寡核苷酸探针膜杂交法检测耐药结核菌

目的应用寡核苷酸探针膜杂交法快速检测临床分离株中结核分枝杆菌对异烟肼(INH)、链霉素(SM)、乙胺丁醇(EMB)的耐药性。方法设计与合成用于检测结核分枝杆菌3种药物常见耐药基因的寡核苷酸探针,点于硝酸纤维素膜上,与结核分枝杆菌临床分离株生物素标记的聚合酶链反应(PCR)产物进行反向斑点杂交。结果26株耐INH菌株中,16株Klb杂交阳性;7株inhla杂交阳性;23株耐SM菌株中,17株rpsl基因突变型探针Slb杂交阳性,2株突变型探针rrslb杂交阳性;31株耐EMB菌株中,13株Elb杂交阳性,1株Elc杂交阳性,5株Eld杂交阳性,1株Ele杂交阳性,11株与El探针杂交。kagG、inhA、rp-sL、rrs和embB基因膜杂交突变检出率分别为61.5%,26.9%,74%,8.7%和64.5%,与PCR-DS分析结果一致。结论寡核苷酸探针膜杂交技术可能成为检测部分结核分枝杆菌耐药基因型简便、快速的方法。     

PCR技术制备ETEC耐热性肠毒素Ⅰ基因探针及其初步应用

产肠毒素性大肠杆菌(ETEC)耐热性肠毒素I(STI)重组质粒pSLM004酶切TaqI片段作为模板,经(PCR)技术扩增STI基因,标以α-~(32)p-dATP作STI基因探针。菌落原位杂交结果表明该探针特异性高、敏感性强。通过对180株婴幼儿腹泻病料和52株仔猪腹泻物分离的大肠杆菌(E.coli),以及28株已知血清型的E.Coli标准菌株菌落原位杂交检测,结果分别有11、2和2株为杂交阳性反应,阳性率分别为6％(11/180),4％(2/52),和7％(2/28);而对84株从犊牛腹泻物分离的E.coli中,未检出杂交阳性株。乳鼠生物学试验比较研究表明,15株探针检测阳性菌株中13株乳鼠试验也为阳性反应,两者符合率为87％(13/15)。     

贵州省部分地区结核分枝杆菌MLVA基因分型研究

目的应用多位点可变串联重复序列分析技术(MLVA)对贵州省结核分枝杆菌进行基因分型分析,为贵州省结核病防控提供科学依据。方法选取贵州省150株临床分离株结核分枝杆菌,采用水煮法提取基因组DNA,PCR扩增15个VNTR位点,统计菌株各VNTR位点的重复数目,通过遗传差异值(h)及Hunter-Gaston指数对VNTR位点进行遗传多态性及分辨力评价,采用BioNumerics 5.0软件对各菌株进行聚类关系和最小间距图(minimum spanning tree,MST)分析。结果 PCR检测结核菌株VNTR各位点呈明显多态性,以Mtub21和MIRU26多态性尤为显著,以h值分别为0.559和0.505;MIRU10和ETRB显示较低基因多态性,h值分别为0.052和0.090。聚类分析显示,150株菌株分为Ⅰ、Ⅱ、Ⅲ、Ⅳ4个基因群,其中Ⅰ群占10.67%(16/150),Ⅱ群占30.67%(46/150),Ⅲ群占40.00%(60/150),Ⅳ群占18.67%(28/150)。4个基因群呈现明显地域分布,毕节市以Ⅰ、Ⅱ群为主要流行菌株,安顺市以Ⅲ群菌株为主要流行菌株,遵义市以Ⅳ群菌株为主要流行菌株。MST分析显示,150株菌株形成3个克隆复合体(clonal complexes,CCs)及若干个独立分支(singleton),遵义、安顺、毕节分离株分别分布于不同的克隆复合体CCs。结论贵州省结核分枝杆菌存在明显的基因多态性和地域分布特性,以Ⅲ群和Ⅱ群菌株为主要流行菌株,应加强对上述两群菌株的监控。     

可视化膜芯片技术用于结核分枝杆菌基因分型的研究

目的建立用于结核分枝杆菌间隔区寡核苷酸分型的膜芯片技术,并评估其应用价值。方法根据结核分枝杆菌直接重复序列43间隔子设计特异的分型探针并制作膜芯片,通过直接重复序列特异引物扩增基因组间隔子相应区段的DNA。扩增引物用生物素标记,扩增标记后的产物与芯片的探针杂交,根据显色后产生的斑点确定标本的基因亚型。对175株临床分离菌株用该方法进行分型,用传统膜杂交法作对照。结果 H37Rv和BCG菌株DNA扩增产物与膜芯片杂交,43条探针均出现杂交斑点。用膜芯片技术杂交获得H37Rv和BCG菌株的基因亚型,与传统固相膜杂交结果一致。用该法对175株临床分离菌株进行分型,其中167株属于4个已知的基因家族,最大的一个为北京家族,占62.9%(110/175)。北京家族中有107株为典型北京家族(1-34间隔区缺失),3株为北京样基因型(Beijing-like),即非典型北京家族(35-43间隔区也有缺失)。T家族占30.9%(54/175),包括34株T1,14株T2,6株T3。2株Manu2和1株LAM9;未匹配8株,占4.6%(8/175)。膜芯片技术杂交结果与传统膜杂交结果完全相同,耗时由膜杂交法的195 min缩短为22 min。方法的灵敏度为10个MTB/ml。结论膜芯片技术用于结核分枝杆菌间隔区寡核苷酸基因分型灵敏度高且简便易行,适用于临床流行病学追踪与分析。     

贵州某结核病高发县结核分枝杆菌流行情况的初步研究

目的分析结核病高发县结核分枝杆菌临床分离株及其基因型特征,探索高疫情地区结核菌株流行情况。方法在罗甸县结核病定点医院门诊收集临床分离的分枝杆菌菌株,用PNB生长试验、结核分枝杆菌散在重复单位(VNTR) 和RD105缺失基因检测法分别进行菌种、结核分枝杆菌DNA多态性和北京基因型鉴定。结果80株菌株中非结核分枝杆菌占12.5%,70株结核分枝杆菌中北京家族占42.9%,非北京家族占57.1%。VNTR结果显示,菌株可分为5个基因群,其中 I群占15.7%,含 11个基因型,Ⅱ群占35.7%,含25个基因型,Ⅲ群占15.7%,含11个基因型,Ⅳ群占30.0%,含 21个基因型,Ⅴ群占2.9%,含2个基因型,未见成簇菌株。结论初步证实罗甸县结核分枝杆菌存在基因多态性,Ⅱ群、Ⅳ群为当地主要流行群,同时存在一定比例的非结核分枝杆菌感染。     

长治地区腹泻患者致泻性大肠杆菌的检测与分析

目的 了解山西省长治地区感染性腹泻患者致泻性大肠杆菌分离情况及菌株特征。方法 收集腹泻患者粪便,EC肉汤增菌后,采用针对致泻性大肠杆菌7个毒力基因eaeA、aggR、ipaH、stx1、stx2、lt、stIb及16S rRNA基因rrs的多重PCR初筛后,阳性者作致泻性大肠杆菌分离,再对分离菌株行生化鉴定、毒力基因检测及血清群测定。结果 170份标本中分离到致泻性大肠杆菌20株,阳性率为12%。其中EPEC 11株,均为非典型EPEC,EAEC 7株,ETEC 2株。结论 长治地区致泻性大肠杆菌感染以5岁以下儿童为主,除EPEC外,EAEC占有很大比例;对致泻性大肠杆菌的检测及判断有赖于血清群与毒力基因检测的分子生物学方法相结合。     

中国肠产毒性大肠杆菌中耶尔森菌强毒力岛的检测

目的　了解耶尔森菌强毒力岛在中国肠产毒性大肠杆菌中的分布及插入位点。方法　使用PCR扩增、DNA打点杂交和DNA测序及分析方法。结果　在 94株分离自中国的肠产毒性大肠杆菌中 ,14株耶尔森菌强毒力岛核心区的 8个基因PCR扩增阳性 ,除 3株菌的整合酶基因外 ,PCR扩增产物长度均与预期一致 ,但天冬酰胺转运RNA(asnTtRNA)位点扩增阴性 ;上述 14株菌进行全菌DNA打点杂交试验 ,均与irp2和 fyuA探针杂交 ;选择上述 3株菌中的 1株 ,对其整合酶基因的PCR产物测序 ,并与鼠疫耶尔森菌强毒力岛的整合酶基因序列比较 ,发现该整合酶基因于 5’端缺失了 347bp的片段。 结论　 14 %肠产毒性大肠杆菌携带的耶尔森菌强毒力岛 ,且均插入在天冬酰胺转运RNA位点处 ;部分菌株所携带的耶尔森菌强毒力岛的整合酶基因发生了缺失。     

恶性疟原虫膜蛋白基因的过度多态性

作者用高密度寡核苷酸探针芯片技术检测恶性疟原虫第 2染色体全长基因组中的单核苷酸多态性 ( SNPs) ,以荷兰分离株 3D7为参照 ,并依照其基因组 DNA设计了 4 1 6 7种长为 2 5nt的寡核苷酸探针 ,用于检测洪都拉斯、东南亚、塞拉利昂和巴西 4个地区的不同分离株 ,在 4种虫株中检测到 SNPs数目分别为 32 4 ,32 0 ,1 0 7和 2 30 ,至少在一个虫株中有 585种探针显示差异。研究发现变异基因的功能分布不是随机的。变异最多的是膜蛋白基因 ,该区探针占全部探针的 2 2 %,占可变探针的 6 9%。这些膜蛋白中有几种性能优良的疫苗靶位 ,包括传播阻断…     

甘肃省临床分离结核分枝杆菌MLVA分型初步研究

目的随机选取2005-2007年间甘肃省肺科医院临床分离结核分枝杆菌菌株,通过多位点可变数目串联重复序列(Multiple locus variable numbers of tandemrepeats analysis,MLVA)分型,了解甘肃结核分枝杆菌流行菌株基因型情况。方法选择标化的15个VNTR位点,对临床分离菌株DNA进行检测,DNA指纹图谱使用Bio Numerics4.5软件进行统计分析,得出聚类分析结果。结果 228株结核分枝杆菌被分为4大基因群,7个大基因类型,分别包含13(5.7%)、3(1.3%)、7(3.1%)、1(0.4%)、171(75%)、31(13.6%)、2(0.9%)个株菌;在株水平基因分型上,93(40.8%)株为单菌株基因型;其余菌株基因型分别包含2-10株结核分枝杆菌,共构成132个基因簇。结论甘肃结核分枝杆菌菌株存在丰富的基因多态性,ML-VA方法具有较高的基因分型能力,可以满足结核分枝杆菌株水平DNA分型的需要。甘肃省结核分枝杆菌流行株主要为北京家族基因型菌株。     

PCR在大肠杆菌ST1b基因扩增及其基因检测上的应用

作者应用聚合酶链反应(PCR)成功地扩增了产肠毒性大肠杆菌ST1b基因,在ST1b产量、活性和纯度上均满足了基因重组的要求。并利用PCR技术建立了一种新型基因探针标记方法(基因扩增标记法),所制备的ST1b基因探针经相关菌菌落杂交试验证实具有良好的特异性和敏感性。通过对180株婴幼儿腹泻大肠杆菌分离株、52株仔猪腹泻大肠杆菌分离株和28株大肠杆菌标准菌株的基因诊断以及96株K99—ST1b基因重组转化菌的基因检测,菌落杂交阳性率分别为11/180、2/52、2/28和5/96。     

致肾盂肾炎大肠杆菌鉴定方法的研究

本文利用带有致肾盂肾炎大肠杆菌（ＵＰＥＣ）Ｐ菌毛粘附基因群的克隆株免疫新西兰白兔，获得抗血清，经吸收后仅保留对ＵＰＥＣ粘附基因群的特异性。继而建立了全菌ＥＬＩＳＡ，用于尿源大肠杆菌的研究，并与血凝试验及菌毛蛋白粗提物的ＳＤＳ－聚丙烯酰胺凝胶电泳和免疫印迹方法进行比较。结果表明，代表ＵＰＥＣ的ＭＲＨＡ＋组有２６．８％阳性，提示尚有不同血清型的ＵＰＥＣ存在；但与非ＵＰＥＣ菌株（ＭＳＨＡ＋和ＨＡ－组）的差异非常显著（Ｐ＜０．００１）。实验证明全菌ＥＬＩＳＡ方法灵敏、特异、经济、便于推广，可用于ＵＰＥＣ菌株的鉴定，填补了国内的空白。     

A comparative study of enterotoxin gene probes and tests for toxin production to detect enterotoxigenic Escherichia coli

Escherichia coli isolated from children with diarrhea were tested for enterotoxin production and for hybridization with gene probes for heat-labile (LT) and heat-stable (ST-H and ST-P) enterotoxin. Fecal specimens were also examined directly for genes coding for enterotoxins. E. coli that hybridized with the cloned enterotoxin gene probes was identified by colony hybridization from 46 children, by enterotoxin production from 38 children, and by specimen hybridization from 37 of 304 children examined. Eighty-six percent (473 of 550) of E. coli that hybridized with the cloned DNA probes produced enterotoxins. Four E. coli that hybridized with the LT and 73 E. coli that hybridized with the ST-H probes were nonenterotoxigenic. These isolates were subsequently shown not to hybridize with other constructions of the same probes and did not hybridize with synthetic single-stranded oligonucleotides directed against the LT or ST genes.     

Tissue culture-adherent Escherichia coli in infantile diarrhea.

To determine the association of tissue culture-adherent Escherichia coli with diarrhea, serotyped E. coli strains isolated in a yearlong case-control study of infantile diarrhea in Bangkok, Thailand, were examined for adherence to HeLa cells and for hybridization with the enteropathogenic E. coli adherence factor, the F1845, and the enteroaggregative E. coli (EAggEC) DNA probes. E. coli that adhered to HeLa cells in a localized adherence (LA) pattern (LA E. coli) was isolated from 26 of 509 infants with diarrhea (cases) and 9 of 509 age-matched controls (P = .006); E. coli with diffuse or aggregative adherence (DA or AA) to HeLa cells or that hybridized with the F1845 or EAggEC probes was not associated with infantile diarrhea. LA E. coli of classical enteropathogenic E. coli (EPEC) serotypes was isolated from 11 cases and 1 control (P = .003). EPEC O44:H18 that adhered to HeLa cells in a DA pattern and hybridized with the F1845 DNA probe was the predominant E. coli (five of five colonies tested) isolated from a 5-month-old girl with diarrhea in whom no other enteric infections were identified. Although LA E. coli was highly associated with infantile diarrhea, the role of DA and AA E. coli was uncertain in this setting.     

Frequency and organization of pap homologous DNA in relation to clinical origin of uropathogenic Escherichia coli

The pap operon encodes the gal alpha 1-4gal beta specific adhesins of Escherichia coli. The presence and organization of pap homologous DNA was determined using two probes specific for pap in 217 uropathogenic E. coli samples by dot blot and Southern blot analysis. The frequency of pap homologous DNA was 76% in pyelonephritis, 69% in cystitis, and 52% in an asymptomatic bacteriuria group. Further, the gal alpha 1-4gal beta binding phenotype among the pap-positive strains was expressed more often in acute pyelonephritis (91%) than the cystitis (60%) or asymptomatic bacteriuria (52%) strains. This was explained in part by difference in organization of pap homologous DNA between the genotypically positive pyelonephritis and asymptomatic bacteriuria strains. The pyelonephritis isolates contained three copies of pap significantly more often than the asymptomatic bacteriuria strains, and the pyelonephritogenic O-antigen types had a general increase in pap copy number. The difference in expression of gal alpha 1-4gal beta adhesins between pyelonephritis and asymptomatic bacteriuria isolates was thus not only a function of the frequency of pap homologous DNA but also of phenotypic expression among genotypically pap-positive strains.     

Properties of Vero cytotoxin-producing Escherichia coli of human origin of O serogroups other than O157.

The 48 Vero cytotoxin-producing Escherichia coli (VTEC) examined for properties associated with virulence were of human origin and represented 17 O serogroups other than O157 and O26. Only Vero cytotoxin production was common to all the strains. About 60% produced enterohemolysin and hybridized with the CVD419 probe derived from plasmid sequences of E. coli O157. Thirteen strains gave localized adherence (LA) to HEp-2 cells. All of these hybridized with the E. coli attaching and effacing (eae) gene probe and were positive in the fluorescence actin staining test, properties characteristic of strains that efface intestinal microvilli. A further 5 strains were eae probe-positive but did not give LA. None of the VTEC hybridized with a probe specific for the enteropathogenic E. coli adherence factor. Seven strains adhered to HEp-2 cells in a diffuse or aggregative pattern but did not hybridize with probes for these phenotypes. Non-O157 E. coli strains are diverse in their properties, although some may share virulence mechanisms with other diarrheogenic E. coli.     

PapG-dependent adherence breaks mucosal inertia and triggers the innate host response

Mucosal pathogens differ from normal flora constituents in that they provoke a host response that upsets mucosal integrity. We investigated whether the elaboration of discrete adherence factors is sufficient to break the inertia of the mucosal barrier. PapG-mediated adherence was selected as an example, because P fimbrial expression characterizes uropathogenic Escherichia coli and because adherence starts the attack on the mucosal barrier. Patients were inoculated intravesically with transformed nonvirulent E. coli strains expressing functional P fimbriae (E. coli pap(+)) or mutant fimbriae lacking the adhesin (E. coli Delta papG). E. coli pap(+) was shown to activate the innate host response, and adherent gfp(+) bacteria were observed on excreted uroepithelial cells. E. coli Delta papG failed to trigger a response and was nonadhesive. We conclude that PapG-mediated adherence breaks mucosal inertia in the human urinary tract by triggering innate immunity and propose that this activation step differentiates asymptomatic carriage from infection.     

肠产志贺样毒素且具侵袭力的大肠埃希氏菌和肠侵袭性大肠埃希氏菌的侵袭基因比较

目的比较肠产志贺样毒素且具侵袭力的大肠埃希氏菌（Ｅｎｔｅｒｏ－ＳＬＴｓ－ＰｒｏｄｕｃｉｎｇａｎｄＩｎｖａｓｉｖｅＥｓｃｈｅｒｉｃｈｉａｃｏｌ－ｉ，ＥＳＩＥＣ）和肠侵袭性大肠埃希氏菌的侵袭基因，阐明它们之间的异同。方法使用质粒酶切图谱分析，侵袭性基因ｉｐａＢ、ｉ－ｐａＣ、ｉｐａＤ的ＰＣＲ和Ｓｏｕｔｈｅｒｎｂｌｏｔ杂交实验，以及利用ＥＩＥＣ８４０１菌株的侵袭性大质粒酶切产物作为探针与ＥＳＩＥＣ大质粒进行Ｓｏｕｔｈｅｒｎｂｌｏｔ杂交等方法。结果发现ＥＳＩＥＣ菌株不具有ＥＩＥＣ、志贺氏菌所特有的ｉｐａＢ、ｉｐａＣ、ｉｐａＤ基因；ＥＳＩＥＣ菌株质粒与ＥＩＥＣ、志贺氏菌质粒同源性很小。结论ＥＳＩＥＣ菌株编码侵袭力的基因与ＥＩＥＣ及志贺氏菌的侵袭基因是完全不同的，证明对ＥＳＩＥＣ的分类是正确的。     

Identification by DNA hybridization of enterotoxigenic Escherichia coli in a longitudinal study of villages in Thailand

Radioactively labeled enterotoxin genes were used to study the epidemiology of enterotoxigenic Escherichia coli infections in two Thai villages. When E. coli that were isolated from 674 specimens were fixed on nitrocellulose paper and examined for hybridization with E. coli enterotoxin gene probes in Bangkok, the technique had a sensitivity of 94% (31 of 33) and a specificity of 100% (641 of 641), when compared with tests of E. coli for enterotoxin production in the Y-1-adrenal cell and suckling-mouse assays. However, when the same specimens were fixed directly onto nitrocellulose paper at a field laboratory and transported to the reference laboratory for assay with the gene probes, 27 specimens that contained enterotoxigenic E. coli did not hybridize with the E. coli gene probes. Enterotoxigenic E. coli that hybridized with the LT, ST-H, and ST-P probes were identified in 10% (17 of 177) of villagers with diarrhea, 7% (8 of 108) of contacts of individuals with diarrhea caused by enterotoxigenic E. coli, and 3% (32 of 1,199) of persons not associated with cases of diarrhea caused by enterotoxigenic E. coli. Enterotoxigenic E. coli that hybridized with the ST-II probe was not a cause of diarrhea. Alternative methods of retaining DNA on filters under field conditions are needed before this technique can be used for direct examination of specimens with enterotoxin gene probes.     

Association of patterns of Escherichia coli adherence to HEp-2 cells with acute and persistent diarrhea]

In Brazil diarrhea is the cause of approximately 15% of death among infants. Enteropathogenic E coli is the most important bacterial agent causing acute diarrhea, which is defined as less than 14 days of duration. About 30% of these cases may evolve to persistent diarrhea, defined as lasting more than 14 days. In this work it was carried out a case-control study including 34 children under 2 years of age, and admitted to hospital facilities in S?o Paulo for rehydration therapy. Thirty-four age matched children hospitalized in the same facilities, and presenting no gastrointestinal symptoms were included as controls. Stool samples were analyzed for the presence of bacterial pathogens (diarrheagenic E coli, Shigella, Salmonella, Yersinia, and Campylobacter), protoparasytes, rotavirus, and enteric adenovirus. The E coli strains isolated were analyzed for their ability to adhere to HEp-2 cultured cells, in a 3 h adhesion assay. Search for homology with DNA probes for localized adherence (EAF, eaeA probes), AA (enteroagregative adherence) (AA probe), and diffuse adherence (F1845, AIDA-I probes) was carried out by the colony hybridization method. Twenty-four of the cases were acute diarrhea and 10 persistent diarrhea. Strains with localized adherence were associated with acute and persistent diarrhea. About 23.5% of E coli were associated with typical Enteropathogenic E coli strains (EAF+, eaeA+). Enteroaggregative E coli (EAggEC) (AA+) was isolated only from cases and in similar frequency for acute and persistent diarrhea. Diffusely adherent E coli (DAEC) which did not hybridize with the diffuse adherence probes were isolated among cases and controls. E coli eaeA+ with localized-like adherence was isolated from cases in a frequency three times higher than in controls, suggesting that it may really have a pathogenic potential.     

Enteroaggregative Escherichia aoliisolated from Chinese diarrhea patients with high-pathogenicity island of Yersinia''''xs involved in synthesis of siderophore yersiniabactin

AIM: To investigate the distribution of 12 high-pathogenicity island(HPI) genes and the relation between HPI genes and expression of yersiniabactin(Ybt) in enteroaggregative E.coli(EAggEC) isolated from Chinese diarrhea patients. METHODS: The distribution of 12 HPI genes was investigated by PCR and DNA hybridization in two prototype strains of EAggEC, EAggEC 17-2, EAggEC 042, and 6 clinical EAggEC isolates from China. The production of siderophore Ybt in HPI-positive strains was detected by reporter gene bioassay to determine the relation between HPI genes and expression of Ybt. Flow cytometry was used to detect fluorescent signal of the reporter strain that could designate production of Ybt. RESULTS: Seven strains were HPI-positive and one strain was HPI-negative. Six of the seven HPI-positive strains were inserted into asnT-tRNA site. Moreover, seven EAggEC HPI-positive strains revealed enhanced fluorescence signal but the EAggEC HPI-negative strain did not. However, there was a difference in Ybt expression condition and level among these seven EAggEC HPI-positive strains. Although UFT073 strain, the prototype strain of uropathogenic E.coli(UPEC), carried the complete HPI core part, we did not detect the expression of Ybt in it. CONCLUSION: EAggEC HPI-positive strains can express the Ybt system, but the presence of HPI core part does not mean the functional expression of Ybt.