常见病原真菌鉴定基因芯片方法的建立

目的 建立临床常见的病原真菌的基因芯片检测体系.方法 选取8种临床常见真菌,包括白色假丝酵母菌、光滑假丝酵母菌、热带假丝酵母菌、近平滑假丝酵母菌、土曲霉、黄曲霉、米根霉和烟曲霉.根据真菌ITS区设计引物和探针,进行扩增和芯片制备.通过将待检真菌的PCR产物变性后与点布探针的芯片杂交,观察并分析荧光信号强度,进行对真菌的快速检测.并选取临床真菌阳性标本和细菌阳性标本共25份进行验证.结果 25份临床阳性标本的芯片检测结果显示,10份细菌阳性的标本,经真菌基因芯片检测,全部为阴性,无荧光信号.其余15份真菌标本中,12份临床真菌阳性标本均鉴定出芯片中的某一种真菌的结果.另外采用克柔假丝酵母菌人为制备的3份待检样本,经检测,芯片中8种真菌位点无荧光信号,为阴性,仅有真菌通用探针得到荧光信号,表明该标本中有真菌存在,但无法确认是哪一种.结论 本研究建立的基因芯片可对常见病原真菌进行快速准确的鉴定,为基因芯片应用于临床奠定了基础.     

基因芯片在细菌及其耐药检测中的应用

广谱抗生素的广泛使用易导致菌株变异和产生耐药性,迫切需要新的技术来解决如何快速、准确诊断病原,正确选择药物和合理用药等问题。基因芯片不仅可以大量快捷地检测出耐药性菌株,进行细菌诊断、鉴别诊断、耐药性监测,而且可以     

金黄色葡萄球菌的基因芯片检测研究

目的用基因芯片对临床标本进行金黄色葡萄球菌快速检测。方法抽取180例临床标本,其中160例常规细菌培养鉴定疑似金黄色葡萄球菌感染和20例非金黄色葡萄球菌感染,金黄色葡萄球菌质控菌株(ATCC25923)、大肠埃希菌(ATCC25922)采用研究制备出金黄色葡萄球菌筛查的基因芯片进行测定,同时进行比对分析。结果金黄色葡萄球菌检出符合率为100%,基因芯片检测的标本需求量少,检验时间短,敏感性、特异性、准确性更高。结论针对细菌培养的临床标本特点,采用高效的基因芯片检测方法,具有现实的临床意义。     

角膜炎常见致病真菌DNA快速提取方法的探索

近年来我国真菌性角膜炎的发病逐年上升，聚合酶链反应（PCR）技术为真菌病原体检测提供了一条快速、敏感的诊断方法。真菌细胞壁较厚不易裂解，传统的DNA提取因其繁琐而成为该技术应用于临床的障碍。本实验使用几种不同的裂解液提取常见角膜致病真菌DNA，并将结果进行比较分析。     

铜绿假单胞菌的基因芯片检测研究

目的用基因芯片对临床标本铜绿假单胞菌进行快速检测。方法结合临床检验经验,抽取98例临床标本,其中78例常规细菌培养鉴定疑似铜绿假单胞菌感染,20例非铜绿假单胞菌感染,铜绿假单胞菌质控菌株(ATCC27853),大肠埃希菌(ATCC25922),用研究制备出铜绿假单胞菌筛查的基因芯片进行测定,同时进行比对分析。结果铜绿假单胞菌检出符合率为100%,基因芯片检测的标本需要量少,检验时间短,敏感性、特异性、准确性高。结论针对细菌培养的临床标本特点,采用高效的基因芯片检测方法,具有现实的临床意义。     

基因芯片技术的最新进展

基因芯片技术在生物医学中的应用在过去几年中发生了巨大的变化。该文概括介绍基因芯片技术,着重阐述了它在疾病机制研究、疾病的诊断和分类及疾病的预测和治疗中的新应用,并探讨了基因芯片技术和其他技术相结合的新进展。     

荧光定量聚合酶链反应检测常见致病真菌方法的建立

目的建立快速的检测临床致病真菌的荧光定量聚合酶链反应(PCR)方法。方法使用真菌的通用引物ITS86和ITS4,检测临床常见的致病性真菌,并观察检测方法的敏感性和特异性。结果建立的荧光定量PCR可以准确地检测念珠菌属、曲霉菌属和新型隐球菌等常见的致病真菌,白色念珠菌的敏感性为0.87fg,光滑念珠菌的敏感性为4.2fg,与细菌和人类基因组DNA以及病毒DNA均无交叉反应,根据融解曲线的Tm值可以进行菌种的初步鉴定。结论荧光定量PCR方法检测真菌的敏感性和特异性好,可以应用于临床进行快速检测。     

建立检测细胞色素P450酶与紫杉醇代谢相关突变位点的基因芯片分型方法

目的 建立针对与临床抗癌药物紫杉醇代谢相关的细胞色素P450(CYP450)酶基因多态性位点的快速、准确、高通量的基因芯片基因分型方法.方法 选取CYP450酶2C8*3、3A4* 18、3A5*3C等3个与紫杉醇代谢相关突变位点,根据基因库中报道的序列,设计每个基因多态性位点的野生型和突变型探针,在突变位点2侧设计PCR扩增引物,PCR片段长度应＜200 bp,并构建标准质粒.探针在3'端氨基修饰,下游引物标记荧光素Cy3,将探针按一定顺序点样于经醛基化处理的玻片制备成基因芯片.人体血液DNA样本分别通过3对引物扩增后与基因芯片上的探针杂交,通过扫描图像和配套软件对结果进行分析和判断.同时,对50份血液样本进行检测.结果 通过标准质粒杂交结果显示,每个位点对应的1对探针均能将野生型和突变型质粒进行准确地区分,无非特异性杂交信号出现;检测50份血液样本,CYP2C8 * 3位点的突变率为2%,CYP3A4 * 18位点均为野生型,CYP3A5 * 3C位点的突变率为62%.同时,通过测序法进行验证,基因芯片方法与测序方法的结果完全一致.结论 建立的同时检测抗癌药物紫杉醇代谢相关的CYP450酶基因多态性位点CYP2C8*3、CYP3A4 * 18、CYP3A5 * 3C的基因芯片分型方法快速、准确,结果可靠,重复性好,可用于指导紫杉醇患者的个体化用药,并为分析个体患者对于紫杉醇药物体内代谢提供了一个高通量技术平台.     

炭疽芽孢杆菌基因芯片探针的制备

目的制备炭疽芽孢杆菌(BA)基因芯片探针,应用于BA基因芯片的检测。方法采用PCR方法,以BA疫苗株A16R基因组DNA为模板,扩增pXO1质粒上的8个特异性片段,连接至pMD19-T Simple载体,构建分别包含8个探针序列的重组质粒,以其为PCR扩增模板获取探针DNA。结果基因测序证明,成功获得BA的3个探针。结论 BA基因探针的成功获取,为炭疽芽孢杆菌基因芯片的制备奠定了基础。     

临床真菌检测及耐药性动态变化特征及分析

目的了解临床标本真菌检出情况,并进行真菌耐药性分析,为临床合理使用抗真菌药物提供依据。方法常规培养分离真菌,用科玛嘉产色培养基和Vitek全自动微生物分析仪鉴定到种。药敏试验采用ATBFungus3微量最低抑菌浓度药敏试验试剂盒。结果共检出476株深部真菌,2006、2007、2008年检出率分别为10.04、14.53、18.08,呈逐年增加的趋势;菌种以白色念珠菌(64.50)、热带念珠菌(15.97)、光滑念珠菌(11.76)为主。真菌检出科室以干部病房(36.76)、重症监护室(14.08)为主;感染标本来源以呼吸道和泌尿道为主,分别占63.24和8.40;真菌对氟胞嘧啶、氟康唑、伊曲康唑、两性霉素B、伏立康唑的耐药率分别为5.7、9.9、32.4、2.3、1.3。结论深部真菌感染逐年增多,对两性霉素B和伏立康唑最为敏感。伊曲康唑、氟康唑耐药率较高,临床用药应根据药敏结果合理选用。     

PCR检测技术在真菌性角膜炎快速诊断中的应用

目的探讨聚合酶链反应(PCR)检测技术在真菌性角膜炎快速诊断中的应用。方法留取攀枝花学院附属医院502例疑似感染性角膜炎患者角膜分泌物分别进行革兰染色镜检、真菌培养和PCR检测,比较3种方法的检出率。结果革兰染色镜检、真菌培养、PCR检出率分别为5.6%(28例)、4.8%(24例)、5.9%(30例),三者比较,差异无统计学意义(χ~2=0.722,P=0.697)。结论 PCR检测、革兰染色镜检和真菌培养3种方法对真菌性角膜炎的检出率无明显差异,PCR检测具有能更快速地诊断真菌性角膜炎的特点。     

Sequence-specific identification of 18 pathogenic microorganisms using microarray technology

We have developed a Multi-Pathogen Identification (MPID) microarray for high confidence identification of eighteen pathogenic prokaryotes, eukaryotes and viruses. Analysis of amplified products from pathogen genomic DNA using microarray hybridization allows for highly specific and sensitive detection, and allows the discrimination between true amplification products and false positive amplification products that might be derived from primers annealing to non-target sequences. Species-specific primer sets were used to amplify multiple diagnostic regions unique to each individual pathogen. Amplified products were washed over the surface of the microarray, and labelled with phycoerythrin-streptavidin for fluorescence detection. A series of overlapping 20-mer oligonucleotide probes hybridize to the entire diagnostic region, while parallel hybridizations on the same surface allow simultaneous screening for all organisms. Comparison to probes that differ by a single mismatch at the central position reduced the contribution of non-specific hybridization. Samples containing individual pathogens were analyzed in separate experiments and the corresponding species-specific diagnostic regions were identified by fluorescence among their highly redundant probe sets. On average, 91% of the 53 660 pathogen probes on the MPID microarray performed as predicted. The limit of detection was found to be as little as 10 fg of B. anthracis DNA in samples that were amplified with six diagnostic primer-pairs. In contrast, PCR products were not observed at this concentration when identical samples were prepared and visualized by agarose gel electrophoresis.     

Development of a microarray for identification of pathogenic Clostridium spp.

In recent years, Clostridium spp. have rapidly reemerged as human and animal pathogens. The detection and identification of pathogenic Clostridium spp. is therefore critical for clinical diagnosis and antimicrobial therapy. Traditional diagnostic techniques for clostridia are laborious, are time consuming, and may adversely affect the therapeutic outcome. In this study, we developed an oligonucleotide diagnostic microarray for pathogenic Clostridium spp. The microarray specificity was tested against 65 Clostridium isolates. The applicability of this microarray in a clinical setting was assessed with the use of mock stool samples. The microarray was successful in discriminating at least 4 species with the limit of detection as low as 10⁴ CFU/mL. In addition, the pattern of virulence and antibiotic resistance genes of tested strains were determined through the microarrays. This approach demonstrates the high-throughput detection and identification of Clostridium spp. and provides advantages over traditional methods. Microarray-based techniques are promising applications for clinical diagnosis and epidemiologic investigations.     

ITS序列鉴定真菌性鼻窦炎病原的方法评价

目的 建立ITS(包括ITSI-5.8 rRNA-ITS2)测序鉴定真菌性鼻窦炎病原的方法.方法 收集北京同仁医院2006-2008年,经临床与CT诊断为真菌性鼻窦炎,并行鼻内镜手术切除的组织标本270份.所有标本分别进行组织病理检查、压片直接镜检、真菌培养鉴定和核糖体RNA转录间隔区测序分析,通过方法比较,评价序列分析直接鉴定病原真菌的可行性,同时分析真菌性鼻窦炎病原学特征.结果 在270份标本中,组织病理阳性率为80.0%(216/270),压片阳性率为80.0%(216/270),真菌培养阳性率为53.0%(143/270),ITS测序阳性率为63.0%(170/270).经培养得到22个种,6个属.ITS测序鉴定32个种.培养与ITS序列种水平符合率为76.1%(102/143).结论 ITS测序可成为真菌鉴定的辅助工具.     

A multiplex PCR assay for the detection of five human pathogenic Vibrio species and Plesiomonas

A multiplex PCR (mPCR) assay was established to detect five pathogenic Vibrio species and Plesiomonas shigelloides. Twelve genes were included: ompW, ctxA, rfbN, and wbfR from V. cholerae; tl, tdh, and trh from V. parahaemolyticus; toxR and vmhA from V. mimicus; toxR from V. fluvialis; vvhA from V. vulnificus; and the 23S rRNA gene from P. shigelloides. The specificity of the mPCR assay was 100% for the detection of 136 strains and the limits of detection (LoD) were 12.5–50 pg/reaction. The assay exhibited higher sensitivity than cultivation methods in the detection of APW cultures of 113 diarrhea samples. In the analysis of 369 suspected Vibrio populations from estuarine water samples, the specificity of the mPCR for V. cholerae and V. parahaemolyticus was 100% for both, while the sensitivities were 100% and 96.1%, respectively. The assay can be applied to screen enrichment cultures and suspected colonies from environmental and clinical samples.     

DNA微阵列芯片法与常规生化法在分枝杆菌菌种鉴定方面的比较

目的通过DNA微阵列芯片法进行分枝杆菌菌种鉴定与常规生化法进行比较,分析其特点,提高分枝杆菌菌种鉴定水平,更好的为临床服务。方法选择我院2010年1月至2013年3月从临床标本中分离培养后所得的结核分枝杆菌复合群12株（其中含1株牛型结核分枝杆菌）,非结核分枝杆菌3l株,分别用DNA微阵列芯片法和常规生化法进行鉴定。结果DNA微阵列芯片法进行分枝杆菌菌型鉴定与普通生化培养法鉴定结果符合率为100％,对常规生化法未定型的两株非结核分枝杆菌也分别定型为1株土分枝杆菌,1株耻垢分枝杆菌。结论DNA微阵列芯片法与常规生化法相比在分枝杆菌菌型鉴定中具有更快速、准确的特点,是分枝杆菌菌型鉴定的有利工具。     

Topical voriconazole as a novel treatment for fungal keratitis

Paecilomyces lilacinus is a fungal pathogen which is generally resistant to amphotericin B and certain other antifungals and is an uncommon cause of devastating fungal keratitis. In the present studies, we evaluated topical voriconazole as therapy for P. lilacinus keratitis in rabbits. Thirty eyes of 15 rabbits were studied. In five animals, the uninfected left eye was treated twice daily with voriconazole (drug control, uninfected eye). In these same animals, the right eye was infected with P. lilacinus but not treated with voriconazole (infection control eye). By day 5, the infection controls had lesions of >2.4 mm in diameter, with conjunctivitis and severe hypopyon, and were sacrificed. In the other 10 rabbits (voriconazole treatment), the right eyes were infected with P. lilacinus and treated with voriconazole beginning on day 3 after infection. Voriconazole therapy caused lesions to decrease during 8 days of therapy, after which rabbits were sacrificed (11 days postinfection). Hyphal masses were present in the control infected eyes and absent in treated infected eyes. Voriconazole was detected in all tissues of treated eyes. Topical voriconazole is effective treatment for P. lilacinus experimental keratitis, and it penetrates more deeply than the corneal tissue.     

Multiplex PCR detection and species differentiation of orthopoxviruses pathogenic to humans

A method for one-stage rapid identification of four orthopoxvirus species pathogenic to humans based on multiplex polymerase chain reaction (MPCR) was developed. Five pairs of oligonucleotide primers--one, genus-specific; and the rest, species-specific for variola, monkeypox, cowpox, and vaccinia viruses, respectively--were used concurrently for MPCR assay of orthopoxvirus DNAs. Specificity and sensitivity of the method developed were evaluated using DNAs of 57 orthopoxvirus strains, including the DNAs isolated from human case clinical materials.     

真菌性角膜炎病人的护理

真菌性角膜炎是角膜病中致盲率较高的感染性眼病。角膜受真菌感染后,真菌在角膜组织内繁殖,真菌毒素、蛋白溶解酶及真菌可溶性抗原等作用产生严重的炎症反应,导致组织坏死及溃疡形成,在短时间内可致视力丧失,甚至眼球毁损。我科2004年2月—2005年5月对14例真菌性角膜炎病人进行综合治疗和护理,取得了良好效果。现将护理体会报告如下。1临床资料本组病人14例(14眼),其中男12例,女2例,年龄20岁～69岁。病因:稻谷刺伤5例,麦芒刺伤4例,农作物枝叶伤3例,原因不明感染2例。症状:患眼视物模糊、畏光、流泪、疼痛、异物感、角膜区病灶呈灰白色或黄色…