甲氧西林耐药溶血性葡萄球菌的基因多态性研究

目的应用随机扩增多态性DNA（RAPD）技术,对甲氧西林耐药溶血性葡萄球菌（MRSH）进行基因多态性研究。方法对81株MRSH通过RAPD技术进行扩增,电泳条带采用SPSS13.0软件分析,根据树状图分型。结果81株MRSH通过RAPD技术可产生相对固定的6条电泳带,经遗传相关系数分析后可分为8型,其中A型占70.37%,而10株血培养阳性的MRSH中有9株为A型。结论通过RAPD分型研究,可以了解MRSH基因流行型的特性,为该菌的感染控制提供分子流行病学依据。     

产超广谱β-内酰胺酶大肠埃希菌同源性分析的3种方法比较

摘要：目的：用随机扩增多态性DNA（RAPD）分析、以基因外重复回文序列（REP）及肠杆菌基因间重复一致序列（ERIC）为模板的重复序列聚合酶链反应3种基因分型方法对产超广谱β-内酰胺酶（ESBLs）大肠埃希菌进行同源性分析,筛选各级医疗机构易于开展的基因分型方法。 方法：从住院患者痰液、尿液、血液等标本中鉴定并筛选产ESBLs大肠埃希菌。以RAPD、ERIC、REP为引物进行扩增的方法,对产ESBLs大肠埃希菌的DNA进行PCR扩增并进行同源性分析。用SPSS 16.0软件进行聚类分析,比较其分辨率系数。 结果：共筛选出产ESBLs大肠埃希菌25株,3种方法均可将大肠埃希菌扩增出丰富的区带,产ESBLs大肠埃希菌分别被分为15、18、20个基因型,分辨率系数分别为0.957、0.970、0.977。 结论：在产ESBLs大肠埃希菌基因同源性分析中,RAPD、ERIC-PCR和REP-PCR 3种方法的分辨力均很好,尤以REP-PCR分辨率最高。     

白假丝酵母菌随机扩增多态DNA分型及其流行趋势分析

目的:探讨采用随机扩增多态性DNA(RAPD)技术分析临床分离的白假丝酵母菌分型结果及其流行趋势。方法:选用不同的引物、Mg~(2+)、模板浓度、Taq聚合酶用量等,获得一优化的反应体系以对白假丝酵母菌进行RAPD分型。结果:经优化的RAPD方法能将105株白假丝酵母菌菌株分成8个基因型。结论:结果显示该方法的可分型性、灵敏度、重复性及特异度均较良好,可对白假丝酵母菌快速、简便地进行基因分型,可广泛应用于流行病学调查与研究。     

金黄色葡萄球菌随机扩增多态性DNA分型及其应用

目的 利用随机扩增多态性DNA（random amplified polymorpohic DNA,RAPD）分析技术，对金黄色葡萄球菌进行基因分型，并观察临床耐药型与其基因型之间的关系。方法 利用自己合成的随机引物，建立RAPD检测分析方法，并利用这一方法对5株金黄色葡萄球菌进行基因分型的研究。结果 5株金黄色葡萄球菌中，2株耐甲氧西林金黄色葡萄球菌（MRSA）为同一基因型，另外3株为两种基因型，均是敏感菌株。结论 从RAPD分析的初步结果可以看出，金黄色葡萄球菌的临床表现型与基因型存在一定的关系，本法有望且于临床金黄色葡萄球菌耐药的快速检测。     

凝固酶阴性葡萄球菌临床分离株的重复片段PCR分型研究

[目的]建立重复片段PCR扩增方法,用于凝固酶阴性葡萄球菌(CNS)临床分离株分型的分子流行病学研究。[方法]选择REPl REP2及ERIC1 ERIC2两对引物,对临床分离的68株凝固酶阴性葡萄球菌进行重复片段PCR扩增,根据扩增模式编制实验菌株之间的相似性矩阵,并利用RAPD、PHYLIP及TREEVIEW等软件绘制实验菌株基于重复片段扩增模式的遗传聚类图。[结果]两对引物的扩增带型比较丰富,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。为感染源的等分子流行病学研究提供依据。[结论]重复片段PCR扩增是一种快速、可靠、具有较高分辨率的基因分型方法,是进行CNS临床分型的有效工具。     

鲍曼不动杆菌随机扩增多态性DNA法基因分型

目的建立鲍曼不动杆菌(A.baumannii)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176株鲍曼不动杆菌进行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176株临床分离鲍曼不动杆菌共得122种RAPD指纹图谱,以第68、58和66型为主要型别,分别为18、15和10株。结论RAPD技术可将临床分离鲍曼不动杆菌分型122种,本院的主要流行基因型为第68、58和66型。     

德尔卑沙门菌的随机扩增多态性DNA分析

[目的] 对2004年湖北省食源性疾病监测中检测出的德尔卑沙门菌进行分型，探讨RAPD技术在沙门菌鉴别和分型中的应用。[方法] 采用RAPD法（random amplified polymorphism DNA），选用11条随机引物，对7株德尔卑沙门菌进行分型，用标准菌株猪霍乱沙门菌（ATCC50019）和鼠伤寒沙门菌（ATCC50115）作为对照。[结果] 在选用的11条随机引物中，不能分析出任何条带的有3条，扩增条带尤多态性的有5条，能够扩增出条带且图谱具有多态性的有3条（WM-1、WM-4、WM-7）。利用3条具有多态性的引物进行分析，扩增出54条DNA片段，片段大小为0．3～1．1kb，其中7个菌株共有的片段有44条，显示多态性的片段有10条，可以将7株德尔卑沙门菌分别分成2～3个亚型。[结论]利用RAPD法对同一血清型的沙门菌进行分析，可区分出不同的亚型菌株，能够起到监测菌株的变异情况的作用。     

多重耐药铜绿假单胞菌随机扩增多态性DNA的分型及应用

目的 应用随机扩增多态性DNA（RAPD）技术，对多重耐药铜绿假单胞菌（MRPA）进行基因多态性研究。方法对临床分离的32株MRPA进行随机引物PCR扩增，扩增产物进行电泳和聚类分析。结果 32株MRPA通过RAPD分型可分为19个基因型。耐药表型相同，基因型可相同；但绝大多数则不同。结论 通过RAPD分析，了解MAPA基因型特性，明确其耐药表型与基因型的关系，并为该菌的感染控制提供分子流行病学依据。     

革兰阴性杆菌中VEB-1型超广谱β-内酰胺酶的分布情况调查

目的　分析上海第二医科大学附属瑞金医院 (以下简称瑞金医院 )和上海交通大学附属第六人民医院(简称市六医院 )分离的 340株革兰阴性杆菌中VEB 1型超广谱 β 内酰胺酶 (ESBL)的分布 ,并分析瑞金医院灼伤科患者标本中分离的产VEB 1型ESBL铜绿假单胞菌是否有同源性。方法　用聚合酶链反应 (PCR)扩增细菌的veb 1基因 ,用PCR RFLP检测并分型整合子 ,以ERIC2为引物 ,采用随机引物扩增多态性DNA分型法(RAPD)对瑞金医院灼伤科分离到的 16株产VEB 1型ESBL的铜绿假单胞菌进行同源性分析。结果　市六医院分离的菌株中未检测到veb 1基因 ,瑞金医院仅在灼伤科来源的铜绿假单胞菌中检出VEB 1型ESBL ,且这些菌株用RAPD分析具有同源性。结论　瑞金医院灼伤科流行携带veb 1型ESBL基因的铜绿假单胞菌。     

革兰阴性杆菌中VEB-1型超广谱β-内酰胺酶的分布情况调查

目的分析上海第二医科大学附属瑞金医院(以下简称瑞金医院)和上海交通大学附属第六人民医院(简称市六医院)分离的340株革兰阴性杆菌中VEB-1型超广谱β-内酰胺酶(ESBL)的分布，并分析瑞金医院灼伤科患者标本中分离的产VEB-1型ESBL铜绿假单胞菌是否有同源性。方法用聚合酶链反应(PCR)扩增细菌的veb-1基因，用PCR-RFLP检测并分型整合子，以ERIC2为引物，采用随机引物扩增多态性DNA分型法(RAPD)对瑞金医院灼伤科分离到的16株产VEB-1型ESBL的铜绿假单胞菌进行同源性分析。结果市六医院分离的菌株中未检测到veb-1基因，瑞金医院仅在灼伤科来源的铜绿假单胞菌中检出VEB-1型ESBL，且这些菌株用RAPD分析具有同源性。结论瑞金医院灼伤科流行携带veb-1型ESBL基因的铜绿假单胞菌。     

Geographic grouping of Cryptococcus neoformans var. gattii by random amplified polymorphic DNA fingerprint patterns and ITS sequence divergence

Eleven reference strains of Cryptococcus neoformans var. gattii were analyzed by random amplified polymorphic DNA (RAPD) patterns using three oligo primers. Three major RAPD pattern profiles (profiles I, II and III) were identified with A-1 oligo primer. Each profile was found to relate to a geographic region. Since the strains which belong to profiles I and II were mainly the isolates from America, and profile III from Asia with A-1 primer, these three profiles were assigned to the geographic grouping of America-1, America-2 and Asia, respectively. Analysis of rDNA sequences coding an internal transcribed spacer (ITS) region of C. neformans var. gattii revealed that the fungus with each RAPD profile has characteristic base sequences at the four positions (10 and 15 positions in ITS1 and 8 and 56 positions at ITS2) of the ITS regions. On the basis of the combinations of the four bases specific for the ITS regions, four ITS types, AAGG (America-1), AAAC (America-2), GGGC (Asia-1) and AGGC (Asia-2) were identified: the geographic group of Asia was further classified into two subgroups of Asia-1 and Asia-2 based on the ITS typing. Clinical isolates from Thailand (6 strains) and Brazil (7 strains) were found to belong to the geographic group of America-1 nd America-2, respectively. Five reference strains of C. neoformans var. gattii from the CBS culture collection were classified into two America-2, one Asia-1 and two Asia-2 groups. This ITS region analysis allowed us to distinguish all isolates of C. neoformans var. gattii into four geographic groups based on the ITS base sequence, and further molecular epidemiological and ecological research on this fungus is recommended.     

重复序列片段基因扩增用于阴沟肠杆菌的分型研究

目的 用重复序列片段基因扩增方法对阴沟肠杆菌进行分型研究。方法 选用肠杆菌科基因间的重复序列(ERIC)进行扩增,对阴沟肠杆菌进行分型研究,并与其抗生素耐药特征进行比较。结果 阴沟肠杆菌分成6种不同基因型,不同基因型有不同的耐药特征。结论 本法简便易行,重复性好,适合于临床分离的病原菌的分型及医院感染的分子流行病学研究。     

Evaluation of human papillomavirus-consensus primers for HPV detection by the polymerase chain reaction

Cervical cancer is one of the most frequently found cancers in women and appears to have a viral aetiology. Substantial evidence points to the human papillomaviruses (HPV) as the infectious agents and there is considerable interest in identifying and accurately typing the viruses. Since HPVs now comprise more than 100 different HPV types, the polymerase chain reaction (PCR) has been the preferred methodology for virus identification and typing on isolated DNA. In that context, five commonly employed PCR consensus primers have been evaluated for the detection and typing of HPV. The five consensus primer pairs were derived from the consensus sequences of either the L1 and E1 open reading frames. All primers exhibited approximately equal sensitivity, as defined by the ability to detect HPV DNA, on a series of standard HPV DNA-containing preparations. However, the five primer pairs performed differently on 24 HPV-positive and 34 HPV-negative samples obtained from cervical scrapes which had been typed by type-specific PCR for HPV 6/11, 16, 18 and 33. The values for agreement between identification of samples by a HPV type-specific PCR and the consensus primer PCR were 78, 84, 91, 93 and 98%. Three samples, which were positive with only one of the five consensus primer pairs and were negative with the PCR for HPV types 6/11, 16, 18 and 33, contained other HPV sequences or HPV-related sequences as determined by DNA sequence analysis. To our knowledge, this report represents the first extensive comparison of five different consensus primers in a polymerase chain reaction for the detection of HPV. Our results suggest that PCR typing for human papillomaviruses requires more than one consensus primer pair to identify all HPV-infected samples.     

Molecular epidemiological study of vertical transmission of vaginal <Emphasis Type="Italic">Lactobacillus</Emphasis> species from mothers to newborn infants in Japanese,by arbitrarily primed polymerase chain reaction

We investigated mother-to-newborn infant transmission of Lactobacillus species in Japanese by the typing of isolates from the vagina of pregnant women and stool specimens from their newborn infants. All infants were born by uncomplicated vaginal delivery and were basically fed breast milk. Lactobacillus strains were fingerprinted by arbitrarily primed polymerase chain reaction (AP-PCR), using ERIC1R and ERIC2 primers. Of 86 pregnant women tested, 71 (82.6%) were positive for vaginal lactobacilli. At 5 days after birth, 24 (33.8%) of the 71 infants whose mothers were lactobacilli-positive had fecal lactobacilli, while only 1 (6.7%) of the 15 infants delivered from the vaginal lactobacilli-negative mothers was lactobacilli-positive (P < 0.01). Lactobacillus crispatus was the most prevalent species of vaginal lactobacilli in mothers and of fecal lactobacilli in infants at 5 days of age, whereas Lactobacillus gasseri was the most common in infants at 1 month of age. Identification to the species level, followed by AP-PCR typing, demonstrated that 23.3% of the 86 infants were likely to be colonized in the intestine by the vaginal lactobacilli of their mothers, and that only 2 of the infants retained the same vaginal lactobacilli until 1 month of age. These results suggest that approximately one-fourth of infants acquire vaginal lactobacilli from their mothers at birth, and that the acquired lactobacilli do not last in the intestine of the infant long-term, but rather, are replaced by ones from milk or unknown sources after birth. AP-PCR with primers ERIC1R and ERIC2 is indicated as a potential tool for the typing of Lactobacillus strains. Received: August 24, 2001 / Accepted: November 15, 2001     

随机扩增多态性DNA分型法用于肠球菌基因分型

目的 研究适于本实验室肠球菌随机扩增多态性NDA分型法（RAPD）分型的最佳实验条件。方法 在改变引物、模板、Mg^2 浓度、Taq多聚酶用量及循环参数等不同条件下,采用RAPD法对肠球菌进行基因分型。结果 经优化后的RAPD方法能很好地对肠球菌进行分子生物学分型。结论 RAPD是从分子水平对微生物感染进行病原学,流行病学研究较理想的一种经济、快速、可靠的基因分型方法。     

Cloning of a novel specific SCAR marker for species identification in Lactobacillus pentosus

Identifying Lactobacillus species using only phenotypic and genotypic (16S rDNA sequence analysis) techniques yields inaccurate results. The objective of this study was to develop species-specific primers based on randomly amplified polymorphic DNA (RAPD) fingerprinting to distinguish species within the closely related Lactobacillus plantarum group. One of these primers, OPD-3, produced a species-specific band that was found only in the tested Lactobacillus pentosus. This specific fragment was isolated from agarose gel and ligated into a vector for DNA sequencing. A pair of primers, SpOPD3Lpen-F1/R1, that were highly specific sequence-characterized-amplified-regions (SCARs) were designed according to the nucleotide sequences of the specific RAPD marker. These primers were used for PCR analysis of the template DNA of the Lactobacillus strains, and a single 542 bp species-specific band was found only in L. pentosus. Using PCR, a novel species-specific primer pair is shown to rapidly, accurately and effectively distinguish L. pentosus from other species in the L. plantarum group of probiotic bacteria.     

New PCR primer pairs specific for Candida dubliniensis and detection of the fungi from the Candida albicans clinical isolates in Japan

The genetic patterns of Candida dubliniensis, including type strain, were analyzed by random amplified polymorphic DNA (RAPD) method in comparison with those of reference Candida albicans genotypes A, B and C strains. This RAPD pattern analysis showed that the patterns of two strains of C. dubliniensis including the type strain are very similar to the three oligo PCR primers used, but different from other C. albicans genotypes A, B and C strains with most of the PCR primers tested. The RAPD band considered to be specific for C. dubliniensis in one oligo primer was extracted and sequenced. Based on the sequence information (417-bp), two new PCR primer pairs specific for C. dubliniensis were prepared. The survey of 58 strains of Candida clinical isolates using these newly prepared PCR primer pairs identified as C. albicans in Japan suggested the presence of one strain of C. dubliniensis. The phenotypic characteristics and the sequence analysis of the variable D1/D2 domain of the large subunit (26S) ribosomal DNA of the strain confirmed that it belongs to C. dubliniensis. The usefulness and specificity of these PCR primer pairs for the identification of C. dubliniensis are discussed.     

肝移植术后阴沟肠杆菌感染耐药性及分子流行病学研究

目的了解肝移植术后阴沟肠杆菌感染的耐药谱和分子生物学特征及两者的关系。方法基因分型采用随机扩增多态性DNA(RAPD)法,耐药谱选用K-B法。结果RAPD分型将28株阴沟肠杆菌分为10种株型,耐药谱则将其分为7种不同的耐药型,28株阴沟肠杆菌中AmpC酶检出率为53.6%,未发现美洛培南耐药株。RAPD的基因分型得的遗传聚类图可分为10类。结论RAPD技术对肝移植术后阴沟肠杆菌感染的流行病学分析是一个有效的方法,肝移植术后阴沟肠杆菌有很高的耐药性,治疗应首选美洛培南,RAPD指纹图谱与耐药谱分型具有一定的相关性。