沙门菌Rep-PCR与PFGE分子分型研究

目的分析比较Rep-PCR和PFGE方法对45株沙门菌的分型能力。方法应用Rep-PCR和PFGE方法对45株沙门菌(其中日常监测菌株29株,食物中毒分离菌株16株)进行分型,并对2种分型方法的分辨力及相关性进行比较。结果 45株沙门菌经Rep-PCR分析,可分为27个型,D值为91.1%;经PFGE方法分析,可分为29个型,D值为93.2%;2种方法的D值均高于血清学分型方法(85.9%)。Rep-PCR和PFGE分型结果表明,大多数菌株具有明显的对应关系,尤其是从食物中毒事件分离得到的菌株PFEG和Rep-PCR带型高度一致,也有部分菌株未显示出明显的对应关系。结论沙门菌Rep-PCR和PFGE方法分型结果相关性较强,但Rep-PCR的分辨力略低于PFGE,二者均可用于疫情溯源研究。     

沈阳市副溶血弧菌重复序列PCR分型

目的 探讨重复序列PCR对临床分离株副溶血弧菌进行分型的可行性;从分子水平了解沈阳市流行的副溶血弧菌菌型特征.方法 利用基因外重复回文序列-PCR(PEP-PCR)和肠细菌基因间共有重复序列-PCR(ERIC-PCR)对40株临床分离副溶血弧菌基因组DNA进行扩增,对扩增的DNA电泳指纹图谱进行分型分析.结果 40株副溶血弧菌呈现不同程度的基因多态性.基因外重复回文序列-PCR分辨力指数可达到0.953,40株菌株分为16个型,优势菌型为G型,占总菌数的20.0%.肠细菌基因间共有重复序列-PCR分辨力指数为0.5;40株菌株可分为4个型,优势菌型为D型,占总菌数的67.5%.结论 重复序列PCR可以用于副溶血弧菌临床分离株的分子分型研究,其分型敏感程度优于血型分型.     

多药耐药纹带棒杆菌的重复序列PCR分型与耐药机制的研究

目的对引起医院感染暴发的多药耐药纹带棒杆菌(MDRCs)进行重复序列PCR研究,为MDRCs的亲缘性分析提供可靠的方法,并对MDRCs的耐药机制进行初步研究。方法用基因外重复回文序列-PCR(REPPCR)和肠细菌基因间共有重复序列-PCR(ERIC-PCR)技术对临床分离的32株MDRCs进行分型研究,根据细菌的耐药表型选择对应的耐药基因进行检测。结果 REP-PCR和改良ERIC-PCR分型技术分别将32株MDRCs分为6型和8型,两种方法的分辨指数分别为0.585和0.696;所有菌株中与大环内酯类抗菌药物相关的核糖体甲基化酶基因(ermX)检出率为90.6%,与四环素耐药相关的核糖体保护蛋白基因(tetW)的检出率为100.0%,菌株耐药基因的检出与耐药表型完全相符。结论重复序列PCR(rep-PCR)分型方法可用于纹带棒杆菌的分型研究及医院感染MDRCs的亲缘性分析,ERIC-PCR的分辨能力优于REP-PCR;中国重庆地区的MDRCs分别介导ermX和tet(W)基因对大环内酯类和四环素类抗菌药物高水平耐药。     

结核分枝杆菌流行病学研究中基因分型的方法

限制性片段长度多态性 (IS6110-RFLP)是结核分枝杆菌(MTB)基因分型的金标准,该方法分辨力高,但操作非常复杂和结果分析困难,因而出现了多种PCR 基础的替代方法,如混合接头PCR、间隔区寡聚核苷酸分型(spoligotyping)、随机扩增多态性DNA( RAPD)和数目可变的串联重复序列和结核分枝杆菌散在分布重复单位(VNTR-MIRU)等.但除近来出现的 MIRU-VNTR 方法外,分辨力均不够强,上述两种或多种 PCR 方法联合可获得高的分辨力.     

三种分子分型技术在嗜肺军团菌分型中的应用与评价

目的对脉冲场凝胶电泳方法 （PFGE）、基因序列分型（SBT）和mip基因分型方法在嗜肺军团菌分型研究中的分辨力和潜在价值进行比较和论述。方法采用PFGE、SBT和mip基因分型方法对29株嗜肺军团菌和1株标准菌株ATCC33153进行分型比较。结果 PFGE可将30株嗜肺军团菌分为5大类群,19个PFGE型,分辨力为0.9586;SBT可分为22个ST型,分辨力为0.9609;mip基因分型可分为9个型别,分辨力为0.8344。血清型LP1和LP14菌株具有相同的PFGE和mip基因型别,相同或相近的SBT型别。结论 SBT较PFGE具有稍高的分辨力,适用于全球数据库比对和进化亲缘关系的研究,结合PFGE和SBT分型方法有利于流行病学溯源分析。     

铜绿假单胞菌采用肠杆菌属重复基因间隔共有序列-PCR的分析

目的探讨铜绿假单胞菌(PAE)多重耐药情况,为临床治疗和防治医院感染提供依据。方法从重症监护病房(ICU)>60岁的患者中分离到的14株PAE,进行传统的生化、PCR方法、药敏分型和质粒、肠杆菌重复基因间隔共有序列(ERIC)-PCR多态性分析。结果传统的生化分为一个型,API 20 NE结果为1344575,药敏则呈现出多重耐药,质粒分型有10株得到约23kb的质粒,其余4株则无,质粒的有无与多重耐药无直接关系;运用ERIC-PCR技术进行基因谱系,聚类分析将株菌主要分为3大类,相似系数从0.62~0.88。结论ICU的PAE在老年患者中呈现出散发的状态,ERIC-PCR指纹图谱基因技术分型方法分辨率高,可重复性好,简便快捷,可用于PAE医院感染流行病学监测。     

结核分枝杆菌流行病学研究中基因分型的方法

限制性片段长度多态性(IS6110-RFLP)是结核分枝杆菌(MTB)基因分型的金标准,该方法分辨力高,但操作非常复杂和结果分析困难,因而出现了多种PCR基础的替代方法,如混合接头PCR、间隔区寡聚核苷酸分型(spoligotyping)、随机扩增多态性DNA(RAPD)和数目可变的串联重复序列和结核分枝杆菌散在分布重复单位(VNTR-MIRU)等。但除近来出现的MIRU-VNTR方法外,分辨力均不够强,上述两种或多种PCR方法联合可获得高的分辨力。     

REP、RAPD和PFGE方法在志贺菌分子分型中的应用评价

目的探索一种简便、准确、快速的分子分型方法用于志贺菌传染源的追溯和调查。方法采用细菌基因组重复序列(REP)、随机扩增多态性分析(RAPD)、脉冲场凝胶电泳(PFGE)分别对62株志贺菌进行分子分型,根据分型结果比较3种方法的优点与不足。结果 REP仅分出A、B 2个型,RAPD可分出A、B、C、D、E、F 6个型,53株福氏志贺菌共分5个PFGE带型,9株宋内志贺菌为1个型,包括3个亚型。结论 REP用于相似度较高的菌株分辨效果较差,不适用于区域内菌株的分型;PFGE分型准确度高,但操作繁琐、时间较长;RAPD分型虽与PFGE分型有所差异,但分辨效果较好,且操作简便、价格低廉。RAPD可用于细菌性痢疾暴发事件的分子流行病学调查。     

改良MLVF与MLVA及PFGE对医院流行耐甲氧西林金黄色葡萄球菌分型研究

目的旨在建立一种操作简便、分型分辨率高以及成本低的耐甲氧西林金黄色葡萄球菌(MRSA)分子分型方法,对多位点可变数量串联重复序列指纹法(MLVF)进行了方法学改良及分型能力比对评估。方法应用42株MRSA参考菌株对改良MLVF分型能力进行验证;应用116株东北三省六家医院的MRSA临床菌株对改良MLVF、脉冲场凝胶电泳(PFGE)及多位点可变数量串联重复序列分析(MLVA)进行了方法学比对。结果改良MLVF对42株参考菌株分型率为100%;改良MLVF、PFGE和MLVA分别将116株临床菌株分成28个型(simpson's多样性系数[SID]=0.855),28个型(SID=0.854)和27个型(SID=0.816)。改良MLVF在区分国内最主要流行克隆CC239和其他克隆方面分辨率优于MLVA。改良MLVF在分群水平上与PFGE分型一致性(adjusted Rand’s,AR系数0.989)优于MLVA(AR指数0.765)。结论改良MLVF具有分辨率高、与金标准PFGE分型一致性好、操作简单、高通量、费用低的优点,适合常规实验室对MRSA进行分子分型,为常规感控工作及分子流行病学研究提供有力工具。     

副溶血性弧菌分型研究进展

副溶血性弧菌（vibrio parahaemolyticus,VP）是一种革兰氏阴性嗜盐性海洋细菌,常导致食物中毒和急性腹泻,本文从传统分型和基因分型角度介绍其分型研究进展。随着分子生物学的发展,各种分型研究,如血清学分型、噬菌体分型、脉冲场凝胶电泳（PFGE）分型、限制性片段长度多态性（RFLP）分型、随机引物扩增DNA多态性（RAPD）分型、核糖分型、肠道细菌重复基因间共有序列PCR（ERIC-PCR）分型等为VP的准确溯源提供了科学依据,可有效预防和控制该菌引起的腹泻及食物中毒事件,对研究VP分子流行病学特征具有重要意义。     

Biofilm formation and genetic diversity of Salmonella isolates recovered from clinical,food, poultry and environmental sources

In the present study, Salmonella isolates (n = 40) recovered from clinical, food, poultry and environmental sources were characterized for serotype identification, genetic diversity and biofilm formation capability. Serotype identification using multiplex PCR assay revealed six isolates to be Salmonella Typhimurium, 14 as Salmonella Enteritidis, 11 as Salmonella Typhi, and the remaining nine isolates unidentified were considered as other Salmonella spp. Most of the Salmonella isolates (85%) produced biofilm on polystyrene surfaces as assessed by microtitre plate assay. About 67.5% isolates were weak biofilm producers and 17.5% were moderate biofilm producers. There was no significant difference in biofilm-forming ability among the Salmonella isolates recovered from different geographical regions or different sources. Among the genetic methods, Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR revealed greater discriminatory power (DI, 0.943) followed by pulsed field gel electrophoresis (PFGE) (DI, 0.899) and random amplification of polymorphic DNA (RAPD) PCR (DI, 0.873). However, composite analysis revealed the highest discrimination index (0.957). Greater discrimination of S. Typhimurium and S. Typhi was achieved using PFGE, while ERIC PCR was better for S. Enteritidis and other Salmonella serotypes. A strong positive correlation (r = 0.992) was observed between biofilm formation trait and clustered Salmonella isolates in composite genetic analysis.     

ERIC PCR and RAPD based fingerprinting of Salmonella Typhi strains isolated over a period of two decades

Salmonella enterica serotype Typhi strains (n = 113) were isolated from typhoid patients over a period of 2 decades, i.e. 1987–2006. RAPD and ERIC PCR methods were used for random whole genome typing of these strains. ERIC PCR was found to be very efficient with the discriminatory index (DI) of 0.9821 with 100% reproducibility. RAPD was satisfactory in discriminating the strains (DI = 0.8978) but with poor reproducibility (40%). However, composite genotypic analysis was still better with DI of 0.9981 but with inherent poor reproducibility due to RAPD. Two major clones were observed to be circulating in the community with few unrelated strains too. The dendrogram constructed based on ERIC PCR banding pattern by involving 89 Typhi strains revealed 71 patterns, indicating that the genome of the bacterium is capable of rapid changes and variations. Thus, the spectrum of biological manifestations of human infection by S. Typhi may be related to its capacity for genetic diversity underlined by its highly plastic hypermutable genome.     

Evaluation of three molecular typing techniques for nonfermentative Gram-negative bacilli.

OBJECTIVE: To evaluate three different DNA techniques for typing nonfermentative gram-negative bacilli isolated from Latin American hospitals. DESIGN: One hundred twenty-six nonfermentative gram-negative bacilli were typed. PARTICIPANTS: Pseudomonas aeruginosa (n = 64) and Acinetobacter baumannii (n = 42) samples were obtained from blood cultures of patients admitted to 10 medical centers in Latin America during 1998 and Stenotrophomonas maltophilia (n = 20) samples were obtained from patients admitted to the Hospital S?o Paulo between 1999 and 2001. METHODS: All samples were typed using automated ribotyping, PFGE, and ERIC-PCR. The discriminatory power for each technique was calculated using Hunter's generalized formula. RESULTS: All strains could be typed by automated ribotyping and ERIC-PCR, but two strains (1.6%) were not typeable by PFGE. All three techniques showed 100% reproducibility. The time to obtain the results was shorter for automated ribotyping and ERIC-PCR compared with PFGE. Likewise, the costs for ERIC-PCR and PFGE were lower than those for automated ribotyping. The interpretation of results was more complicated and more difficult with ERIC-PCR than with both PFGE and automated ribotyping. All techniques presented excellent discriminatory power for P. aeruginosa (0.98). PFGE presented the highest discriminatory power (0.94) for A. baumannii, and both PFGE and ERIC-PCR showed higher discriminatory power (0.90 for both) than automated ribotyping (0.82) for S. maltophilia. CONCLUSIONS: PFGE showed the highest discriminatory power for typing these nonfermentative gram-negative bacilli. However, automated ribotyping and ERIC-PCR can provide results in a shorter time period with similar discriminatory power.     

重复片段PCR检测葡萄球菌DNA指纹在医院感染中的分析

目的 应用重复片段PCR，对凝固酶阴性葡萄球菌(CNS)进行基因分型，用于医院感染中分子流行病学研究。方法 选择REPl REP2和ERICl ERIC2两对引物，对临床分离的68株(CNS)菌株进行重复片段PCR，根据扩增产物的电泳模式编制菌株问的相似矩阵，并利用RAPD、PHYLIP及TREEVIEw等软件，绘制各株间的遗传聚类图。结果 两对引物的扩增带型均比较丰富，对于实验CNS菌株具有较好的分辨率，根据聚类结果，可以确定某些菌株之间的同源性，为感染源的确认等分子流行病学研究提供依据。结论 重复片段PCR分辨率高，根据聚类结果确定菌株的同源性，为医院感染确认感染源提供了分子流行病学的依据。     

Subtyping of Clostridium difficile polymerase chain reaction (PCR) ribotype 001 by repetitive extragenic palindromic PCR genomic fingerprinting

Fifty isolates of the most common UK strain of Clostridium difficile [polymerase chain reaction (PCR) ribotype 001] were analysed by three PCR-based typing methods in order to determine genomic diversity within this strain that may form the basis of a subtyping method. The three methods used were repetitive extragenic palindromic elements (REP), conserved repetitive DNA elements (BOX), and enterobacterial repetitive PCR intergenic consensus sequences (ERIC). The performance of each typing method was assessed by comparing powers of discrimination, typeability and reproducibility. All methods had satisfactory levels of typeability and reproducibility as determined by blind-coded repeats, but REP-PCR typing proved to be the most discriminatory method, yielding seven distinct amplicon profiles consisting of up to eight major bands. BOX-PCR generated between two and five major amplicons with four distinct BOX profiles. ERIC-PCR primers, however, could not discriminate between isolates. These results suggest that PCR ribotype 001 is not clonal in nature at present, and that REP-PCR subtyping methods offer promise to further our understanding of the epidemiology of C. difficile PCR ribotype 001 disease in UK hospitals.     

Molecular subtyping of Staphylococcus aureus from an outbreak associated with a food handler

On 6 May 2000, a staphylococcal food poisoning outbreak occurred at a high school, affecting 10 of the 356 students who attended the breakfast. Twenty-seven Staphylococcus aureus isolates, producing enterotoxin A (SEA), SEB-, or non-SEA-E, were recovered from 7 patients, 2 food handlers and left-overs. To investigate the outbreak, we genotyped the isolates by using pulsed-field gel electrophoresis (PFGE) and three PCR-based techniques: inter-IS256 PCR typing, protein A gene (spa) typing, and coagulase gene restriction profile (CRP) analysis. Our results show that PFGE was the most discriminatory technique, whereas the three PCR-based techniques were insufficient in the discriminatory power to distinguish the S. aureus isolates from the outbreak. Based on the enterotoxin-producing types and the results of genotyping, three distinct types of strains (A1111, B2221 and N3221) were designated. Both the A1111 and B2221 strains were found in the specimens from the patients and a hand lesion of a food handler, suggesting that the source of contamination for the outbreak was most likely originated from a food handler.     

Molecular characterization of Salmonella isolates by REP-PCR and RAPD analysis

Eighteen Salmonella isolates from both human and food (non-human) sources (fish, meat, and poultry) were characterized using conventional culture methods, biochemical, serological, and molecular analyses. REP-PCR and RAPD produced DNA profiles for differentiation purposes. Enterobacterial repetitive intergenic consensus (ERIC), repetitive extragenic palindronic (REP) and BOXAIR primers were selected for REP-PCR and two arbitrary primers, namely OPP-16 and OPS-11 were used for RAPD to generate DNA fingerprints from the Salmonella isolates. REP-PCR method showed greater discriminatory power in differentiating closely related strains of the related strains of Salmonella and produced more complex banding patterns as compared with RAPD. A dendogram was constructed with both sets of profiles using SPSS Version 13.0 computer software and showed that most human isolates were separately clustered from the non-human isolates. Two of the human isolates were closely related to some of the non-human isolates. A good correlation was also observed between the serogrouping of the O antigen and the molecular profiles obtained from REP-PCR and RAPD data of the Salmonella isolates. The results of a principal coordinate analysis (PCA) corresponded to the clustering in the dendrogram.     

Typing of Acinetobacter baumannii isolated from hospital-acquired respiratory infections in a tertiary care centre in southern India

In an attempt to define the epidemiology of Acinetobacter baumannii infection, 27 isolates, obtained from hospital-acquired respiratory infections, were typed using random amplified polymorphic DNA (RAPD) profile and antimicrobial susceptibility patterns. Ten different patterns were obtained with ERIC2 primer: 14 isolates had a similar profile representing a single strain. Within RAPD types, isolates could be further classified based on their antibiogram; however, strains of different types had similar antibiograms. This study showed that many different genetic types of A. baumannii are prevalent in our hospital. While antibiograms alone are not sufficiently discriminatory, RAPD typing helps in identifying outbreaks and in assessing infection control procedures within a hospital.     

Molecular typing of Mannheimia (Pasteurella) haemolytica serotype A1 isolates from cattle in Japan

Pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) methods were applied for molecular typing of 130 Mannheimia (Pasteurella) haemolytica serotype A1 isolates obtained from 13 prefectures in Japan. These isolates were divided into 15 ApaI PFGE profiles that formed six distinct clusters (clusters A-F). Fifty-three (40.7%) isolates were classified in cluster B, and 20.0, 13.8, 12.3, 6.9 and 6.1% of isolates were in clusters E, A, F, D and C, respectively. The isolates of cluster B were differentiated into seven subtypes (B1-B7) and subtype B5 contained 63% (34/53) of isolates. RAPD revealed four banding patterns (types I-IV), and among 130 isolates 60.7% (79/130) of isolates were RAPD type I. All of the RAPD type I isolates were grouped into clusters A-C by PFGE. There was no relationship between molecular typing and geographic origin of these isolates. These results indicate that isolates of M. haemolytica A1 strain with various molecular profiles have already spread in Japan and may have caused sporadic infections.     

Multi-locus variable number tandem repeat analysis for Clostridium botulinum type B isolates in Japan: Comparison with other isolates and genotyping methods

Clostridium botulinum produces botulinum neurotoxin (BoNT) and causes botulism in humans and animals. Recently, 15-loci multi-locus variable number tandem repeat analysis (MLVA) for C. botulinum was developed for high-resolution and inter-lab comparative genotyping. This study examines the relation between MLVA and other genotyping methods such as pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), BoNT/B subtyping and bont/b gene location to evaluate MLVA as a method applicable to the genetic markers for C. botulinum type B. Japanese isolates were genotyped using MLVA and were compared with strains from other sources reported previously. Results show that the discriminatory power of MLVA was comparable to that of PFGE and higher than that of MLST. The topology of the minimum spanning tree (MST) constructed using MLVA data was very consistent with the phylogenetic classifications of PFGE and MLST. The MST topology also represented genetic diversity between the strains possessing bont/b gene on chromosomes and plasmids. Some Japanese isolates including those associated with infant botulism were inferred to be related to isolates of Europe origin from MLVA genotyping results. The MLVA scheme used for this study is apparently useful not only for high-resolution molecular typing, but also for phylogenetic characterization of C. botulinum type B.