阴沟肠杆菌随机扩增多态性DNA法基因分型

目的建立阴沟肠杆菌(ECL)随机扩增多态性DNA(RAPD)法基因分型图谱.方法以优化的随机引物和反应体系对临床分离的159株ECL进行RAPD法基因分型,并按DNA条带数及片段大小绘制指纹图谱.结果 159株临床分离ECL共得121种RAPD指纹图谱.结论 RAPD技术可将临床分离ECL分型121种.     

上海市浦东地区淋病奈瑟菌分型与耐药性的相关分析

目的了解上海市浦东地区淋病病原体奈瑟双球菌的不同基因分型及各基因型与耐药性之间的对应关系。方法运用随机扩增多态性DNA(RAPD)指纹图谱对78株分离自不同患者淋球菌菌株进行区分，从分子水平对淋球菌进行基因分型，并在此基础上探讨不同的基因分型与耐药性之间的关系。结果78株淋球菌分离株RAPD图谱上相似，但各菌株基因图谱之间有明显不同DNA多态性，可将菌株区分为Ⅰ、Ⅱ、Ⅲ三种基因分型，对此三种基因分型与A、B、C、D四种不同的耐药类型进行对应分析，发现耐药类型与基因型别之间存在对应关系。结论通过研究发现浦东地区淋球菌流行株有着不同的耐药特点及基因型别，耐药性与不同的基因分型之间存在着一定相关性。     

102株单核细胞增生李斯特菌随机扩增多态性DNA基因分型研究

目的:对单核细胞增生李斯特菌进行随机扩增多态性DNA(RAPD)基因分型研究。方法:以优化的随机引物和反应体系对生肉、熟肉、生奶、乳制品、水产品、冷冻食品、生食蔬菜共七大类食品中分离的102株单核细胞增生李斯特菌进行RAPD法基因分型,并按DNA条带数及片段大小绘制指纹图谱。结果:102株单核细胞增生李斯特菌共得26种RAPD指纹图谱,以第12、18、23和10型为主要型别,分别为15、14、8和6株。结论:RAPD技术可将单核细胞增生李斯特菌分型26种,我省食品中分离的单核细胞增生李斯特菌主要基因型为第12、18、23和10型。     

肺炎克雷伯菌随机DNA多态性分型研究

目的 建立肺炎克雷伯菌随机扩增DNA多态性分型技术,以应用于临床菌株流行病学调查。方法 对临床分离的29株肺炎克雷柏菌进行Etest检测,并用酚氯仿法提取其DNA后行RAPD分型。结果 29株临床分离菌有7株ESBL阳性,24株有稳定的RAPD带型,其中ICU分离的5株ESBL阳性株的4株RAPD指纹图谱相同。结论 RAPD基因分型技术不需已知核酸序列,分型率高,分辨力强,简便快捷可为肺炎克雷伯菌株提供分型标志,是分子流行病学研究的有效方法。     

重庆市老年中心铜绿假单胞菌流行病学分型研究

目的对重庆市老年中心引起医院感染的铜绿假单胞菌(PAE)进行流行病学分型研究,了解其流行与耐药,以便更好地控制其引起的医院感染.方法采用随机扩增多态性DNA(RAPD)基因分型方法对重庆市老年中心分离的24株PAE进行RAPD基因分型.结果24株PAE共得RAPD 14型,分型率100%;同一病区不同患者分离出的PAE未检出相同基因型的PAE,3个不同病区间未检出相同RAPD型别的PAE;7例感染个体连续收集的PAE有5例RAPD指纹图谱完全一致,2例出现跳跃性指纹图谱,且与其他分型指纹图谱不相同.结论RAPD能满足对PAE进行分子流行病学分型;通过分型可判断内原性、外源性或突变PAE引起的感染.     

随机引物扩增技术对变形菌DNA多态性分型研究

目的建立变形菌随机扩增DNA多态性分型技术,应用于临床菌株流行病学调查. 方法优化随机扩增DNA多态性指纹技术(RAPD)的实验条件,利用RAPD技术成功地检测21株变形菌DNA指纹图谱. 结果 21株变形菌分为18型别,其中16株奇异变形菌分为13个型,5株普通变形菌分为5个型. 结论 RAPD分型技术可简便、快速为变形菌提供分型标志,是分子流行病学研究的有效方法.     

随机扩增多态性DNA分型法在铜绿假单胞菌流行病学研究方面的应用

目的摸索适于铜绿假单胞菌随机扩增多态性DNA分型法(RAPD)分型的最佳实验条件,并对我院临床分离到的34株铜绿假单胞菌进行分型研究。方法从随机引物筛选、镁离子浓度、模板量、引物浓度4方面对RAPD反应体系进行优化。结果不同病区分离到的铜绿假单胞菌的指纹图谱互不相同;从烧伤病房分离到的8株高度耐药的铜绿假单胞菌中,其中7株为同一指纹图谱,另外1株为独特的指纹图谱,而外科重症监护病房、肺科病房、干部病房同一病区内分离到的铜绿假单胞菌均为同一指纹图谱。结论铜绿假单胞菌在病区内存在交叉感染的情况,但未出现病区间感染同一流行株。     

随机扩增多态性DNA在淋球菌基因分型中的应用

目的 建立随机扩增多态性DNA(RAPD)对淋球菌进行基因分型的方法。方法 用4种不同的预处理方法提取淋球菌基因组DNA,对其优劣进行比较;并运用RAPD对淋球菌菌株进行区分及对经性伴传播的淋病病例进行RAPD指纹图谱比较。结果 运用经典的十六烷基三乙基溴化铵法可以抽提较完整的基因组DNA,获得良好的RAPD指纹图谱;各菌株的RAPD指纹图谱间有明显不同DNA多态性;性伴传播的病例中获得了非常相似的RAPD指纹。结论 选择最佳的DNA抽提技术,运用RAPD可以对淋球菌进行有效基因分型,并可用于分子流行病学对传染源的追踪。     

白色假丝酵母菌的随机扩增多态性DNA分型研究

目的建立医院感染白色假丝酵母菌的基因多态性DNA分型方法,了解白色假丝酵母菌医院感染的分子流行病学情况,并探讨其基因多态性与真菌药物敏感性检测结果的可能关系。方法收集临床各病区送检的住院患者白色假丝酵母菌感染标本30份和其相应患者资料、抗真菌药物敏感性检测结果,采用经优化的随机扩增多态性(RAPD)DNA方法,对30株白假丝酵母菌进行基因多态性分型,利用Phylip3.67聚类分析软件分别用邻接法和算术平均数的非加权成组配对法,对RAPD扩增条带结果进行聚类分析。结果随机引物OPE-03进行RAPD扩增的指纹图谱清晰,带型稳定,多态性丰富,可以作为分型引物;30株不同来源的菌株可大致分为3种亲缘关系;两种聚类分析结果相同,提示该实验结果可靠。结论白色假丝酵母菌基因多态性分布可能与菌株的来源有关;RAPD多态性可能与其耐药性有关;不同聚类分析方法的RAPD图谱分析结果相同。     

嗜麦芽窄食单胞菌随机扩增多态性DNA基因分型及同源性分析

目的采用一种快速,准确的基因分型方法,针对嗜麦芽窄食单胞菌导致的医院内感染进行流行病学调查,为预防院内交叉感染提供理论依据。方法收集南京市某医院住院患者痰液样本,并分离嗜麦芽窄食单胞菌,采用随机扩增多态性DNA(RAPD)技术获得指纹图谱,并利用SAS9.2软件对其进行聚类分析。结果 39株嗜麦芽窄食单胞菌菌株根据其DNA条带聚类分析,可分为A、B、C、D、E、F和G 7个型,A、B、D、E均有2个亚型,其中A型为优势型别。同源性分析结果表明在神经内科与呼吸内科,ICU与呼吸内科分别有同一克隆株的传播,科室之间可能存在交叉感染。结论 RAPD是一种简单快速,低成本的基因分型方法,适合临床病原菌的分型及医院感染分子流行病学研究。     

重症监护室内鲍曼不动杆菌随机扩增DNA多态性分型研究

目的：建立鲍曼不动杆菌随机扩增DNA多态性（RAPD）分型技术,以用于临床菌种鉴定及流行病学调查。方法：收集6个月内从ICU内分离出的14株鲍曼不动杆菌,提取DNA后进行RAPD分型;同时进行生物学分型和抗生素敏感性实验,结果：14株菌株被分为5种RAPD型,其中有5株属于同一种类型（A型）,有6株属于另外同一种类型（B型）,其他3株分别属于另3种不同类型（C,D,E型）,而生物学分型只有两种类型（生物型1,9）,抗生素敏感性实验表明14株菌株均为仅对亚胺培南／西司他丁（泰能）,头孢哌酮/舒巴坦（舒普深）等少数几种抗生素敏感的多重耐药菌株。结论：ICU内存在鲍曼不动杆菌暴发流行,RAPD分型技术分型率高,分辨力强,简便快速,具有分子流行病学研究的应用价值。     

隐源性食酸丛毛单胞菌血流感染调查研究 FREE

目的调查某医院临床科室分离的8株食酸丛毛单胞菌的同源性,探讨其导致医院血流感染的途径。方法对分离的食酸丛毛单胞菌,采用随机引物扩增多态性DNA技术检测并进行同源性分析,同时采集病房环境和医疗用品样本进行培养。结果8株食酸丛毛单胞菌为同一基因型,而病房环境和医疗用品中未分离出此菌。结论此起食酸丛毛单胞菌血流感染为集中发病,病原菌为同一基因型,但感染源未明。     

Genetic typing of human and food isolates of Listeria monocytogenes from episodes of listeriosis

Ten clinical and food Listeria monocytogenes strains isolated during the epidemiological investigations of episodes of listeriosis (one outbreak and two sporadic cases) that occurred in northern Italy during 1993–1995 have been examined by DNA macrorestriction pattern analysis obtained by PFGE and RAPD typing, in order to confirm the food vehicle of infections. The same DNA profiles within the isolates from the three episodes were obtained by both techniques. The ApaI and SmaI PFGE profiles and RAPD patterns with primer OPM-01 confirmed the close relationship between strains from two distinct episodes. However, RAPD analysis with primer UBC-127 distinguished between these L. monocytogenes isolates.     

Molecular subtyping by genome and plasmid analysis of Campylobacter jejuni serogroups O1 and O2 (Penner) from sporadic and outbreak cases of human diarrhoea.

Ribosomal RNA gene patterns, randomly amplified polymorphic genomic DNA (RAPD) profiles and plasmid profiles were used to discriminate between 28 strains of Campylobacter jejuni serogroups O1 and O2 (Penner). Most isolates were biotype I (Lior). The strains were representative isolates from a UK school outbreak of enteritis (7 cases) and from 21 sporadic human cases of enteritis in 4 countries. The molecular techniques discriminated to various degrees between strains in each of the serogroups. The outbreak strains were homogeneous in most molecular features but a variety of types was detected amongst the isolates from the sporadic cases. Five groups of two or more strains with identical ribopatterns were identified and within each, strains from different patients were homogenous with respect to serogroup. RAPD profile typing based on numerical analysis generally matched ribotyping. Plasmid profiling overall gave least discrimination but was useful in separating some strains similar in other features. We concluded that optimal discrimination of C. jejuni could best be achieved using a combination of phenotypic and genotypic properties. Hae III ribotyping was the single most discriminatory and reproducible technique investigated. Several strains of C. jejuni from sporadic infections had similar molecular profiles which have potential for general typing purposes.     

Epidemiological study of invasive pulmonary aspergillosis in a haematology unit by molecular typing of environmental and patient isolates of Aspergillus fumigatus

In order to determine the possible relationship between environmental contamination by Aspergillus fumigatus and occurrence of invasive aspergillosis, a one-year prospective study was carried out in the haematology ward of Hautepierre Hospital, Strasbourg, France. During the study period, 21 environmental isolates and 26 clinical isolates of A. fumigatus were collected. Each was genotyped using a random amplification of polymorphic DNA (RAPD) technique. Thirty-four distinct profiles were identified by RAPD analysis, indicating the great genetic diversity of A. fumigatus isolated from infected patients and from the environment. For two patients, RAPD analysis demonstrated concurrent infection by at least two different strains. In two cases, a genetic similarity was noted between isolates obtained from a patient and from the environment.     

新生儿铜绿假单胞菌随机扩增多态性DNA分子流行病学研究

对在６个月内从１２１名产科新生儿中的３０名检出的３１株铜绿假单胞菌进行ＲＡＰＤ指纹图谱分析和血清学分型。２４株为Ｏ：６血清型，６株属于另外３个血清型，１株未能分型。ＲＡＰＤ分型率为１００％。３１个菌株分成５个ＲＡＰＤ谱型。２５株为Ｒ：１型，其它６株为另外的４个谱型。血清型不同的新生儿菌株的ＲＡＰＤ谱型亦不相同。２株Ｏ：１型的菌株，其ＲＡＰＤ谱型却不同。同期分别来自４个其他科室的５名患者的５株Ｏ：６型ＰＡ菌的ＲＡＰＤ谱型各自不同，与新生儿Ｏ：６型的ＲＡＰＤ谱型也不同。结果表明，铜绿假单胞菌在产科新生儿的暴发流行，Ｏ：６／Ｒ：１型为暴发流行菌株；ＲＡＰＤ分型率高，分辨力强，快速简便，颇具医院感染分子流行病学研究的应用价值。     

Molecular epidemiological survey of Citrobacter freundii misidentified as Cronobacter spp. (Enterobacter sakazakii) and Enterobacter hormaechei isolated from powdered infant milk formula

A total of 75 powdered infant milk formula (PIF) samples collected from pharmacies and drugstores in Western Sicily, Italy, and representative of 12 different brands were analyzed in this study to evaluate their microbiological quality. According to the U.S. Food and Drug Administration protocol, 32 samples out of 75 were contaminated by enterobacteria. Commercial biochemical API(r) 20E-system identification method indicated that six PIF samples were presumptively contaminated by Cronobacter spp., but further characterization by alpha-glucosidase based polymerase chain reaction (PCR) assay identification strongly suggested that these strains did not belong to the genus Cronobacter. Phylogenetic analysis of partial 16S rRNA (rrs) sequences combined with the results of biochemical tests allowed to identify the six strains as Citrobacter freundii. Similarly, rrs sequence analysis identified as Enterobacter hormaechei 23 strains originally ascribed to Enterobacter cloacae by the API 20E system. Characterization of C. freundii and E. hormaechei PIF isolates by the DiversiLab(r) repetitive sequence-based PCR (rep-PCR) typing method revealed a variety of amplification patterns, but the recovery of the same rep-PCR genotype in several products might indicate a special adaptation of genetic clones to this food or cross-contamination through common ingredients. Antibiotic-resistance profiles were also determined, but none of the strains tested was resistant to third-generation cephalosporins or fluoroquinolones and extended-spectrum beta-lactamase activity was not detected. Our results confirm that E. hormaechei contamination of PIF is widespread, thus making it a cause for concern. Similarly to what was demonstrated for E. hormaechei, we suggest that C. freundii also may be an under-reported cause of bacterial infection, especially in high-risk neonates, due to misidentification.     

吸毒人群口腔念珠菌的DNA多态性及其与耐药性的分析

目的 了解吸毒人群口腔念珠菌的DNA多态性以及与耐药性的关系.方法 选择4种随机引物对75株吸毒人群口腔念珠菌进行DNA多态性分析,并优化试验条件,用聚类分析方法对其进行基因分型.结果 筛选出两条随机引物进行RAPD,念珠菌基因组DNA在100～200 ng/μL,引物浓度在5～10 μmoL/L时,得到的PCR指纹图最丰富和清楚.聚类分析显示,吸毒人群口腔念珠菌同种不同株间存在着基因多态性,不同种间具有一定的基因型特点,某些耐唑类和耐5-氟胞嘧啶药物菌株单独聚为特殊的基因型.结论 RAPD基因分型可作为念珠菌种间鉴定的参考方法.吸毒人群口腔念珠菌中某些菌株的耐药性与其DNA多态性有一定关系,某些特殊PCR指纹图可能与耐唑类药物基因和5-FC基因有一定关系.