羊泰勒虫吉林省分离株18S rRNA基因的克隆和序列分析

为分析羊泰勒虫吉林省分离株的18S rRNA基因序列,根据羊泰勒虫18S rRNA基因的保守区序列设计1对引物,对吉林省的绵羊血液基因组DNA进行PCR扩增,结果扩增出长度为459 bp的基因片段,并成功地将该基因克隆到pMD18-T载体。将经纯化、筛选及酶切鉴定和PCR鉴定为阳性的重组质粒进行测序,与已发表的羊泰勒虫基因序列进行比较。序列分析显示,羊泰勒虫吉林省分离株与羊泰勒虫China 1的同源性为100%。     

黑龙江省猫旋毛虫18S rRNA基因分子克隆及序列分析

本文利用GenBank中发表的(Trichinella spiralis)18S rRNA序列为参考设计引物,对分离自黑龙江省猫体内的旋毛虫及本地毛形线虫(Trichinella nativa)的18S rRNA基因进行扩增,克隆后测序,序列分析结果表明:猫旋毛虫与旋毛形线虫基因同源性更高。     

多浆旱生植物霸王18SrRNA基因的克隆及序列分析

为探讨多浆旱生植物霸王(Zygophyllum xanthoxylum)的生物进化历程及与其他植物的亲缘关系,本研究以霸王叶基因组DNA为模板,使用通用引物扩增其18S rRNA 基因片段,并克隆到pGEM T载体,阳性克隆经鉴定后进行测序。核苷酸序列分析结果表明,该片段长1 808 bp,所得序列与GenBank中注册的18S rRNA基因序列的同源性均在96%以上。可见,高等植物18S rRNA 的基因非常保守。同源性分析与Blast比较结果表明,霸王与小盘木(Galearia filiformis)、驱虫苋(Cnidoscolus aconitifolius)及橡胶树(Hevea brasiliensis)同源性最高。系统进化树分析表明,霸王与三七(Panax notoginseng)的亲缘关系最近。     

18S rRNA序列差异在马梨形虫分类学中的应用

为揭示18S rRNA V4高变区在物种分类学的本质意义,及进一步阐述马泰勒虫(之前称马巴贝斯虫)的分类学地位,本试验参考马泰勒虫(DQ287951)和驽巴贝斯虫(FJ209026) 18S rRNA基因序列,在其V4高变区设计引物.将获得的片段克隆至pMD19-T进行测序,正确的结果与其他种梨形虫的相应序列进行分析.系统发生树结果显示,泰勒虫和巴贝斯虫处于明显的两个分支,而称之为马巴贝斯虫的物种和泰勒虫有着较为密切的关系,他们处于同一分支,其亲缘关系较巴贝斯虫更远.序列对齐分析表明,巴贝斯虫核苷酸序列相对于泰勒虫种序列存在多个位点的缺失和突变.而马巴贝斯虫和泰勒虫18S rRNA V4高变区核苷酸序列的相似程度较高,与巴贝斯虫在该序列上的差异较大.以上结果表明,泰勒虫种和巴贝斯虫种18S rRNA V4高变区核苷酸序列间的这种缺失和突变可作为泰勒虫和巴贝斯虫分类依据的本质因素之一.马巴贝斯虫应隶属于泰勒虫科,泰勒虫属.     

Intraspecific sequence variation in 16S rRNA gene of Ureaplasma diversum isolates

Ureaplasma diversum infection in bulls may result in seminal vesiculitis, balanoposthitis and alterations in spermatozoids. In cows, it can cause placentitis, fetal alveolitis, abortion and the birth of weak calves. U. diversum ATCC 49782 (serogroups A), ATCC 49783 (serogroup C) and 34 field isolates were used for this study. These microorganisms were submitted to Polymerase Chain Reaction for 16S gene sequence determination using Taq High Fidelity and the products were purified and bi-directionally sequenced. Using the sequence obtained, a fragment containing four hypervariable regions was selected and nucleotide polymorphisms were identified based on their position within the 16S rRNA gene. Forty-four single nucleotide polymorphisms (SNP) were detected. The genotypic variability of the 16S rRNA gene of U. diversum isolates shows that the taxonomy classification of these organisms is likely much more complex than previously described and that 16S rRNA gene sequencing may be used to suggest an epidemiologic pattern of different origin strains.     

基于18S rRNA基因测序基础上的锥虫分子分类学

利用18S rRNA基因序列分析方法,对我国湖北、广西、新疆和浙江4省的伊氏锥虫及1株布氏锥虫进行分子分类学研究.用分离的锥虫感染实验动物,自感染小鼠的红细胞或全血中提取基因组DNA,根据GenBank中已发表的雏虫18S rRNA序列设计1对锥虫通用引物,通过PCR扩增目的基因片段,将扩增产物连接到pGEM-T载体中,经酶切、PCR确定,进行序列测定.结果显示,7个锥虫分离株的18S rRNA基因大小为2 188 bp.利用DNAS-tar对试验所获得的这7株锥虫和GenBank中部分锥虫的18S rRNA基因进行比较分析,建立2个进化系统发生树.核苷酸同源性比较及系统发生树显示:自我国上述4省分离的伊氏锥虫来源于同一株系,布氏锥虫和广西分离的伊氏锥虫株的18S rRNA核苷酸与其他锥虫分离株仅有较小差异,与国外的6株锥虫的同源性为99%～100%,与另外7株的同源性为62%.     

套式PCR扩增山羊吕氏泰勒虫18S rRNA基因

泰勒虫是一种危害动物的重要血液寄生性原虫,常寄生于牛、羊、骆驼和其他野生动物,在中国的东北、西北等地较为流行。采集安徽定远县某山羊场疑似焦虫病的山羊血样,首先采用血涂片法检查,再用Blood DNA&Tissue kit提取血液基因组,参照Kim和Wei的方法设计2对引物用于泰勒虫18S rRNA基因的扩增,并对该方法进行敏感性和特异性分析。结果显示:血涂片检查明显可见红细胞内有典型的虫体,大小为0.6~2μm,初步怀疑为泰勒虫(Theileria);PCR扩增可扩增出359 bp大小目的片段,与预期结果相一致;基因序列同源性表明,该泰勒虫分离株基因与已报道泰勒虫毒株(JQ926740.1)核苷酸同源性最高,可达99.72%;在遗传进化方面,所分离的泰勒虫和吕氏泰勒虫(Theileria luwenshuni)Nanjingdong(JQ926740.1)最接近,而且在一个分支上。本试验所建立的套式PCR具有良好的特异性,且敏感性较高,最低测出量是3.8 fg/μL。本次分离的羊血液原虫为吕氏泰勒虫。     

牛附红细胞体16SrRNA基因的克隆测序及分析

无菌采集自然感染附红细胞体的牛血液,提取全血基因组,用血营养菌16S rRNA基因的通用引物进行PCR扩增,得到长约1 500 bp的扩增片段,将其克隆到pMD18-T载体后进行测序和分析.结果,所克隆的牛附红细胞体基因片段大小为1 454 bp,GenBank登录号为FJ375309(丰都株).序列比对结果显示,牛附红细胞体丰都株与武汉株(AY946266)最高,达99.7%,与支原体科代表种同源性为60.7%～76.2%,而与立克次氏体科的立克次氏体和无形体科的无形体同源性仅为51.4%～56.4%,表明牛附红细胞体应归为支原体科,附红细胞体属,而不应属于立克次氏体目,无浆体科.对国内外牛温氏附红细胞体的亲缘关系分析表明,牛温氏附红细胞体无明显的地域性差别趋势.     

应用16S rRNA基因测序法鉴定禽多杀性巴氏杆菌的研究

应用 16SrRNA基因测序法对以疫苗标准强毒株C4 8_1和弱毒疫苗代表株G190E4 0为阳性对照株和分离鉴定的 2株禽源多杀性巴氏杆菌Chicken/guangxi/ 2 0 0 0_1、Chicken/guangdong / 2 0 0 2_1株 ,进行 16SrRNA基因序列分析。结果表明 ,2株分离菌与对照的强弱毒株之间的同源性为 10 0 %。后经Blastn分析Chicken/guangxi/ 2 0 0 0_1、Chicken/guangdong /2 0 0 2_1、C4 8_1、G190E4 0株与已发表 11株多杀性巴氏杆菌同源性高达 10 0 % ,与已发表的 34株多杀性巴氏杆菌同源性均超过 98% ,同时与其它巴氏杆菌种如溶血巴氏杆菌等同源性均低于 96 % ,进一步证实Chicken/guangxi/ 2 0 0 0_1、Chicken/guangdong / 2 0 0 2_1分离株均为多杀性巴氏杆菌。生化实验鉴定表明Chicken/guangxi/ 2 0 0 0_1、Chicken/guangdong / 2 0 0 2_1、C4 8_1、G190E4 0菌株均为subsp .Multocida亚种。     

Phylogenetic analysis of Mycoplasma suis isolates based on 16S rRNA gene sequence in China

To perform phylogenetic analysis of Mycoplasma suis isolates derived from China to define the nature of this pathogen, nearly complete of 16S rRNA genes from Chongqing, Sichuan, Henan and Guangdong isolates were amplified by PCR and sequenced. The four sequences from the blood samples in this study, with other 17 Hemoplasmas sequences and related 3 mycoplasma sequences available in the GenBank, were aligned using Clustal X (version 1.83) sequences alignment program. Maximum parsimony, neighbor-joining and minimum evolution (MEGA 4.0) algorithms were used to create phylogenetic trees. Phylogenetic analysis of these sequences showed that all hemoplasma species were located within a single clade and were most closely related to M. pneumoniae group. The hemoplasma species were further subdivided into two distinct groups, one containing M.wenyonii, M.suis and Candidatus M. haemominutum and the other containing M. haemofelis and M. haemocanis. Within the former clade, four M.suis isolates from Mainland China and other M.suis species formed a monophyletic group in the tree. A tendency of clear geographical grouping of the isolate was evident.     

Avian malaria in captive psittacine birds: detection by microscopy and 18S rRNA gene amplification

A cross-sectional survey was conducted to estimate the occurrence of malaria infection among captive psittacine birds (n=127) from three zoological gardens in Brazil. Malaria infection was evaluated by the association of direct examination of blood smears with amplification of the 18SSU rRNA gene of the Plasmodium genus, demonstrating an overall occurrence of 36%. Most infected bird species were Amazona aestiva (28/73), Ara ararauna (6/10), and Amazona amazonica (3/10). The low parasitemias observed among the infected birds suggest a chronic infection. The sequence analyses of 10 isolates indicate a potential occurrence of four distinct Plasmodium lineages. These findings provide new data on malarial infection in captive psittacine birds, and emphasize the need for better control of importation and exportation of these birds.     

鸡奇异变形杆菌的分离鉴定和16S rRNA基因序列测定与系统进化分析

从山东泰安一鸡场发病雏鸡眼中分离到1株致病菌(编号为QY),通过细菌形态学等常规鉴定符合奇异变形杆菌(Proteus mirabilis)特性。用奇异变形杆菌阳性血清诊断结果呈阳性,人工感染证明该菌株是造成该鸡场雏鸡大批发病死亡的致病菌。药敏试验结果显示对头孢类、恩诺沙星等高度敏感,而对青霉素和复合磺胺等不敏感。以细菌16SrRNA基因通用引物进行PCR扩增,得到QY的16SrRNA基因序列,长约1 453bp(GenBank,登录号为GU477712)。将该序列与GenBank中序列进行Blast比对,发现与其匹配度最高的均是奇异变形杆菌各株系的16SrRNA序列,均高达98%以上。运用DNAStar软件与其中10株奇异变形杆菌分离株构建系统进化树,结果表明,分离株(QY菌株)与10个代表菌株的同源性均为98.9%～99.9%,其中与AB272366同源性最高为99.9%。从分子水平证明该菌是奇异变形杆菌并分析了其遗传进化规律,为鸡奇异变形杆菌的鉴定及其引起的疾病的诊断与治疗提供了参考。     

Identification of Theileria parva and Theileria sp. (buffalo) 18S rRNA gene sequence variants in the African Buffalo (Syncerus caffer) in southern Africa

Theileria parva is the causative agent of Corridor disease in cattle in South Africa. The African buffalo (Syncerus caffer) is the reservoir host, and, as these animals are important for eco-tourism in South Africa, it is compulsory to test and certify them disease free prior to translocation. A T. parva-specific real-time polymerase chain reaction (PCR) test based on the small subunit ribosomal RNA (18S rRNA) gene is one of the tests used for the diagnosis of the parasite in buffalo and cattle in South Africa. However, because of the high similarity between the 18S rRNA gene sequences of T. parva and Theileria sp. (buffalo), the latter is also amplified by the real-time PCR primers, although it is not detected by the T. parva-specific hybridization probes. Preliminary sequencing studies have revealed a small number of sequence differences within the 18S rRNA gene in both species but the extent of this sequence variation is unknown. The aim of the current study was to sequence the 18S rRNA genes of T. parva and Theileria sp. (buffalo), and to determine whether all identified genotypes can be correctly detected by the real-time PCR assay. The reverse line blot (RLB) hybridization assay was used to identify T. parva and Theileria sp. (buffalo) positive samples from buffalo blood samples originating from the Kruger National Park, Hluhluwe-iMfolozi Park, the Greater Limpopo Transfrontier Park, and a private game ranch in the Hoedspruit area. T. parva and Theileria sp. (buffalo) were identified in 42% and 28%, respectively, of 252 samples, mainly as mixed infections. The full-length 18S rRNA gene of selected samples was amplified, cloned and sequenced. From a total of 20 sequences obtained, 10 grouped with previously published T. parva sequences from GenBank while 10 sequences grouped with a previously published Theileria sp. (buffalo) sequence. All these formed a monophyletic group with known pathogenic Theileria species. Our phylogenetic analyses confirm the distinction between Theileria sp. (buffalo) and T. parva and indicate the existence of a single group of T. parva and two Theileria sp. (buffalo) 18S rRNA gene variants in the African buffalo. Despite the observed variation in the full-length parasite 18S rRNA gene sequences, the area in the V4 hypervariable region where the RLB and real-time PCR hybridization probes were developed was relatively conserved. The T. parva specific real-time PCR assay was able to successfully detect all T. parva variants and, although amplicons were obtained from Theileria sp. (buffalo) DNA, none of the Theileria sp. (buffalo) 18S rRNA sequence variants were detected by the T. parva-specific hybridization probes.     

10株火鸡组织滴虫18S rRNA基因的克隆及进化分析

应用聚合酶链反应(PCR)鉴定湖南娄底(简称HMLD1-3)、湘潭(HMXT1-2)、永州(HMYZ1-3)和宁乡(HMNX1-2)共10株火鸡组织滴虫,并用18S rRNA部分序列重构火鸡组织滴虫与其它相近原虫的种群遗传关系.作者通过PCR扩增火鸡组织滴虫虫株的18S rRNA部分序列,将PCR扩增出的片段纯化后克隆...     

Sequence and phylogenetic analysis of the 16S rRNA gene of Ehrlichia canis strains in dogs with clinical monocytic ehrlichiosis

The purpose of this study was to characterize, at the molecular level, the Ehrlichia canis strains involved in naturally occurring canine monocytic ehrlichiosis (CME) in Greece, and to investigate if any sequence diversity exists between the 16S rRNA genes of those involved in the mild non-myelosuppressive or the severe myelosuppressive form of CME. To this end, amplification of the ehrlichial 16S rRNA gene was attempted by nested polymerase chain reaction (PCR) assays in bone marrow (BM) aspirates from 20 dogs tentatively diagnosed as having non-myelosuppressive (n=10, group A) or myelosuppressive (n=10, group B) CME. PCR assay using E. canis-specific primers revealed that 15 BM samples, including all group A and 5 group B dogs, were positive. Using universal PCR primers, a nearly full-length 16S rRNA gene could be amplified from 13 BM samples, including 9 group A and 4 group B dogs. The 16S rDNA analysis based on secondary structure revealed that all sequences of the Greek strains were identical to each other and indicated 100% identity among some American (Venezuelan and Brazilian), European (Greek), Middle Eastern (Turkish) and Asiatic (Thailand) strains. The results of this study suggest that the E. canis strains involved in the non-myelosuppressive and myelosuppressive forms of CME in Greece share an identical 16S rRNA genotype.     

基于18S rRNA和HSP70基因序列的隐孢子虫种系发育分析

为阐明河南区域隐孢子虫分子流行病学特点,用PCR技术扩增分离虫株的18S rRNA基因全序列和HSP70基因序列,并对扩增片段进行测序。用PAUP 4.0和TREEPUZZLE 4.1构建进化树,试图从分子水平证明河南省不同地区不同宿主来源隐孢子虫的遗传特征,以阐明隐孢子虫病的分子流行病学特点。通过18S rRNA基因全序列和HSP70基因序列分析,其结果：河南人源隐孢子虫分离株为Cryptosporidium parvum鼠基因型;河南鹿源隐孢子虫分离株为C. parvum鹿基因型;河南猪源隐孢子虫的2个分离株均为C. parvum猪基因I型,即C. suis;河南鹌鹑源的隐孢子虫2个分离株分别为C. baileyi和C. meleagridis;河南乌鸡源隐孢子虫和鸵鸟源隐孢子虫分离株均为C. baileyi;河南牛源隐孢子虫分离株为C.andersoni。     

牛瑟氏泰勒虫吉林株18S rRNA基因的扩增及系统发育研究

本研究根据GenBank上发表的牛瑟氏泰勒虫18S rRNA基因的核苷酸序列设计并合成1对特异性引物,对寄生于牛体内的瑟氏泰勒虫基因组DNA进行扩增,得到1 356 bp的18S rRNA基因片段,测序后blast分析表明该虫种属牛瑟氏泰勒虫。将该基因片段序列与GenBank中8种已知泰勒虫的相应序列进行比较分析,建立系统发育树。结果表明,牛瑟氏泰勒虫吉林分离株与水牛泰勒虫亲缘关系最近,与小泰勒虫亲缘关系较远。这一结果说明宿主因素对泰勒虫的基因型影响较大。     

Detection of Renibacterium salmoninarum in tissue samples by sequence capture and fluorescent PCR based on the 16S rRNA gene

The 16S rRNA genes from eight isolates of Renibacterium salmoninarum with different origins and dates of isolation were sequenced to evaluate the possibility to construct a diagnostic PCR system with target sites within this gene. The sequences were found to be identical but for one single position in one of the isolates, and two regions with an adequate number of nucleotide differences as compared to closely related species were identified. Species-specific fluorescent PCR primers complementary to these regions were constructed as well as oligonucleotides for DNA preparation by sequence capture. A mimic molecule was constructed to be used as an internal control. The PCR was specific and allowed the detection of DNA equivalent to 1-10 R. salmoninarum genomes per reaction. The DNA preparation with sequence capture and analysis by PCR with a mimic was found to be a reliable method for analysis of kidneys from fish with BKD. The amount of PCR inhibiting substances present in the tissue was reduced, and the relevant DNA was concentrated in the capture step. Furthermore, the use of the mimic molecule in the system assured that false negative results could be identified.     

A PCR-based epidemiological survey of Hepatozoon canis in dogs in Nigeria

The prevalence of Hepatozoon canis infections in dogs in Nigeria was surveyed using molecular methods. DNA was extracted from blood samples obtained from 400 dogs. A primer set that amplified the Babesia canis 18S rRNA gene, which has high similarity to the H. canis 18S rRNA gene, was used for the PCR. As a result, samples from 81 dogs (20.3%) produced 757 bp bands, which differed from the 698 bp band that corresponded to B. canis infection. The sequence of the PCR products of 10 samples were determined, all of which corresponded with the H. canis sequence.     

湖南省珍稀雉类组织滴虫核糖体18S rRNA系统进化分析

为分析珍稀雉类组织滴虫核糖体18S rRNA序列遗传变异情况,并利用18S rRNA序列分析组织滴虫与其他原虫的种群遗传关系,应用PCR技术扩增珍稀雉类异刺线虫体内组织滴虫核糖体18S rRNA基因序列,并进行测序与分析。结果表明:异刺线虫雄虫与异刺线虫雌虫体内均含有火鸡组织滴虫,且9个组织滴虫分离株核糖体18S rRNA序列片段大小基本一致,约为590 bp,各分离株之间核糖体18S rRNA序列同源性均高于99.4%,与Gen Bank中收录其他原虫相应序列同源性均低于66.8%;通过建立NJ系统发育树,9个组织滴虫分离株与Gen Bank收录的火鸡组织滴虫位于同一分支,得到明显的鉴定。核糖体18S rRNA序列种内相对保守,但种间差异明显,可作为火鸡组织滴虫的种间遗传变异研究的分子遗传标记。