运用PCR 扩增技术检测解脲脲支原体生物群的临床应用

目的:探讨运用酶链聚合反应(PCR)技术检测泌尿生殖道解脲脲原体(Uu)的生物群,分析Uu生物群与临床症状的相关性。方法:以支原体16S rRNA保守区域基因为扩增靶序列设计引物,采用PCR方法扩增Uu 16S rRNA基因检测125例临床标本,并将检测结果与临床症状进行相关性分析。结果:PCR法检出Uu阳性率44.8%,其中35例Uu生物1群阳性,其中有16例有症状;27例Uu生物2群阳性,其中有18例有症状。Uu生物1群感染与症状的相关性无统计学差异(P>0.05),而Uu生物2群感染与症状相关性有统计学差异(P<0.05)。结论:PCR检测Uu 16S rRNA基因可用于Uu生物群的检测,Uu生物2群可能是非淋菌性尿道炎(NGU)的一个致病菌,而Uu生物1群与NGU无明显相关性。     

溶脲脲原体及其两个生物群与非淋菌性尿道炎相关性的研究

目的 阐明溶脲脲原体及其2个生物群与非淋菌性尿道炎的关系。方法 使用通用引物-PCR-毛细管电泳法对淋菌性尿道炎组,非淋菌性尿道炎组和对照组中的溶脲脲原体的2个生物群进行检测。结果 溶脲脲原体生物群2在非淋菌性尿道炎中的检出率高于对照组（P〈0．05）,溶脲脲原体生物群1在淋菌性尿道炎中的检出率低于对照组（P〈0．05）,而在非淋菌性尿道炎和对照组中,溶脲脲原体生物群1的检出率差异无显著性（P〉0．05）。结论 溶脲脲原体生物群2是和非淋菌性尿道炎有一定关系的,溶脲脲原体生物群2可能才是引起非淋尊性尿道炎的病原体之一,而生物群1不引起非淋菌性尿道炎,淋球菌的增殖有可能抑制尿道中的溶脲脲原体生物群1的生长。     

A method of genotyping clinical isolates of Ureaplasma urealyticum biovar Parvo

A new method for typing clinical isolates of U. urealyticum (Parvo biovar) is based on SSCP analysis of amplicons of mba gene 5' region and upstream region. The mba gene is coding for MB gene of U. urealyticum. This method allows genotyping of U. urealyticum isolates using vaginal and cervical swabs without culturing. Sixty-two clinical specimens from patients with a history of chronic cystitis, chronic pyelonephritis, chronic salpingo-oophoritis, erosion of the cervix uteri, and spontaneous abortions were tested for U. urealyticum. The bacterium was detected in 64% (40 specimens), 83% (33) of which belonged to Parvo biovar. Parvo biovar isolates were analyzed and genotyped as follows: first genotype 52%, second genotype 33%, and third genotype 16%. Further sequencing of the first and second genotype amplicons showed that the first genotype belonged to serotype 3 and second genotype to serotype 6.     

男性泌尿道来源的脲原体对氟喹诺酮类药物耐药性研究*

目的:运用酶链聚合反应(PCR)技术分析和比较不同生物群的脲原体对氟喹诺酮类药物的耐药情况。方法:以脲原体16Sr RNA保守区域基因为扩增靶序列检测脲原体的不同生物群,采用PCR方法扩增拓扑异构酶gyr A和parC基因并进行测序,分析基因突变与耐药的关系。结果:脲原体生物一群对左旋氧氟沙星的耐药性高于生物二群,二者差异有统计学意义(t=2.071,P=0.044)。gyr A基因主要为112号编码蛋白D112E的变异,parC基因主要为编码蛋白S83L的变异,即83号位丝氨酸(TCA)到亮氨酸(TTA)的变异的变异。与未突变株相比,拓扑异构酶基因突变株对环丙沙星MIC存在统计学差异(P0.001)。结论:不同生物群的脲原体对部分氟喹诺酮类耐药存在差异,拓扑异构酶基因突变与脲原体对喹诺酮类耐药存在相关性。     

反相斑点杂交法对解脲脲原体分型的研究

目的研究以聚合酶链反应为基础的快速检测与鉴定解脲脲原体基因型的方法。方法选择2003年11月至2005年11月在中山大学附属第二医院门诊就诊的有外阴阴道炎症状和体征的患者601例,设为病例组,同期无自觉症状的正常体检人群306例,设为对照组,分别取宫颈分泌物待检测。将解脲脲原体不同基因型的特异探针固定在硝酸纤维素膜上,临床标本按常规方法提取解脲脲原体DNA,采用生物素标记的解脲脲原体特异通用引物PCR扩增DNA,然后分别与解脲脲原体不同基因型特异探针杂交、显色。结果病例组解脲脲原体阳性421例占70.0%,对照组解脲脲原体阳性126例占41.2%。病例组中单型别感染的U.parvum占65.4%,其中1型、3型、6型和14型分别占28.8%、43.3%、26.0%和1.9%,U.urealyticum占18.4%;对照组中单型别感染的U.parvum占79.3%,其中1型、3型、6型和14型分别占63.2%、21.1%、15.7%和0.0%,U.urealyticum占13.8%。18例阳性标本随机DNA测序鉴定,均为相应的解脲脲原体基因型。结论U.parvum群,尤其是其中的1、3、6型别是正常人群携带的可能性较大,U.urealyticum则有可能和1型起协同作用或独自导致疾病。用反相斑点杂交进行解脲脲原体基因分型,方法简单、实用,适用于临床。     

Biovar Diversity Is Reflected by Variations of Genes Encoding Urease of Ureaplasma urealyticum

Five oligonucleotide primers derived from the gene encoding urease of Ureaplasma urealyticum were designed to evaluate the relationship between the urease gene and biovar diversity of this organism. Five combinations of these primers were tested by PCR and the result revealed that there were variations in urease genes among different serovars of U. urealyticum. This result, in agreement with other PCRs based on other functionally unrelated (rRNA and MB antigen) genes, may reflect the phylogenetic relationship among organisms taxonomically classified as U. urealyticum.     

环介导等温扩增技术对解脲脲原体的临床检测

目的建立并优化环介导等温扩增(LAMP)技术对解脲脲原体(U.urealyticum)的检测,并应用于临床样本分析。方法针对U.urealyticum的urease基因设计LAMP引物;研究LAMP的最适温度、最佳检测时间及灵敏度和特异度;与传统PCR检测进行方法学比对。结果 LAMP技术检测U.urealyticum的最适温度和最佳时间分别是61℃和60 min,并且具有良好灵敏度和特异度,较普通PCR检测的灵敏度高出1 000倍。临床样本检测中,PCR和LAMP技术达到的灵敏度分别为25.00%和87.50%。两种方法的特异度均为100.00%。结论 LAMP与PCR相比在基层检测和大规模筛查方面有显著的优势和巨大的利用价值。     

光动力抗微生物化学疗法对解脲脲原体体外活性的影响

解脲脲原体是一种重要的病原微生物,近年来其耐药形势十分严峻,因此寻找一种全新的有效替代治疗方案尤为重要。本研究旨在探索光动力抗微生物化学疗法对解脲脲原体体外活性的影响。选取解脲脲原体两种生物群（Parvo生物群及T960生物群）代表菌株,包括标准株及临床株,与系列稀释的2.5~0.039 062 5 mmol/L光敏剂甲苯胺蓝孵育20 min或60 min,再以（633±10）nm红光照射,设置48、102、204和408 mJ/cm²共4组能量密度,48 h后判读结果。观察不同解脲脲原体与甲苯胺蓝孵育时间、甲苯胺蓝浓度、光照能量密度对光动力抗微生物化学疗法灭活解脲脲原体效果的影响,并观察两种生物群对光动力抗微生物化学疗法敏感性的差异。结果显示,光动力抗微生物化学疗法在体外对解脲脲原体有明显灭活作用。在光照能量密度及解脲脲原体与甲苯胺蓝孵育时间固定的前提下,这种灭活作用随甲苯胺蓝浓度的增加而增强;单一633 nm红光光源在408 J/cm²及以下的能量密度对解脲脲原体的活性无明显影响。在甲苯胺蓝浓度及解脲脲原体与甲苯胺蓝孵育时间固定的条件下,光动力抗微生物化学疗法对解脲脲原体的灭活作用随光照能量密度（48~408 mJ/cm²）的增加而增强;随甲苯胺蓝孵育时间（30~60 min）延长,光动力抗微生物化学疗法对解脲脲原体的灭活作用有增强的趋势。结果提示,解脲脲原体两种生物群对光动力抗微生物化学疗法的敏感性相似。本研究证实,光动力抗微生物化学疗法在体外能有效灭活解脲脲原体,有望成为解脲脲原体感染的有效替代治疗方法。     

Rapid and specific identification of four Agrobacterium species and biovars using multiplex PCR

On the basis of 23S rRNA gene sequences, 1 universal forward and 4 taxon (species/biovar)-specific reverse primers were designed for multiplex PCR to aid in identification and differentiation of Agrobacterium rubi, Agrobacterium vitis and Agrobacterium biovars 1 and 2. In reactions with DNA of 119 bacterial strains belonging to: Agrobacterium, Allorhizobium, Mesorhizobium, Rhizobium, Sinorhizobium and Phyllobacterium, as well as phytopathogenic bacteria representing various genera, the primers developed for identification of A. vitis, A. rubi or Agrobacterium biovar 1 amplified only DNA of strains belonging to these taxa, producing fragments of the expected sizes: 478, 1006 and 184bp, respectively. However, in the case of the primer developed for identification of Agrobacterium biovar 2, the characteristic 1066bp PCR product was obtained not only with DNA of this biovar, but also with DNA of 3 atypical biovar 1 strains and some rhizobial strains. Differentiation between Agrobacterium biovar 2 and the other strains was possible using the restriction analysis of this product with endonuclease Alw26I. The method developed is an excellent tool for rapid classification of these 4 taxa of Agrobacterium.     

Comparison of polymerase chain reaction assay and Mycoplasma IST 2 test with culture for detection of infections caused by Ureaplasma urealyticum and Mycoplasma hominis

The polymerase chain reaction (PCR) technique and commercial Mycoplasma IST 2 test were compared with culture for the detection of U. urealyticum and M. hominis in 173 clinical samples obtained from patients without clinical symptoms from genito-urinary tract. The presence of U. urealyticum was diagnosed by culture in 24 samples, by PCR in 33 samples and by Mycoplasma IST 2 test in 39 samples. The presence of M. hominis was diagnosed in 26 samples only by Mycoplasma IST 2 test--culture and PCR were negative. The study showed the excellent sensitivity (100%) and good specificity (appropriately 94.0% and 90.0%) for U. urealyticum in PCR and Mycoplasma IST 2 test. The discrepancy of results obtained in Mycoplasma IST 2 test and culture as well as in PCR may suggest the over sensitivity of the commercial test for detection of M. hominis.     

Recognition of biovar C of Fusobacterium necrophorum (Flügge) Moore and Holdeman as Fusobacterium pseudonecrophorum sp. nov., nom. rev. (ex Prévot 1940)

The cellular morphology, colonial morphology, biochemical properties, DNA base compositions, and DNA-DNA homolgies of three biovars of Fusobacterium necrophorum were examined. Some differences were found among the three biovars in cellular morphology, colonial morphology, and biochemical properties. The guanine-plus-cytosine contents of DNAs from biovar C strains Fn521T (T = type strain), Fn522, and Fn520 were 30.4, 29.3, and 28.0 mol%, respectively, and the guanine-plus-cytosine contents of DNAs from strains VPI 2891 (biovar A) and VPI 6161 (biovar B) were 31.3 and 32.0 mol%, respectively. Labeled DNA from biovar C strain Fn521T exhibited 96 and 82% relatedness to DNAs from biovar C strains Fn522 and Fn520, respectively; however, it exhibited only about 10% relatedness to DNAs from strains of biovars A and B. Labeled DNAs from strains VPI 2891 and VPI 6161 exhibited more than 70% relatedness to each other, but about 6 to 20% relatedness to DNAs from biovar C strains. Therefore, Fusobacterium pseudonecrophorum sp. nov., nom. rev. (ex Prévot 1940) is proposed for Fusobacterium necrophorum biovar C. The type strain is strain Fn521 (= JCM 3722).     

Physiological studies on the utilization of oleic acid by Saccharomyces cerevisiae in relation to microbody development

Twenty one Chlamydia trachomatis reference strains and 40 clinical isolates belonging to the lymphogranuloma venerum (LGV) and trachoma biovars were genotyped by differential restriction mapping of the major-outer-membrane-protein gene (MOMP) obtained by the polymerase-chain reaction (PCR). AluI digestion of the PCR product distinguishes eight MOMP-genotypes corresponding to 8 serovars. Six additional enzymes (NlaIII, CfoI, EcoRI, HinfI, DdeI and FokI) further permit the discrimination of 10 MOMP-genotypes corresponding to the 10 remaining serovars of the species. AluI alone allows direct typing of 78% of the clinical isolates. AluI digestion patterns of mouse C. trachomatis biovar, a C. pneumoniae and two C. psittaci strains, studied for comparison, were clearly distinguishable from one another and from the C. trachomatis LGV and trachoma strains. These results indicate that MOMP genotyping by PCR is a valuable molecular tool for studying C. trachomatis epidemiology.     

Proposal of two subspecies of Fusobacterium necrophorum (Flügge) Moore and Holdeman: Fusobacterium necrophorum subsp. necrophorum subsp. nov., nom. rev. (ex Flügge 1886), and Fusobacterium necrophorum subsp. funduliforme subsp. nov., nom. rev. (ex Hallé 1898)

The biological and biochemical properties, DNA base compositions, and levels of DNA-DNA homology of two biovars of Fusobacterium necrophorum were examined. Some differences were found between the two biovars in biological and biochemical properties. The G + C contents of DNAs from biovar A strains VPI 2891T (T = type strain), NCTC 10576, N167, Fn47, and Fn43, were 32, 30, 29, 28, and 31 mol%, respectively. The G + C contents of DNAs from biovar B strains Fn524T, 606, Fn49, Fn45, and 1260 were 30, 31, 27, 31, and 30 mol%, respectively. Labeled DNA from biovar A strain VPI 2891T exhibited 100 to 80% relatedness to DNAs from biovar A strains and 59 to 51% relatedness to DNAs from biovar B strains. Labeled DNA from biovar B strain Fn524T exhibited 100 to 81% relatedness to DNAs from biovar B strains and 71 to 60% relatedness to DNAs from biovar A strains. Therefore, the names Fusobacterium necrophorum subsp. necrophorum subsp. nov., nom. rev. (ex Flügge 1886), and Fusobacterium necrophorum subsp. funduliforme subsp. nov., nom. rev. (ex Hallé 1898), are proposed for Fusobacterium necrophorum biovars A and B, respectively. The type strain of F. necrophorum subsp. necrophorum is strain VPI 2891 (= JCM 3718 = ATCC 25286), and the type strain of F. necrophorum subsp. funduliforme is strain Fn524 (= JCM 3724).     

Molecular characterization of beta-lactamase genes blaA and blaB of Yersinia enterocolitica biovar 1A

The beta-lactamase genes blaA and blaB were detected by PCR amplification in strains of Yersinia enterocolitica biovar 1A isolated from India, Germany, France and the USA. Both genes were detected in all strains. Polymerase chain reaction-restriction fragment length polymorphism revealed genetic heterogeneity in blaA but not in blaB. Cluster analysis of blaA restriction profiles grouped the strains into three groups. The blaA gene of Y. enterocolitica biovar 1A showed a high degree of sequence homology to that of Y. enterocolitica 8081 (biovar 1B) and Y. enterocolitica Y-56 (biovar 4), whereas homology was low with class A beta-lactamase genes of other members of the family Enterobacteriaceae. The pI 8.7 of enzyme Bla-A of Y. enterocolitica biovar 1A was similar to that of biovars 2, 3 and 4. The enzyme Bla-B focused at 6.8 and 7.1, indicating that biovar 1A strains produced a 'B-like' enzyme. This is the first study to have investigated the genetic heterogeneity of the beta-lactamase genes of Y. enterocolitica.     

Species of Pseudomonas obtained at 7°C and 30°C during aerobic storage of lamb carcasses

A total of 268 strains of Pseudomonas isolated during storage life of lamb carcasses was identified to species level. One-hundred and thirteen strains obtained at 30°C were Ps.fragi (51), Ps. lundensis (17), Ps. fluorescens biovars I (10), III (9) and VI (1), Ps. putida biovar A (8 strains) and unidentified (17 strains). Species and biovars isolated at 7°C (155) were Ps. fragi (101), Ps. lundensis (32), Ps. fluorescens biovar I (6), Ps. putida biovar A (8) and unidentified (8). Numerical analysis (82% S _SM, UPGMA) of 'psychrotrophic' and 'mesophilic' strains resulted in the formation of nine and eight clusters respectively. The dendrograms obtained exhibited similar structures. Most of the strains of Ps. lundensis and Ps. fragi clustered together. Strains of this latter species also joined the type strain of Ps. testosteroni and appeared included with phenons containing the Ps. putida strains. There were clusters made up exclusively of strains assigned to one biovar or group ( Ps. fluorescens biovars I and II and unidentified). A high level of similarity was observed between clusters of Ps. fluorescens biovar I and those containing the Ps. fragi-Ps. lundensis complex (>74% S _SM) and Ps. lundensis (>80%). The recovery of pseudomonads seemed to be affected by the sampling day.     

Distribution of virulence-associated genes in Yersinia enterocolitica biovar 1A correlates with clonal groups and not the source of isolation

Yersinia enterocolitica, an important food- and water-borne enteric pathogen, is represented by six biovars viz. 1A, 1B and 2-5. Some biovar 1A strains, despite lacking virulence plasmid (pYV) and chromosomal virulence genes, have been reported to cause symptoms similar to that produced by isolates belonging to known pathogenic biovars. Virulence-associated genes viz. ail, virF, inv, myfA, ystA, ystB, ystC, tccC, hreP, fepA, fepD, fes, ymoA and sat were studied in 81 clinical and nonclinical strains of Y. enterocolitica biovar 1A by PCR amplification. All strains lacked ail, virF, ystA and ystC genes. The distribution of other genes with respect to clonal groups revealed that four genes viz. ystB, hreP, myfA and sat were associated exclusively with strains belonging to clonal group A. The clonal groups A and B were differentiated previously based on rep (REP-/ERIC) - PCR genomic fingerprinting. The distribution of virulence-associated genes, however, did not differ significantly between clinical and nonclinical strains. In strains of Y. enterocolitica biovar 1A, clonal groups seem to reflect virulence potential better than the source (clinical vs. nonclinical) of isolation.     

Development of real-time PCR for the differential detection and quantification of Ureaplasma urealyticum and Ureaplasma parvum

Ureaplasma parvum and Ureaplasma urealyticum are recently recognized species of the genus Ureaplasma. In humans, Ureaplasma spp. can be found on mucosal surfaces, primarily in the respiratory and urogenital tracts. They have been implicated in various human diseases such as nongonococcal urethritis, intrauterine infections in association with adverse pregnancy outcome and fetal morbidity, and pneumonitis in immunocompromised hosts. We have developed two quantitative real-time PCR assays to differentially detect U. parvum and U. urealyticum. Based upon the sequence information of the urease gene (ureB), we designed two TaqMan primer and probe combinations specific for U. parvum and U. urealyticum, respectively. The assays did not react with nucleic acid preparations from 16 bacterial species commonly encountered in relevant clinical specimens, including seven urease-producing species. Each assay had a detection limit of approximately five copies per reaction of the respective gene target. The results suggest that these assays are both sensitive and specific for U. parvum and U. urealyticum. Further investigation of both assays using clinical specimens is appropriate.     

Organization of Ureaplasma urealyticum urease gene cluster and expression in a suppressor strain of Escherichia coli.

Ureaplasma urealyticum is a pathogenic ureolytic mollicute which colonizes the urogenital tracts of humans. A genetic polymorphism between the two biotypes of U. urealyticum at the level of the urease genes was found. The urease gene cluster from a biotype 1 representative of U. urealyticum (serotype I) was cloned and sequenced. Seven genes were found, with ureA, ureB, and ureC encoding the structural subunits and ureE, ureF, ureG, and a truncated ureI) gene encoding accessory proteins. Urease expression was not obtained when the plasmid containing these genes was incorporated into an opal suppressor strain of Escherichia coli, although this enzymatic activity was found in the same E. coli strain transformed with pC6b, a plasmid with previously cloned urease genes from the U. urealyticum T960 strain of biotype 2 (serotype 8). Although there are 12 TGA triplets encoding tryptophan within urease genes, the level of expression obtained was comparable to the levels reported for other bacterial genes expressed in E. coli. Nested deletion experiments allowed us to demonstrate that ureD is necessary for urease activity whereas another open reading frame located downstream is not. The promoter for ureA and possibly other urease genes was identified for both serotypes.     

Comparison of various PCR methods for the detection of Chlamydia trachomatis, Ureaplasma urealyticum, and Mycoplasma hominis in women with impaired reproductive function

The comparative evaluation of the PCR test "Polimik" (Research and Production Firm "Litekh", Moscow) and the PCR test of the Novosibirsk Institute of Bioorganic Chemistry (NIBC) was carried out. The results obtained with the use of the PCR test "Polimik" and the PCR test of the NIBC of the detection of C. trachomatis and M. hominis coincided in 97.8% and 97.4% of cases. For U. urealyticum, the coincidence of the results of both PCR tests was 81.2%. Among women who visited gynecologists for reproductive function disturbances, the use of the PCR tests made it possible to detect C. trachomatis in 19 (5.5%) out of 343 cases, U. urealyticum in 96 (39.0%) out of 246 cases and M. hominis in 25 (16.9%) out of 148 cases. The results of the investigation revealed that the occurrence of C. trachomatis infection in Novosibirsk was comparable with that in other regions of the world among the low-risk groups of the population. The detection frequency of M. hominis and U. urealyticum with the use of the PCR tests showed that the occurrence of infections caused by these causative agents coincided with the data obtained in other countries.