用PCR扩增tim基因检测蓝氏贾第鞭毛虫

采用聚合酶链反应(PCR)对蓝氏贾第鞭毛虫(Giardia lamblia)的磷酸丙糖异构酶(triose phosphate isomerase,缩写为tim)基因进行特异性扩增,结果扩增出1条683bp的DNA片段.此方法的特异性可高达100%,而其它DNA样本,如日本血吸虫(Schistosoma japonicum)、刚地弓形虫(Toxoplasma gondii)、微小隐孢子虫(Cryptosporidium parvum)、溶组织内阿米巴(Entamoeba histolytica)、旋毛虫(Trichinella spiralis)和阴道毛滴虫(Trichomonas vaginalis),以及人体血细胞等均未出现扩增反应.本法的敏感性也很高,可检测到0.4pg贾第虫包囊的DNA.13株来自不同地理位置和/或宿主的贾第虫DNA样本在PCR中均各产生1条长为683bp的目的片段.上述结果表明本实验建立的检测贾第虫的PCR方法有效.     

用PCR扩增tim基因检测蓝氏贾第鞭毛虫

采用聚合酶链反应(PCR)对蓝氏贾第鞭毛虫(Giardia lamblia)的磷酸丙糖异构酶(triose phosphate isomerase,缩写为tim)基因进行特异性扩增,结果扩增出1条683bp的DNA片段.此方法的特异性可高达100%,而其它DNA样本,如日本血吸虫(Schistosoma japonicum)、刚地弓形虫(Toxoplasma gondii)、微小隐孢子虫(Cryptosporidium parvum)、溶组织内阿米巴(Entamoeba histolytica)、旋毛虫(Trichinella spiralis)和阴道毛滴虫(Trichomonas vaginalis),以及人体血细胞等均未出现扩增反应.本法的敏感性也很高,可检测到0.4pg贾第虫包囊的DNA.13株来自不同地理位置和/或宿主的贾第虫DNA样本在PCR中均各产生1条长为683bp的目的片段.上述结果表明本实验建立的检测贾第虫的PCR方法有效.     

实验感染C57 BL/6N小鼠粪内蓝氏贾第鞭毛虫tim基因检测

建立蓝氏贾第鞭毛虫感染动物模型 ,用PCR技术对感染动物粪内该虫tim基因进行检测 ,探讨蓝氏贾第鞭毛虫基因检测方法。用人源蓝氏贾第鞭毛虫河北株 (CD)和江苏株 (XZ)的包囊分别接种两组C5 7BL 6N小鼠 ,于接种后第 6天、1 0天和 1 4天收集粪便标本 ,采用酚 氯仿法提取总DNA。将提取液分别按 1 0 0 、 1 0 - 1 、 1 0 - 2 和 1 0 - 3稀释并纯化。根据该虫磷酸丙糖异构酶 (Triosephosphateisomerase,tim)基因合成 1对特异性引物 ,采用PCR技术分别扩增各个稀释度的样本。结果贾第虫阳性样本均被扩增出相应基因的 1条 683bp长的片段 ,纯化后的各个稀释度样本的PCR检测阳性率随稀释倍数的增加而增高。本研究结果为贾第虫感染基因检测方法的进一步研究提供了实验资料。     

Contracaecum ogmorhini复合种分类鉴定特异PCR方法的建立

根据作者已测得的Contracaecumogmorhini复合种 (包括Contracaecumogmorhini和Contracaecummargolisi)线粒体DNAcox1基因的部分核苷酸序列 ,设计、合成了一对针对C .margolisi的特异性引物(CHL1,CHL2 ) ,通过PCR条件优化和扩增 ,C .margolisi扩增出 1条清晰的 30 6bp大小的特异DNA片段 ,而其他虫体如C .ogmorhini、拟地新线虫、鲁道夫对盲囊线虫、猪蛔虫、犬弓首蛔虫、猪有齿食道口线虫等 14个对照样本均未扩增出特异性片段。敏感性实验可检测到C .margolisiDNA的最低浓度为 0 34ng μL ,从而初步建立了鉴定C .ogmorhini复合种的特异PCR方法。该方法特异性强、敏感度高、重复性好 ,不仅可用于C .ogmorhini复合种的分类鉴定 ,也可用于它们所导致的寄生虫病的诊断、防制和流行病学调查 ,同时也为C .ogmorhini复合种在中国的分布及其进一步的分类学研究提供了重要依据和手段     

牛瑟氏泰勒虫病PCR诊断方法的建立

为建立一种快速、敏感的牛瑟氏泰勒虫病诊断方法,本实验分离提取牛瑟氏泰勒虫全血基因组DNA,并设计一对编码牛瑟氏泰勒虫32 kDa (P32) 主要表面蛋白基因的引物,进行PCR扩增,预期扩增的片段为875 bp.并对该实验进行特异性、敏感性及临床检测实验.结果成功扩增出了长为875 bp的基因片段,且具有高特异性和很好...     

应用多聚酶链反应技术检测疟原虫的初步研究

根据编码恶性疟原虫(P．f)红细胞结合抗原(EBA-175)的部分DNA片段和间日疟原虫(P．v．)小亚基核糖体RNA基因序列设计合成恶性疟原虫和间日疟原虫的特异性引物各一对并进行多聚酶链反应(PCR)检测恶性疟和间日疟病人标本。扩增产物经琼脂糖电泳分析，可见在P．f.样本中扩增出特异的492bp大小的DNA片段，P．v．样本中扩增出特异的714bp大小的DNA片段。而在健康人血的白细胞、伯氏疟原虫样本和食触猴疟原虫样本中均不能扩增出以上片段。其结果与镜检符合率分别达95％和93.3％以上，表明该方法是一种特异、敏感的检测方法。     

直接PCR法检测血液中伊氏锥虫感染

为建立伊氏锥虫动基体DNA(KinetoplastDNA,kDNA)的PCR扩增技术,血液直接PCR法检测伊氏锥虫的感染,本文以伊氏锥虫kDNA片段为靶扩增DNA设计引物,确定最适PCR反应条件,建立kDNA片段的PCR扩增技术,应用直接扩增缓冲液(Ampdirect)从感染小鼠的滤纸血标本直接PCR检测伊氏锥虫。结果发现,PCR扩增伊氏锥虫kDNA片段分子量为364bp,能检测的最小DNA量是0·06pg,与布氏锥虫、活动锥虫没有交叉反应。应用直接扩增缓冲液不需进行样本的DNA抽提,直接PCR能检测感染小鼠的早期滤纸血样本,敏感性高于镜检法。本实验建立的直接PCR法检测伊氏锥虫具有较高的敏感性和特异性,方法快捷,可进一步应用于伊氏锥虫病的早期诊断以及现场调查。     

巢式PCR检测日本血吸虫感染的研究

本研究通过设计2对巢式引物扩增日本血吸虫高拷贝的Sjα1基因片段,建立检测日本血吸虫感染的巢式PCR技术,并对感染小鼠血清、全血样本以及实验和现场钉螺样本进行检测.建立的巢式PCR方法特异性扩增日本血吸虫420 bp的Sjα1片段,和曼氏血吸虫没有交叉,基因组DNA作为模板时最低检测量为0.1fg.小鼠感染日本血吸虫后2周的血清样本中即能检测出特异性DNA,建立的方法能同时检测血清和全血标本.钉螺实验感染4 h后能检测到日本血吸虫DNA,现场采集钉螺的检测结果显示比传统的镜检方法敏感性高.建立的巢式PCR检测日本血吸虫感染具有较高的敏感性和特异性,为疾病诊断和媒介调查提供了新的分子生物学检测技术. PCR技术,并对感染小鼠血清、全血样本以及实验和现场钉螺样本进行检测.建立的巢式PCR方法特异性扩增日本血吸虫420 bp的sja1片段,和曼氏血吸虫没有交叉,基因组DNA作为模板时最低检测量为0.1fg.小鼠感染日本血吸虫后2周的血清样本中即能检测出特异性DNA,建立的方法能同时检测血清和全血标本.钉螺实验感染4 h后能检测到日本血吸虫DNA,现场采集钉螺的检测结果显示比传统的镜检方法敏感性高.建立的巢式PCR检 日本血吸虫感染具有较高的敏感性和特异性,为疾病诊断和媒介调查提供了新的分子     

涎腺淋巴上皮癌中EB病毒潜伏膜抗原-1基因30碱基缺失的研究

目的 了解潜伏膜抗原(LMP)-1基因3’端DNA的30碱基在涎腺淋巴上皮癌中的缺失率及LMP-1的基因亚型。方法 用聚合酶链反应(PCR)方法对46例涎腺淋巴上皮癌中EBV编码LMP-1基因3’端DNA进行扩增,观察有无30碱基(bp)缺失。为减少从石蜡组织中提取DNA失败产生的影响,同时对样本作B-肌动蛋白基因扩增,并对PCR产物进行测序。结果 46例标本中4例经β-肌动蛋白基因扩增证实为DNA提取失败。剩余42例样本中35例(83．3％)检出LMP-1 DNA。阳性样本PCR产物电泳条带位置有2种,分别位于300bp上下,其中高于300bp有31例(88．6％),低于300bp有4例(11．4％)。DNA测序证实二者碱基相差30bp,分别为316bp和286bp。结论 涎腺淋巴上皮癌中存在EBV编码LMP-1基因3’端DNA的30bp缺失,但大多数为非缺失型LMP-1。     

阿尔茨海默病患者血液细胞线粒体内细胞色素C氧化酶亚基Ⅱ基因存在缺失

目的 调查25例老年痴呆患者血细胞线粒体中细胞色素C氧化酶亚基Ⅱ基因的突变情况,探讨线粒体DNA突变与阿尔茨海默病(AD)性老年痴呆发生发展的关系。方法 采用微量法提取血细胞总DNA直接作为PCR模板,进行巢式PCR扩增,最后采用DNA序列分析鉴定突变。结果在正常老人组,只扩增了280bp的线粒体细胞色素C氧化酶亚基Ⅱ基因(COX2)扩增片段,而在老年痴呆患者中出现了464bp和280bp的多态性扩增片段,并且发现了1个320bp的新的线粒体DNA缺失片段,1位AD患者的COX2基因存在一种新的缺失后重组现象,COX2基因的1条单链在np7503处断裂,另1条单链在np7785处断裂,这两个断裂的游离片段通过插入1个额外的胞嘧啶核苷酸重新连接起来。结论 老年痴呆患者中的COX2基因存在片段扩增多态性。     

Real-time PCR assay for detection and genotype differentiation of Giardia lamblia in stool specimens

Real-time PCR, using dual-labeled fluorescent probes targeting the beta-giardin gene, was used to detect Giardia lamblia in human stool specimens and to discriminate between isolates from the two major genetic assemblages of G. lamblia infective to humans, assemblages A and B.     

Comparison of two target genes for detection and genotyping of Giardia lamblia in human feces by PCR and PCR-restriction fragment length polymorphism

A PCR assay targeting the tpi gene was developed to detect and to genotype Giardia lamblia in human feces. Our assay was specific and discriminated between G. lamblia assemblages A and B. G. lamblia cysts isolated from human feces were also analyzed with two previously described PCR-restriction fragment length polymorphism (RFLP) assays, which are based on the detection of tpi or gdh genes. These RFLP analyses distinguished groups I and II within assemblage A or groups III and IV within assemblage B. Among 26 fecal samples from patients with sporadic giardiasis diagnosed by hospital laboratories, the tpi gene was amplified from 25 (96%) with our PCR assay, whereas only 21 (81%) samples were positive when the gdh gene was targeted. Of the 25 positive samples, nine (36%) contained assemblage A and 16 (64%) contained assemblage B. Thus, RFLP analysis classified eight samples (32%) in assemblage A group II, eight (32%) in assemblage B group III, and five (20%) in assemblage B group IV. The group could not be specified for four samples. The tpi and gdh genes of G. lamblia assemblage B were amplified from 14 (93%) of 15 samples collected only from French soldiers coming back from the Ivory Coast. All of these contained assemblage B group III. The PCR method developed is sensitive, simple, and specific and shows that the tpi gene is well adapted for G. lamblia genotyping.     

Classification of subgroups of Giardia lamblia based upon ribosomal RNA gene sequence using the polymerase chain reaction.

A sensitive and specific polymerase chain reaction-based assay has been developed to detect and analyze polymorphism in the Giardia lamblia 18S ribosomal RNA gene. Efficient amplification required the inclusion of cosolvents (glycerol and dimethyl sulfoxide) in the reaction. Following the optimization of conditions for amplification and subsequent hybridization of amplified product with radiolabeled oligonucleotide probe, a detection limit of less than one organism's worth of DNA was achieved. Thirty-five different G. lamblia strains obtained from various human and animal host types and geographic locations were analyzed by this method. The strains could be divided into 3 groups on the basis of defined nucleotide substitutions within the 183-bp amplified DNA fragment of the 18S ribosomal RNA gene. The groupings based upon the 18S ribosomal RNA gene sequence correlated with groupings previously assigned based upon patterns of surface antigens and restriction enzyme analysis. Analysis of the G. lamblia 18S ribosomal RNA gene sequences present in fecal specimens obtained from giardiasis patients revealed the presence of the different sequence types in these specimens. Some specimens contained more than one sequence type. The identification of subgroups of G. lamblia may facilitate studies of virulence, infectivity, and the epidemiology of giardia infection.     

Identification of Mycobacterial Species by Comparative Sequence Analysis of the RNA Polymerase Gene (rpoB)

For the differentiation and identification of mycobacterial species, the rpoB gene, encoding the beta subunit of RNA polymerase, was investigated. rpoB DNAs (342 bp) were amplified from 44 reference strains of mycobacteria and clinical isolates (107 strains) by PCR. The nucleotide sequences were directly determined (306 bp) and aligned by using the multiple alignment algorithm in the MegAlign package (DNASTAR) and the MEGA program. A phylogenetic tree was constructed by the neighbor-joining method. Comparative sequence analysis of rpoB DNAs provided the basis for species differentiation within the genus Mycobacterium. Slowly and rapidly growing groups of mycobacteria were clearly separated, and each mycobacterial species was differentiated as a distinct entity in the phylogenetic tree. Pathogenic Mycobacterium kansasii was easily differentiated from nonpathogenic M. gastri; this differentiation cannot be achieved by using 16S rRNA gene (rDNA) sequences. By being grouped into species-specific clusters with low-level sequence divergence among strains of the same species, all of the clinical isolates could be easily identified. These results suggest that comparative sequence analysis of amplified rpoB DNAs can be used efficiently to identify clinical isolates of mycobacteria in parallel with traditional culture methods and as a supplement to 16S rDNA gene analysis. Furthermore, in the case of M. tuberculosis, rifampin resistance can be simultaneously determined.     

Performance of three enzyme immunoassays and two direct fluorescence assays for detection of Giardia lamblia in stool specimens preserved in ECOFIX

ECOFIX is a single-vial stool preservative that is both formalin- and mercury-free. We evaluated the abilities of three commercial Giardia lamblia-specific enzyme immunoassays (EIAs) (ProSpecT Giardia Microplate Assay [Alexon-Trend Inc.], Giardia Test [Techlab], and Premier Giardia lamblia [Meridian Diagnostics, Inc.]) and two commercial direct fluorescent-antibody (FA) assays for G. lamblia (Crypto/Giardia IF Test [Techlab] and Merifluor Cryptosporidium/Giardia [Meridian Diagnostics, Inc.]) to detect G. lamblia in 34 G. lamblia-positive and 44 G. lamblia-negative stool specimens (determined by traditional examination for ova and parasites) preserved in ECOFIX compared to their abilities to detect G. lamblia in the same specimens preserved in formalin as the "gold standard" for each assay. Of the 34 formalin-fixed positive specimens, the number detected by each assay was as follows:, Alexon EIA, 34; Meridian EIA, 27; Techlab EIA, 29; Meridian FA assay, 31; and Techlab FA assay, 28. Both FA tests and the Alexon EIA performed well with ECOFIX, but the other two EIAs detected fewer positive specimens (the difference was statistically significant with the Techlab EIA) when ECOFIX was the preservative. Use of G. lamblia cyst antigen from cultured organisms preserved in formalin and ECOFIX demonstrated that the Alexon EIA could detect smaller amounts of antigen in ECOFIX than the other two EIAs could and suggested that cyst antigen is more stable in formalin. We recommend that laboratories use an FA assay or the Alexon EIA if they plan to use ECOFIX as their stool preservative.     

Detection of Bordetella bronchiseptica by the polymerase chain reaction.

Polymerase chain reaction (PCR) assays were developed that enabled not only discriminative detection of three Bordetella species, B. pertussis, B. parapertussis, and B. bronchiseptica (Bspp PCR), but also specific detection of B. bronchiseptica (Bb PCR). An upstream sequence of the flagellin gene was used as a target DNA region. This sequence contained differences in B. pertussis, B. parapertussis, and B. bronchiseptica DNA. These species could then be differentiated using two different sets of primers, Bspp and Bb. When oligonucleotide Bspp primers were used, PCR products were obtained from the three species of Bordetella. A fragment of the expected size (164 bp) was amplified using B. bronchiseptica and B. parapertussis DNA, but a fragment with a distinct molecular weight was amplified with B. pertussis DNA (195 bp). This Bspp PCR was specific and sensitive, but it could not differentiate between B. parapertussis and B. bronchiseptica. When Bb primers were used, a 237-bp PCR product was detected only from B. bronchiseptica DNA. No PCR products were identified after Bb PCR amplification of DNAs either from B. parapertussis isolates or B. pertussis isolates, nor from other respiratory pathogen DNAs tested. This second PCR assay had a sensitivity limit of less than 10 organisms of B. bronchiseptica after detection with a specific probe. The specificity and the sensitivity of the fla PCR assay were evaluated with purified DNA, as was its capacity for detecting the bacteria in human clinical samples and in lungs of infected mice.     

Detection of Helicobacter pylori in gastric mucosa of patients with gastroduodenal diseases by PCR-restriction analysis using the RNA polymerase gene (rpoB)

A novel PCR restriction analysis method using the RNA polymerase beta-subunit- coding gene (rpoB) was employed to both detect and identify Helicobacter pylori in biopsy specimens and culture isolates. The rpoB DNAs (458 bp) were specifically amplified by PCR with the Helicobacter-specific primers (HF and HR). Based on the determined rpoB sequences of the culture isolates, an H. pylori-specific restriction site, Tru9I, was found. H. pylori can be identified by observing two discernible DNA fragments (288 and 138 bp) after Tru9I digestion and agarose gel electrophoresis. The rpoB PCR and subsequent restriction analysis (PRA) enabled the specific detection and identification of H. pylori in biopsy specimens from patients with gastroduodenal diseases. The rpoB PRA conferred a compatible or a slightly higher positive rate (53.7%) than did the Campylobacter-like organism (CLO) test (50.4%) and glmM PCR (48.8%), suggesting that it is useful for diagnosing an H. pylori infection without culture in the clinical laboratory.     

实时荧光PCR检测蓝氏贾第鞭毛虫和隐孢子虫方法的建立

目的建立贾第鞭毛虫和隐孢子虫的基因检测方法。方法根据GenBank中贾第鞭毛虫和隐孢子虫相对保守序列,设计引物及其相应的Taqman探针。通过对引物和探针浓度、Taq酶以及反应条件等优化筛选后,建立检测贾第鞭毛虫和隐孢子虫的荧光PCR方法。结果贾第鞭毛虫和隐孢子虫的检测结果为阳性,对其它DNA样本如日本血吸虫、刚地弓形虫、溶组织内阿米巴、旋毛虫和阴道毛滴虫的检测均为阴性。本法的敏感性高,可检测到10～102copies/μl的DNA浓度。结论建立的基因检测方法,对贾第鞭毛虫和隐孢子虫的检测具有高度的特异性和敏感性,可用于饮用水和临床样本等的快速检测。