不同来源O∶3血清型小肠结肠炎耶尔森氏菌的生物特性研究

使用随机引物ＲＡＰＤ２，设计自小肠结肠炎耶尔森氏菌Ｏ∶９血清型肠毒素基因的引物，设计自小肠结肠炎耶尔森氏菌毒力质粒ｖｉｒＦ基因上的引物及堪萨斯分枝杆菌Ｍ．Ｋａｎｓａｓｉ１６ＳｒＲＮＡ基因的序列和小肠结肠炎耶尔森氏菌Ｏ∶３血清型２３ＳｒＤＮＡ序列的引物...     

致泻性大肠杆菌中发现小肠结肠炎耶尔森菌毒力岛

目的　研究肠产志贺样毒素且具侵袭力的大肠杆菌 (ESIEC)菌株是否含有耶尔森菌的HPI(毒力岛 )基因。方法　采用PCR扩增和Southern杂交的方法。结果　从 35 %的ESIEC菌的染色体上同时扩出irp1、irp2和fyuA 3个片段 ,片段大小分别与小肠结肠炎耶尔森菌WA菌株的相应片段一致 ,鼠疫耶尔森菌HPI上的 6对引物均未扩出目的片段。 6 5 %的ESIEC使用以上 9对引物未扩出目的片段。ESIEC菌的染色体EcoRI酶切产物电泳后与小肠结肠炎耶尔森菌WA菌株的fyuA探针杂交出大小一致的条带 ,与irp1和irp2探针杂交出不同的带型。结论　ESIEC菌含有小肠结肠炎耶尔森菌的HPI,且有变异现象。ESIEC和EAEC的关系还有待于进一步的研究     

铜绿假单胞菌随机扩增多态性DNA指纹法基因分型研究

建立了铜绿假单胞菌（ＰＡ）随机扩增多态性ＤＮＡ（ＲＡＰＤ）指纹图基因分型方法，并应用于流行病学相关或不相关的７５个ＰＡ菌株的基因分型。分型率为１００％。３２株新生儿暴发感染菌株，可分成４个血清型和５个ＲＡＰＤ谱型，其中２５株属于同一谱型。在８例患者的垂直追踪菌株中，除有１例其第１和第３个分离株的血清型和ＲＡＰＤ谱型均相同，但与第２个分离株的血清型和ＲＡＰＤ谱型不同外，其余７例前后菌株的谱型相同。１３株散发菌株可分成３个血清型和１０个ＲＡＰＤ谱型；１２株流行病学无关菌株的ＲＡＰＤ谱型均不相同。本研究结果表明，ＲＡＰＤ指纹图基因分型法具有分型率高、分辨力强、比较快速简便等优点，并且不需要已知核酸序列，是在分子水平上对微生物感染的病原学、发病机理及流行病学研究的较理想的方法。     

两种耶尔森氏菌对动物的致病和交叉免疫性试验

目的了解广东省鼠疫静息期小肠结肠炎、假结核耶尔森氏菌的致病和交叉免疫性。方法致病性试验：菌株分皮下注射、饮水感染昆明小鼠,观察其发病和死亡情况,然后剖检。交叉免疫性试验：提取假结核鼠株冻溶抗原加佐剂研磨免疫家兔,然后分别用小肠结肠炎0：3猪株和鼠疫菌EV76株攻毒。试验均设对照组。结果0：3猪株皮下注射小鼠5只全部死亡,饮水感染死亡3只,2只发病;0：9鼠株皮下注射死亡2只,饮水感染仅发病;假结核鼠株两种途径感染仅发病。假结核鼠株免疫的家兔能抵抗0：3猪株攻毒,用EV76株攻毒未能产生鼠疫F1抗体。结论0：3猪株毒力强,0：9鼠株弱,耶尔森氏菌对动物有交叉免疫力。     

应用抑制削减杂交技术进行鼠疫耶尔森菌的比较基因组研究

目的确定古典型鼠疫耶尔森菌的特有序列，为建立和完善该菌基因组分型系统提供可靠数据。方法通过抑制削减杂交技术发现差异片段并应用PCR验证。结果发现了5个在不同生物型鼠疫菌问存在差异的DNA片段，其中一个383bp的片段在来自天山山地灰旱獭、长尾黄鼠鼠疫自然疫源地的30株鼠疫菌全部阳性，而来自其它鼠疫自然疫源地的菌株全部阴性，5株假结核耶尔森菌中有2株(生物Ⅰ型和Ⅱ型)阳性。结论该383bp片段为天山山地灰旱獭、长尾黄鼠鼠疫自然疫源地的鼠疫菌所特有，此疫源地的菌株可能是我国较古老的鼠疫菌。     

携耶尔森菌HPI大肠埃希菌的致泻性调查

ＨＰＩ(ｈｉｇｈ ｐａｔｈｏｇｅｎｉｃｉｔｙｉｓｌａｎｄ)为高致病力群耶尔森菌 (包括鼠疫耶尔森菌、假结核耶尔森菌和小肠结肠炎耶尔森菌生物群 1Ｂ)中发现的一种毒力岛 ,介导铁载体耶尔森杆菌素 (ｓｉｄｅｒｏｐｈｏｒｅｙｅｒｓｉｎｉａｂａｃｔｉｎ)的生物合成和摄取 ,为菌株致小鼠死亡所必需。近年来国内外学者在许多大肠埃希菌分离菌株中先后也发现了此ＨＰＩ〔1,2〕,然而 ,目前尚不明了大肠埃希菌中检出的ＨＰＩ是否与菌株的致病性有关。我们对分离自杭州各类人群的大肠埃希菌菌株 ,应用菌落原位杂交技术和ＰＣＲ技术检测ＨＰＩ的…     

RAPD分析在Hp菌株鉴定中的应用

目的建立一种快速准确地鉴定不同幽门螺杆菌（Ｈｐ）菌株的ＲＡＰＤ分析方法，并评价它在区分Ｈｐ复发和再感染中的应用价值。方法用苯酚－氯仿方法提取ＨｐＤＮＡ，以一个１０ｎｔ的寡核苷酸引物，建立ＨｐＰＣＲ扩增体系，所得ＰＣＲ产物以２％琼脂糖凝胶电泳分析（ＲＡＰＤ分析）。同一菌株验证方法的重复性，对４例十二指肠溃疡伴Ｈｐ感染者三联治疗前后及根除后一年内再现的Ｈｐ菌株进行ＲＡＰＤ分析，鉴别复发和再感染。结果（１）同一Ｈｐ菌株于不同时间进行的２次ＲＡＰＤ分析，所得的ＰＣＲ产物完全一致，说明该方法具有良好的可重复性。（２）５０株不同的Ｈｐ菌株各产生不同的ＲＡＰＤ图谱，可按此图谱的不同将它们区分开来。（３）对４例十二指肠溃疡伴Ｈｐ感染治疗前及治疗后１年再现的菌株的ＲＡＰＤ分析，发现３例患者的菌株具有相同的ＲＡＰＤ图谱，证实为原菌株的复发，而１例患者的菌株则产生不同的ＲＡＰＤ图谱，推测为新菌株的再感染，４例患者随访１年均为十二指肠溃疡复发。结论ＲＡＰＤ分析只需单一的随机引物，方法简单、快速、经济、重复性好，可用于区分不同来源的Ｈｐ菌株，特别对治疗前后再现的菌株复发和再感染的鉴别更具临床应用价值     

PCR指纹图技术筛选鼠疫耶尔森菌种特异性探针的研究

目的 利用PCR指纹图技术筛选细菌种特异性探针，探索利用PCR指纹图技术实现病原菌通用检测的可能性。方法 以鼠疫耶尔森菌为实验对象，利用REP－1和REP－2引物对在非严谨的条件下进行PCR指纹图扩增；将所有扩增片段纯化后，克隆至pGEM-T载体，转化大肠杆菌JM109；用生物素化的载体引物扩增克隆化片段，进行末端标记；在硝酸纤维素膜上固定鼠疫杆菌的染色体DNA以及相应对照菌株的染色体DNA，以末端标记的扩增产物进行杂交，通过生物素－链霉亲和素－辣根过氧化物酶系统显色，判断扩增片段的特异性。结果 经杂交筛选，发现1个长度约为900bp的扩增具有较好的杂交特异性片段，将序列与鼠疫耶尔森菌已完成的基因组序列比较，仅发现4个核苷酸的差异；根据这段序列设计的引物对可以特异性地扩增鼠疫耶尔森菌的模板。结论 利用PCR指纹图技术成功筛选到一段鼠疫耶尔森菌的种特异性探针，为细菌通用检测技术的研究开辟了新的思路。     

用PCR SSCP方法检测念珠菌和隐球菌的实验研究

ng of Cryptococcus and Candida with PCRSSCP念球菌属,隐球菌属,聚合酶链反应,单链构象多态性,核糖体Candida,Cryptococcus r R N A Polymerade chain reactionR446.62目的　根据医学真菌大亚单位核糖体（ｌｓｕ ｒ Ｒ Ｎ Ａ） 基因的高度保守区设计广谱扩增引物,建立应用 Ｐ Ｃ Ｒ Ｓ Ｓ Ｃ Ｐ 技术检测医学重要真菌的方法,并应用于临床。方法　首先用 Ｐ Ｃ Ｒ 技术扩增白色念珠菌、热带念珠菌、假热带念珠菌、近平滑念珠菌、光滑念珠菌、解脂念珠菌、新型隐球菌的ｌｓｕｒ Ｒ Ｎ Ａ 基因,然后根据７ 种标准菌株 Ｄ Ｎ Ａ 的单链构象多态性特征将上述医学真菌鉴定到菌种;观察引物对常见细菌和哺乳动物细胞 Ｄ Ｎ Ａ 的扩增结果; 于生理盐水和血液中加入念珠菌,观察方法的灵敏度;对扩增产物进行序列分析;方法建立后用于临床标本的检测。结果　不同真菌菌种的 Ｄ Ｎ Ａ 具有可以区分的单链构象多态性,扩增片段长度为２１９ ～２８５ｂｐ ,可将白色念珠菌、热带念珠菌和新型隐球菌等医学重要真菌鉴定到菌种;此引物对大肠埃希氏菌、铜绿假单胞菌和金黄色葡萄球菌等１２ 种常见细菌和病毒 Ｄ Ｎ Ａ 扩增均为阴性结果, 人血液白细胞、 Ｈｅ Ｌａ 细胞、 Ｋ Ｂ 细胞、 Ｓ１８０ 细胞和３ Ｔ３ 细胞亦无阳性 Ｄ Ｎ Ａ 带出现。于生理盐水和血液中加入白色念珠菌,检测灵敏度分别为５ 个和１０ 个真菌细胞。 Ｐ Ｃ Ｒ 产物的序列分析结果表明,白色念珠菌和新     

登革2型病毒43株膜蛋白前体基因片段的克隆与表达

依照国际标准株ＮＧＣ的序列，设计合成了一对引物，用于扩增登革２型病毒中国分离株Ｄ２－４３的ＰｒＭＣ端抗原区的基因片段，结构分析及特异性分析的结果表明引物合乎要求，反转录（ＲＴ）－ＰＣＲ扩增出一３８５ｂｐ的目的片段，经ＨａｅⅢ酶切得到２１９、１６６ｂｐ的两条预期片段，表明ＲＴ－ＰＣＲ扩增出ＰｒＭ基因片断。扩增产物经ＢａｍＨＩ酶切后插入经ＳｍａＩ、ＢａｍＨＩ双酶切的ｐＵＥｘ１中，转化ＭＣ１０６１菌，表达与β－半乳糖苷酶的融合蛋白，ＳＤＳ－ＰＡＧＥ结果表明有一约１３０ｋＤ的目的蛋白带，表达量占菌体总蛋白的３０％，Ｗｅｓｔｅｒｎｂｌｏｔ及ＥＬＩＳＡ结果表明表达产物能与Ｄｅｎｇｕｅ－２多抗血清结合，为ＰｒＭ的免疫学及生物学性质研究打下了基础。     

Rapid diagnostic test that uses isocitrate lyase activity for identification of Yersinia pestis.

The presence of high levels of isocitrate lyase activity in Yersinia pestis grown on blood agar base medium, as compared with low levels of this enzyme in Yersinia pseudotuberculosis and Yersinia enterocolitica, suggested that the differences in the levels of this enzyme could be used for the presumptive identification of Y. pestis. A modified, semiquantitative assay for isocitrate lyase activity is described which requires no expensive instrumentation, utilizes readily available chemicals and substrates, and requires only 20 min for completion. This test yielded positive results with all 108 isolates of Y. pestis tested and negative results with all strains of Y. pseudotuberculosis (68 isolates) and Y. enterocolitica (202 isolates) tested. Less than 2% of the approximately 1,300 non-Yersinia isolates from the family Enterobacteriaceae and none of the 93 isolates from the family Pseudomonadaceae yielded positive results. We conclude that this test provides for rapid identification of Y. pestis and should be useful in the initial screening of isolates from rodent and flea populations and in the presumptive identification of this organism from suspected cases of human plague.     

Characterization of common virulence plasmids in Yersinia species and their role in the expression of outer membrane proteins.

The virulence plasmids pYV019, pYV8081, and pIB1 from Yersinia pestis, Yersinia enterocolitica, and Yersinia pseudotuberculosis, respectively, were characterized by restriction endonuclease analysis. The three plasmids exhibited a region of common DNA previously shown to encode determinants which confer Ca2+ dependence. The plasmids from Y. pestis and Y. pseudotuberculosis were similar throughout their genomes. In contrast, a region of the plasmid from Y. enterocolitica which contained an origin of replication differed from the other two plasmids as determined by DNA homology and replication properties. Plasmid-associated outer membrane proteins from all three species of Yersinia were characterized by polyacrylamide gel electrophoresis. There were no differences in the outer membrane protein profiles between plasmid-containing and homogenic strains lacking the plasmid after growth at 28 degrees C. After growth at 37 degrees C, both Y. enterocolitica and Y. pseudotuberculosis showed at least four major plasmid-associated outer membrane proteins. Y. pestis did not show any discernible changes after growth at 37 degrees C. It was shown by using E. coli minicell analysis that the plasmid DNA from all three species of Yersinia contained the coding capacity for production of the novel outer membrane proteins.     

The difference in the lcrV sequences between Y. pestis and Y. pseudotuberculosis and its application for characterization of Y. pseudotuberculosis strains.

We have sequenced the lcrGVH operon from Y. pseudotuberculosis plasmid pYV995 and compared its sequence with that of Y. pestis. The sequences were highly homological, however, six base pair substitutions were found in one short 14 bp region termed variable sequence. Two oligonucleotides corresponding to variable sequence of Y. pestis (pes-V) or Y. pseudotuberculosis (ptb-V) were synthesized and were used as molecular probes in hybridization experiments with sets of Y. pestis and Y. pseudotuberculosis strains. All 17 Y. pestis strains tested were positive only with the pes-V probe, 18 of 21 Y. pseudotuberculosis strains were positive with the ptb-V probe, while three Y. pseudotuberculosis strains reacted with the pes-V probe but not the ptb-V probe. The 200 bp fragment including variable sequence was sequenced in seven Y. pseudotuberculosis strains. The Y. pseudotuberculosis strains which were positive with the pes-V probe possessed the 200 bp fragment sequence almost identical with that from Y. pestis. No correlation between the Y. pestis-like lcrV sequence and virulence was found for these strains. Moreover, the Y. pseudotuberculosis strains with Y. pestis-like sequences in contrast to Y. pestis possessed unaltered yadA gene. However, we have found the yadA frameshift mutation characteristic for Y. pestis in one Y. pseudotuberculosis strain 312.     

Evaluation of DNA colony hybridization and other techniques for detection of virulence in Yersinia species.

The virulence of yersiniae varies according to (i) species and biotype and (ii) possession of a 67- to 72-kilobase virulence plasmid. Y. pestis, Y. pseudotuberculosis, and biotypes 1B, 2, 3, 4, and 5 of Y. enterocolitica are inherently virulent but express full virulence only when in possession of a virulence plasmid. Other Yersinia species and biotypes 1A and 3B of Y. enterocolitica are seldom implicated in disease. In this study, we prepared DNA probes from eight nonoverlapping regions of the virulence plasmid of a strain of Y. enterocolitica and from the inv and ail chromosomal loci responsible for the invasive capacity of Y. enterocolitica and Y. pseudotuberculosis. The probes were used in colony hybridization experiments to investigate 156 yersiniae of various species and biotypes and of differing virulence. Probes prepared from the inv gene of Y. pseudotuberculosis hybridized with Y. pseudotuberculosis and Y. pestis only, whereas an analogous probe prepared from Y. enterocolitica hybridized with all species and biotypes of yersiniae (but not with other bacteria) regardless of virulence or potential virulence. Probes prepared from the ail region of Y. enterocolitica reacted almost exclusively with Y. enterocolitica strains of pathogenic biotypes. Probes prepared from the virulence plasmid of a serogroup O:8, biotype 1B isolate of Y. enterocolitica identified virulent yersiniae in all species with a high degree of sensitivity and specificity. These probes did not react with yersiniae of avirulent biotypes or species. Of the other assays of virulence evaluated (calcium dependence, binding of crystal violet, and pyrazinamidase activity), binding of crystal violet provided a simple means for identifying plasmid-bearing strains.     

Rapid and specific identification of Yersinia pestis by using a nested polymerase chain reaction procedure.

We developed a 4-h nested polymerase chain reaction assay that detected a region of the plasminogen activator gene of Yersinia pestis in 100% of 43 Y. pestis strains isolated from humans, rats, and fleas yet was unreactive with the closely related species Yersinia enterocolitica and Yersinia pseudotuberculosis.     

Correlation of autoagglutination and virulence of yersiniae.

Virulent strains of Yersinia pestis, Y. pseudotuberculosis and Yersinia enterocolitica invariably autoagglutinated in tissue culture media when grown at 36 degrees C. Avirulent strains did not possess this property.     

Multilocus sequence typing for studying genetic relationships among Yersinia species

The intra- and interspecies genetic relationships of 58 strains representing all currently known species of the genus Yersinia were examined by multilocus sequence typing (MLST), using sequence data from 16S RNA, glnA, gyrB, recA, and Y-HSP60 loci. Yersinia aldovae, Y. bercovieri, Y. intermedia, Y. pestis, Y. pseudotuberculosis, Y. rohdei, and Y. ruckeri were genetically more homogeneous than were Y. enterocolitica, Y. frederiksenii, Y. kristensenii, and Y. mollaretii. The MLST data concerning the genetic relatedness within and among various species of Yersinia support the idea that Y. pestis and Y. pseudotuberculosis are two lineages within the same species rather than two distinct species. Y. ruckeri is the genetically most distant species within the genus. There was evidence of O-antigen switching and genetic recombination within and among various species of Yersinia. The genetic relatedness data obtained by MLST of the four housekeeping genes and 16S RNA agreed in most, but not all, instances. MLST was better suited for determining genetic relatedness among yersiniae than was 16S RNA analysis. Some strains of Y. frederiksenii and Y. kristensenii are genetically less related to other strains within those species, compared to strains of all other species within the genus. The taxonomic standing of these strains should be further examined because they may represent currently unrecognized Yersinia species.     

Analysis of clinical and environment Yersinia isolates in the Republic of Georgia.

The Center for Infectious Diseases Control, Georgian Ministry of Health, isolated 2,493 Yersinia enterocolitica and Y. enterocolitica-like strains, 22 Y. pestis strains, and 21 Y. pseudotuberculosis strains from 130,574 clinical and environmental samples. Analysis of 100 Y. enterocolitica and Y. enterocolitica-like strains showed none to be within traditional pathogenic biogroups or serogroups, and none carried genetic markers for virulence. However, some strains were enterotoxigenic in infant mice, while others were associated with prolonged carriage in adult mice.     

Pyrazinamidase activity in Yersinia enterocolitica and related organisms.

Pyrazinamidase activity was tested in 381 Yersinia strains from various ecological and geographical origins and belonging to the following species: Y. enterocolitica (five biogroups), Y. intermedia, Y. frederiksenii, Y. kristensenii, Y. aldovae, Y. pseudotuberculosis, and Y. pestis. The pyrazinamidase test was negative (Pyz-) in all bioserogroups of Y. enterocolitica, in which is usually harbored the virulence plasmid, and was involved in human or animal diseases. Y. pseudotuberculosis and Y. pestis were also Pyz-. The more ubiquitous bioserogroups of Y. enterocolitica, without naturally occurring virulence plasmid, and related species were all Pyz+. Pyrazinamidase activity allowed the separation of the pathogenic North American Y. enterocolitica isolates from other nonpathogenic strains within biogroup 1. Similarly, environmental biogroups 3A and 3B were clearly distinguished from pathogenic biogroup 3. However, the pyrazinamidase test was not linked to the presence of the virulence plasmid itself and should not replace the pathogenicity tests to assess the actual virulence of an individual strain. This test proved to be a valuable tool to distinguish potential pathogenic from nonpathogenic strains of Y. enterocolitica in epidemiological surveillance programs.