度人为2．5pg,鸡、鸭均为200Pg;混合DNA中任一种DNA的含量只要高于检测灵敏度即可被准确检出．不受另一种DNA量的干扰：盲测结果准确。结论本方法可以利用同一种PCR反应条件鉴定多个物种的种属。     

黄芪-丹参对动脉粥样硬化大鼠肠道菌群的影响

目的 研究黄芪-丹参对动脉粥样硬化（atherosclerosis,AS）大鼠肠道菌群的影响。方法 将大鼠随机分为空白组、模型组、辛伐他汀组（1 mg/kg）、黄芪-丹参组（0.75 g/kg）,采用高脂饲料喂养联合腹腔注射维生素D3 8周的方法复制AS模型,同时灌胃给药。采用自动生化分析仪检测大鼠血清总胆固醇（total cholesterol, TC）、三酰甘油（Triacylglycerol, TG）、低密度脂蛋白胆固醇（low-density lipoprotein cholesterol, LDL-C）水平,采用酶联免疫吸附试验检测血清肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）、白细胞介素-6（interleukin-6,IL-6）、白细胞介素-1β（interleukin-1β,IL-1β）水平,光学显微镜下观察胸主动脉病理变化,采用生物信息分析方法观察大鼠结肠内容物肠道菌群变化。结果 与空白组比较,模型组大鼠血清TC、TG、LDL-C、TNF-α、IL-6、IL-1β水平明显升高（P<0.05）,HDL-C水平明显降低（P<0.05）,胸主动脉血管内皮损伤严重,可见明显蓝色钙状斑块,结肠内容物肠道菌群Berger-Parker、Simpson指数明显降低（P<0.05）,放线菌门（Actinobacteria）、乳杆菌属（Lactobacillus）、双歧杆菌属（Bifidobacterium）和杜氏杆菌属（Dubosiella）群落丰度明显降低（P<0.05）,变形菌门（Proteobacteria）、疣微菌门（Verrucomicrobia）以及布劳特氏菌属（Blautia）、unclassified_f_Ruminococcaceae和Lachnospiraceae_NK4A136_group群落丰度明显升高（P<0.05）。与模型组比较,黄芪-丹参组大鼠血清血脂和炎症因子水平明显改善（P<0.05）,胸主动脉血管内皮无明显损伤及增厚,大致恢复正常,结肠内容物肠道菌群Berger-Parker、Simpson指数明显升高（P<0.05）,放线菌门、乳杆菌属、双歧杆菌属和杜氏杆菌属群落丰度明显升高（P<0.05）,变形菌门、疣微菌门以及布劳特氏菌属、unclassified_f_Ruminococcaceae和Lachnospiraceae_NK4A136_group群落丰度明显降低（P<0.05）。〖JP〗结论 黄芪-丹参治疗AS的作用机制可能是通过调控肠道菌群改善血脂代谢和抑制炎症水平实现的。     

Forensic analysis of hallucinogenic fungi: a DNA-based approach

Hallucinogenic fungi synthesize two controlled substances, psilocin and psilocybin. Possession of the fungal species that contain these compounds is a criminal offence in North America. Some related species that are morphologically similar, do not contain the controlled substances. Therefore, unambiguous identification of fungi to the species level is critical in determining if a mushroom is illegal. We investigate a phylogenetic approach for the identification of species that contain the psychoactive compounds. We analyzed 35 North American specimens representing seven different genera of hallucinogenic and non-hallucinogenic mushrooms. We amplified and sequenced the internal transcribed spacer region of the rDNA (ITS-1) and a 5′ portion of the nuclear large ribosomal subunit of rRNA (nLSU rRNA or 28S). ITS-1 locus sequence data was highly variable and produced a phylogenetic resolution that was not consistent with morphological identifications. In contrast, the nLSU rRNA data clustered isolates from the same species and separated hallucinogen containing and non-hallucinogen containing isolates into distinct clades. With this information, we propose an approach that combines the specificity of PCR detection and the resolving power of phylogenetic analysis to efficiently and unambiguously identify hallucinogenic fungal specimens for legal purposes.     

羊源巴贝斯虫未定种甘肃敦煌株的发现

采用动物接种法将采自甘肃敦煌的15只绵羊血液混合后感染1只除脾绵羊,感染后逐日做血涂片检查。结果显示,在感染后第10天的血片上出现了1种大型巴贝斯虫。虫体形态以双梨子形、单梨子形、圆形及卵圆形为主。在感染后第12天,感染羊体温升至41.1℃。染虫率达到0.125%,之后逐渐下降。无菌采集该羊血液,提取全血基因组,用梨形虫通用引物进行PCR扩增全长18SrRNA基因,得到长约1 700bp的片段,将其克隆并测序(登录号:HQ730762),与GenBank中的其他巴贝斯虫的序列进行比较,发现其与新疆巴贝斯虫未定种的同源性最高,达到99.8%。本研究发现甘肃敦煌又是一个新疆羊巴贝斯虫未定种的疫源地。