RAPD基因指纹图谱在解脲脲原体分型研究中的应用…

采用随机扩增多态性ＤＮＡ（ＲＡＰＤ）技术，以美国Ｏｐｅｒｏｎ公司生产的ＯＰＬ１￣２０十本脱氧寡核苷酸为引物，对解脲脲原体８和１０血清型标准株基因指纹图谱进行了构建。通过比较发现，两型解脲脲原体ＤＮＡ间存在同源性和多态性。初步研究结果表明，ＲＡＰＤ可用于解脲脲原体分型。     

女性NGU与单纯解脲脲原体感染的分群分型初步研究

目的探讨女性非淋菌性尿道炎或宫颈炎（NGU）与解脲脲原体（Uu）分群和分型的关系。方法采用液体培养,A7固体分离,液体纯化的方法获得Uu纯菌株,分别对114例女性单纯Uu感染的NGU和113例单纯Uu感染的女性携带者的Uu做PCR分群和生长抑制试验分型。结果NGU组1群56例（49.12%）,2群58例（50.88%）。正常组1群51例（45.13%）,2群62例（54.87%）;NGU组Uu分型阳性率最高依次为2型、7型、6型,正常人组最高依次为7型、2型、14型。结论Uu感染所致NGU与Uu分群关系不大。     

反相斑点杂交技术在解脲脲原体分型中的应用

解脲脲原体（UU）被认为是盆腔及生殖器炎症的主要病原体之一。然而，女性下生殖道内查到UU者，是否皆须按非淋菌性阴道炎进行治疗，近年来一直困扰广大妇产科医生。有学者研究认为UU的感染与其群别和亚型的型别相关，但目前临床中尚缺乏一种简单易行的进行UU分型的方法，为此我们对UU基因型的分型方法进行了一些研究，报道如下。     

RAPD基因指纹图谱在解脲脲原体分型研究中的应用初探

采用随机扩增多态性DNA(RAPD)技术,以美国Operon公司生产的OPL1～20十聚体脱氧寡核苷酸为引物,对解脲脲原体8和10血清型标准株基因指纹图谱进行了构建。通过比较发现,两型解脲脲原体DNA间存在同源性和多态性。初步研究结果表明,RAPD可用于解脲脲原体分型。     

PCR法解脲脲原体分群和四环素耐药检测

目的 建立解脲脲原体(Ureaplasma urealyxicum，Uu)的分群和四环素耐药PCR检测方法。方法 1．根据Uu的多带抗原(multi—banded antigen，MBA)基因序列自行设计引物(P15′-ATT，TGC，AAT，CTT，TAT，ATG，TT；P25′-TTC，AGC，TGA，TGT，AAG，TGC，AGC，ATT，AAA，T)，并建立分群PCR反应体系。2．根据TetM基因序列自行设计引物(P15′-TTA，TCA，ACG，GTT，TAT，CAG，G；P25′-GCT，ATA，TAT，GCA，AGA，CG)，并建立四环素耐药反应体系。结果 1．MBA基因PCR能将Uul4个血清型正确分为二个生物群，其中生物一群(1、3、6、14等4个血清型)出现404bp的产物带，生物二群(2、4、5、7、8、9、10、11、12、13等10个血清型)出现448bp的产物带。60株Uu临床分离株经MBA基因PCR检测，生物一群53例、生物二群6例、生物一／二群同时存在1例。2.Uu典型株14个血清型，TetM基因PcR扩增的不能出现任何条带；60株Uu临床分离株经TetM基因PCR检测41例出现目的条带。结论 PCR法进行Uu生物分群和四环素耐药检测简单、快速，为相关研究提供了手段。     

解脲脲原体Parvo和T960生物群msr基因的检测

目的 检测解脲脲原体(Uu)是否携带介导对红霉素耐药的msr基因,并分析其在Uu两生物群间分布的差异.方法 采用微量肉汤稀释法测定72株Uu临床株对红霉素的体外耐性,PCR检测msrA、msrB、msrG、msrD基因,并对Uu进行PCR分群.结果 72株Uu的最低抑菌浓度(MIC)范围是≤0.125 μg/ml≥128 μg/ml,MIC_(50)为32 μg/ml,MIC_(90)≥128μg/ml.分群结果示Parvo生物群51株,占70.83%,T960生物群21株,占29.17%.共检测到msrD基因的Uu24株,msrB基因12株,msrA基因1株,没有发现Uu菌株携带msrC基因.5株Uu同时检测到msrB和msrD基因,1株Uu同时检测到msrA、msrB和msrD基因.以MIC≥8μg/ml为耐药判定值时,两生物群对红霉素耐药性无显著差异,msrB基因主要分布在T960生物群.结论 Uu临床菌株携带对大环内酯类耐药的msr基因(包括msrA、msrB、msrP),msrB基因主要分布在T960生物群.     

妇科疾病中的解脲脲原体、沙眼衣原体Nesed-PCR检测

目的探讨UU、CT在本地区妇科疾病中的发病率及其致病作用,方法收集535例妇科阴道炎、不孕症、宫颈炎、附件炎等疾病患者的阴道分泌物,应用nested PCR(nPCR)技术检测解脲脲原体(UU)及沙眼衣原体(CT)核酸.结果393人检测解脲脲原体(UU),UU-DNA阳性192例(48.8%);142人检测沙眼衣原体(CT),CT-DNA阳性32例(22.5%).结论解脲脲原体(UU-DNA)、沙眼衣原体(CT-DNA)与妇科疾病的发生关系密切,特别是阴道炎的妇科患者.     

解脲脲原体的实验室检测方法的研究现状

解脲脲原体是一种条件致病菌，多与宿主共存，仅在某些条件下引起机会性感染，感染部位常为男女泌尿道，其中更易致病的为U．urealyticum型。近年来检测解脲脲原体的方法主要有液体培养法、固体培养法、免疫学方法和分子生物学方法等等，这些检测方法各有优劣。本文就临床使用较多的液体培养法、固体培养法和分子生物学方法进行重点分析，希望能给临床提供更多的帮助。     

免疫组化法检测解脲脲原体的研究

目的　建立检测解脲脲原体 (UU)的实验方法。方法　应用亲和素 生物素 酶复合物技术 (ABC法 )观察UU感染者 ,并与培养法和PCR法相比较。结果　ABC法对 75例临床标本的检出率为 5 2 % ,与培养法和PCR法比较 ,在统计学上差异无显著性 (P >0 .0 5 )。结论　ABC法可作为诊断UU感染的快速检测方法 ,在临床应用中有一定实用价值。     

128例原发性不育患者精液解脲脲原体的检测

采用解脲脲原体检测试剂对１２８例原发性不育患者的精液进行解脲脲原体病原学诊断，阳性率为３１．２５％（４０／１２８）。本文结果提示在山西省大同地区，解脲脲原体感染可能是引起男性原发性不育的重要原因之一。     

大肠埃希菌尿液分离株中检出gyrA基因新变异株

目的 调查大肠埃希菌尿液分离株中喹诺酮类耐药相关基因的存在与变化状况.方法 收集宁波市第一医院2008年10月到2009年3月患者尿液标本中分离的大肠埃希菌共28株,采用聚合酶链反应(PCR)及序列分析的方法分析1种染色体介导的喹诺酮类耐药相关基因(gyrA基因)和5种质粒介导的喹诺酮类耐药相关基因[qnrA、qnrB、qnrS、aac(6＇)-Ⅰb、qepA].结果 28株大肠埃希菌检测到1株aac(6＇)-Ⅰb-Cr基因阳性株(经测序比对证实),qnrA、qnrB、qnrS、qepA基因均未检出.gyrA基因83位密码子28株菌都有突变(100.0%),其突变方式为TCG-83→HTG,导致氨基酸从丝氨酸(S)-83→亮氨酸(L);87位密码子22株菌(78.6%)有突变,可分为两种突变方式:21株(75.0%)突变方式为GAC-87→AAC,导致氨基酸从天冬氨酸(D)-87→天冬酰胺(N);5号株gyrA基因(3.6%)为新亚型,其突变方式为GAC-87→TAC,导致氨基酸从天冬氨酸(D)-87→脯氨酸(Y),另6株菌87位密码子无突变.结论 本组大肠埃希菌gyrA基因突变率为100.0%,是喹诺酮类耐药的主要原因.其他耐药相关基因阳性率很低.     

Geographically dependent distribution of gyrA gene mutations at codons 83 and 87 in Salmonella Hadar, and a novel codon 81 Gly to His mutation in Salmonella Enteritidis

In all, 90 nalidixic acid-resistant clinical strains of Salmonella Hadar and Salmonella Enteritidis isolated in Norway but of predominantly foreign origin were subjected to sequencing of the gyrA, gyrB, parC and parE genes. All the isolates contained at least one mutation in gyrA codon 83 or codon 87. A highly significant correlation between mutations in gyrA codon 83 and strains originating from Southeast Asia was found in S. Hadar but not in S. Enteritidis. A novel gyrA codon 81 Gly to His mutation was discovered in one S. Enteritidis isolate. One amino-acid (aa) changing mutation was found outside the quinolone resistance-determining region (QRDR) of S. Hadar parC at codon 57, which has previously only been observed once in Salmonellae.     

Detection of Shigella gyrA mutations and correlations between gyrA mutations and quinolones resistance

Bacterial DNAgyrase is constituted by 2 A-sub-units and 2 B-subunits ,whichare encoded bygy-rAgene andgyrBgene ,respectively. Resistanceto quinolones in bacteriais mainly due tothe vari-ations of the bacterial DNAtopoisomerases ,typeⅡ(including DNAgyrase andtopoisomeraseⅣ) .This is because quinolones exert their antibacterialactivity by approachingthe bacterial DNAtopoiso-merases ,type Ⅱand disturbingtheir physiologicalfunctions. As variations occur in the bacterialDNA topoisomerase…     

Liquid-based urine cytology as a tool for detection of human papillomavirus, Mycoplasma spp., and Ureaplasma spp. in men

Liquid-based urine cytology (LB-URC) was evaluated for cytological diagnosis and detection of human papillomavirus (HPV), Mycoplasma, and Ureaplasma. Midstream urine samples were collected from 141 male patients with urethritis and 154 controls without urethritis, and sediment cells were preserved in liquid-based cytology solution. Urethral swabs from urethritis patients were tested for the presence of Neisseria gonorrhoeae and Chlamydia trachomatis. Papanicolaou tests were performed for cytological evaluation. HPV, Mycoplasma, and Ureaplasma genomes were determined by PCR-based methods, and localization of HPV DNA in urothelial cells was examined by in situ hybridization (ISH). The β-globin gene was positive in 97.9% of LB-URC samples from urethritis patients and in 97.4% of control samples, suggesting that high-quality cellular DNA was obtained from the LB-URC samples. HPV DNA was detected in 29 (21.0%) urethritis cases and in five (3.3%) controls (P < 0.05). HPV type 16 (HPV 16) was most commonly found in urethritis patients. Cytological evaluations could be performed for 92.1% of urethritis patients and 64.3% of controls. Morphological changes suggestive of HPV infection were seen in 20.7% of the HPV-positive samples, and ISH demonstrated the presence of HPV DNA in both squamous and urothelial cells in HPV-positive samples. Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum were detected in 14.5%, 10.9%, 6.5%, and 12.3% of urethritis patients, respectively. The prevalence rates of these microorganisms (except Ureaplasma parvum) were significantly higher in urethritis cases than controls (P < 0.05). LB-URC is applicable for detection of HPV, Mycoplasma, and Ureaplasma. HPV infection occurs in urothelial cells, especially in gonococcal urethritis.     

Isolation and detection of urease genes in Ureaplasma urealyticum.

Urease from ureaplasmas was purified by immunoaffinity chromatography, and the N-terminal amino acid sequence was determined for two of the three subunits. These sequences were used to design primers for a polymerase chain reaction (PCR) that amplified most of the gene coding for one of the subunits. By using a novel "PCR walking" technique, we synthesized almost the complete locus on two overlapping PCR products. We present here a partial nucleotide sequence of the urease locus from Ureaplasma urealyticum (serotype 8), which agrees with our N-terminal amino acid data but differs slightly from the sequence previously reported (A. Blanchard, Mol. Microbiol. 4:669-676, 1990). Also described are PCR primers, intended for diagnostic use, that amplify a sequence from all Ureaplasma strains tested but not from any other mycoplasmas or urease-positive bacteria.     

Development of a real-time fluorescence resonance energy transfer PCR to identify the main pathogenic Campylobacter spp.

A simple real-time fluorescence resonance energy transfer (FRET) PCR, targeting the gyrA gene outside the quinolone resistance-determining region, was developed to identify Campylobacter jejuni and Campylobacter coli. These species were distinguished easily, as the corresponding melting points showed a difference of 15 degrees C. A second assay using the same biprobe and PCR conditions, but different PCR primers, was also developed to identify the less frequently encountered Campylobacter fetus. These assays were applied to 807 Campylobacter isolates from clinical specimens. Compared to phenotypic identification tests, the FRET assay yielded the same results for all except three of the isolates. Analysis by standard PCR and 16S rDNA sequencing demonstrated that two of these isolates were hippurate-negative C. jejuni strains, resulting in an erroneous phenotypic identification, while the third was an isolate of C. coli that contained a gyrA gene typical of C. jejuni, resulting in misidentification by the FRET assay. The FRET assay identified more isolates than standard PCR, which failed to yield amplification products with c. 10% of isolates. It was concluded that the FRET assays were rapid, reliable, reproducible and relatively cost-efficient, as they require only one biprobe and can be performed directly on boiled isolates.     

Rapid detection of point mutations of the Neisseria gonorrhoeae gyrA gene associated with decreased susceptibilities to quinolones.

Mutations in the gyrA gene resulting in amino acid changes at Ser-91 and Asp-95 are significantly associated with decreased susceptibilities to quinolones in Neisseria gonorrhoeae. To detect these mutations, we developed a rapid and simple assay based on amplification of the region of the gyrA gene containing the mutation sites by PCR and digestion of the PCR product with a restriction enzyme. A naturally occurring HinfI restriction site was present in the region containing the Ser-91 codon, and an artificial HinfI restriction site was created in the region containing the Asp-95 codon by the method of primer-specified restriction site modification. The mutations generating alterations at Ser-91 and Asp-95 were detected as restriction fragment length polymorphisms of the PCR products digested with HinfI. Fifty-five clinical strains of N. gonorrhoeae were examined for mutations in the gyrA gene by this method. Mutations at Ser-91 and/or Asp-95 were detected in all the 31 strains in which the mutations had been confirmed by DNA sequencing. Our method allows simultaneous testing of a large number of strains and provides results within 8 h. This rapid and simple assay could be a useful screening device for genetic alterations associated with decreased susceptibilities to quinolones in N. gonorrhoeae and could facilitate epidemiological studies on clinical isolates of N. gonorrhoeae with decreased susceptibilities to quinolones.     

Ureaplasma spp. in male infertility and its relationship with semen quality and seminal plasma components

Objective

We investigated the prevalence of Ureaplasma spp. in semen samples of infertile men in Shanghai, China and evaluated the correlation between the sperm parameters (seminal volume, sperm concentration, progressive motility and non-progressive) and the secretary function in these infectious populations.

Ureaplasma urealyticum biovar specificity and diversity are encoded in multiple-banded antigen gene.

Ureaplasma urealyticum is a commensal organism of the lower genital tract of females, but in a subpopulation of individuals, it can invade the upper genital tract. It is a significant cause of chorioamnionitis and neonatal morbidity and mortality. There are 14 recognized serovars of U. urealyticum; these can be divided into two distinct clusters or biovars. Biovar 1 is composed of serovars 1, 3, 6, and 14, Biovar 2 is composed of serovars 2, 4, 5, 7, 8, 9, 10, 11, 12, and 13. We previously identified a surface antigen, the multiple-banded (MB) antigen, which contains both serovar-specific and cross-reactive epitopes. Genotypic characterization of the C-terminal region of the MB antigen of serovar 3 indicates that serovar specificity and MB antigen size variation reside in that domain. In the present study, we used PCR analysis with primers derived from the serovar 3 MB antigen gene DNA sequence to determine if the MB antigen gene was present in the remaining 13 reference serovars as well as in invasive clinical isolates. The results indicated that not only was the MB antigen gene present in all serovars but that the genes' 5' regions were markers of biovar specificity and diversity. Further analysis of this region should reveal the phylogenetic relationship among serovars of U. urealyticum and, possibly, their invasive potential.     

Rapid detection of clindamycin resistance in Bacteroides spp.

High-level resistance to clindamycin can be accurately detected by the Wadsworth disk identification test. Of the 98 isolates of the Bacteroides fragilis group that were tested, 90 were inhibited by the 60-micrograms erythromycin disk and had clindamycin minimal inhibitory concentrations of less than or equal to 3.2 micrograms/ml. Of the remaining eight isolates, all were resistant to the erythromycin disk and had clindamycin minimal inhibitory concentrations of greater than or equal to 100 micrograms/ml.