某养殖场羔羊突然死亡,为了查明死亡原因,采集病死羔羊病变肺脏等进行细菌的分离培养,分离到1株革兰阳性、短链状排列的球菌,在血琼脂培养基上呈β溶血。通过对该菌的16S r RNA进行扩增和序列分析,其与屎肠球菌的同源性高达99%,应用通...某养殖场羔羊突然死亡,为了查明死亡原因,采集病死羔羊病变肺脏等进行细菌的分离培养,分离到1株革兰阳性、短链状排列的球菌,在血琼脂培养基上呈β溶血。通过对该菌的16S r RNA进行扩增和序列分析,其与屎肠球菌的同源性高达99%,应用通用引物对16S^23S rRNA的序列进行PCR扩增产生2个条带,分别为522 bp和619 bp。用Vitek 2全自动微生物分析仪鉴定为屎肠球菌,可能性为98%。该菌对万古霉素和米诺环素敏感,对青霉素、庆大霉素等药物表现耐药。结果显示,分离到的细菌为屎肠球菌,且该菌具有多重耐药性,本实验为兽医临床肠球菌的分离和鉴定提供了参考。展开更多
The aim of this study was to develop and validate an oligonucleotide suspension array for rapid identification of 15 bacterial species responsible for bacteremia, particularly prevalent in Chinese hospitals. The multi...The aim of this study was to develop and validate an oligonucleotide suspension array for rapid identification of 15 bacterial species responsible for bacteremia, particularly prevalent in Chinese hospitals. The multiplexed array, based on the QIAGEN LiquiChip Workstation, included 15 oligonucleotide probes which were covalently bound to different bead sets. PCR amplicons of a variable region of the bacterial 23S rRNA genes were hybridized to the bead-bound probes. Thirty-eight strains belonging to 15 species were correctly identified on the basis of their corresponding species-specific hybridization profiles. The results show that the suspension array, in a single assay, can differentiate isolates over a wide range of strains and species, and suggest the potential utility of suspension array system to clinical laboratory diagnosis.展开更多
AIM： To characterize the types of mutations present in the 23S rRNA genes of Malaysian isolates of clarithromycin-resistant Helicobacter pylori （H pylorl~. METHODS： Clarithromycin susceptibility of H pylori isolate...AIM： To characterize the types of mutations present in the 23S rRNA genes of Malaysian isolates of clarithromycin-resistant Helicobacter pylori （H pylorl~. METHODS： Clarithromycin susceptibility of H pylori isolates was determined by E test. Analyses for point mutations in the domain V of 23S rRNA genes in clarithromycin-resistant and -sensitive strains were performed by sequence analysis of amplified polymerase chain reaction products. Restriction fragment length polymorphism was performed using Bsa I and MboI enzymes to detect restriction sites that correspond to the mutations in the clarithromycin- resistant strains. RESULTS： Of 187 isolates from 120 patients, four were resistant to clarithromycin, while 183 were sensitive. The MIC of the resistant strains ranged from 1.5 to 24 pg/mL. Two isolates had an A2142G mutation and another two had A2143G mutations. A T2182C mutation was detected in two out of four clarithromycin-resistant isolates and in 13 of 14 clarithromycin-sensitive isolates. Restriction enzyme analyses with Bsa I and Mbo I were able to detect the mutations. CONCLUSION： Clarithromycin resistance is an uncommon occurrence among Malaysian isolates of Hpylori strains and the mutations A2142G and A2143G detected were associated with low-level resistance.展开更多
Objectives: To investigate possible sources of Stenotrophomonas maltophilia(S. maltophilia) in the clinical environment.Methods: Different samples were collected from Amol City of Iran. Steps for the identification of...Objectives: To investigate possible sources of Stenotrophomonas maltophilia(S. maltophilia) in the clinical environment.Methods: Different samples were collected from Amol City of Iran. Steps for the identification of S. maltophilia included culturing, biochemical tests, polymerase chain reaction(PCR) of 16 S r RNA gene and 23 S r RNA gene. In addition, production of melanin pigment and patterns of motility of the bacteria, were also investigated.Results: In our study, 20 S. maltophilia strains were isolated from clinical sources,oxygen manometer apparatus of hospitals were 7/110(6.36%), blood was 1/777(0.13%),sputum was 4/40(4%), urine was 1/2 947(0.03%), tap water was 1/240(0.42%) and dental suction was 6/120(5%). The isolated bacteria showed production of melanin pigment with rates of strong, moderate, weak, and lack of pigment. Types of motilities were seen in isolates.Conclusions: The highest percentage of bacteria is isolated of oxygen manometer system and dental suction, yet has not been reported from oxygen manometer system. These bacteria have also been associated with patients who have respiratory problems, so it is essential for staffs of hospitals to draw attention to this source of bacteria.展开更多
目的观察幽门螺杆菌(HP)相关萎缩性胃炎对常用抗生素的耐药性,并就克拉霉素、左氧氟沙星耐药菌株的耐药基突变点位进行分析,为临床治疗Hp相关萎缩性胃炎敏感抗生素的选择提供参考。方法选取2019年6月至2020年6月在成都市双流区中医医院...目的观察幽门螺杆菌(HP)相关萎缩性胃炎对常用抗生素的耐药性,并就克拉霉素、左氧氟沙星耐药菌株的耐药基突变点位进行分析,为临床治疗Hp相关萎缩性胃炎敏感抗生素的选择提供参考。方法选取2019年6月至2020年6月在成都市双流区中医医院消化内科经胃镜检查确诊的Hp相关萎缩性胃炎患者210例,分离菌株,用体外药敏试验观察其对常用抗生素的耐药性,采用RT-PCR法检测Hp23s rRNA基因V区A2142G、A2143G突变和gyrA基因N87K(C261A)、D91N(G271A)突变。结果对分离的200株菌株进行7种常用抗生素的药敏试验,甲硝唑耐药140株(70.0%),左氧氟沙星耐药77株(38.5%),克拉霉素耐药31株(15.5%),阿莫西林耐药20株(10.0%),呋喃唑酮、庆大霉素和四环素耐药各2株(1.0%);N87K(C261A)突变耐药菌株15株,敏感组0株,差异具有统计学意义(P<0.05);D91N(G271A)突变耐药菌株13株,敏感组0株,差异具有统计学意义(P<0.05);A2142G突变耐药菌株17株,敏感菌株2株,差异具有统计学意义(P<0.05);A2143G突变耐药菌株13株,敏感菌株1株,差异具有统计学意义(P<0.05);T2182C突变耐药菌株15株,敏感菌株4株,差异无统计学意义(P>0.05)。结论本地区分离的HP耐药菌株中,对克拉霉素耐药率最高的以23S r RNA基因V区中的A2143G位点突变为主,对左氧氟沙星耐药率最高的以gyrA基因C261A、G271A位点突变为主。展开更多
To study the genetic characterization of four strains of Borrelia burgdorferi isolated in China. PCR technique was used to amplify the 5S-23S rRNA intergenic spacer DNA from the whole cellular DNA of isolated GXLD-4, ...To study the genetic characterization of four strains of Borrelia burgdorferi isolated in China. PCR technique was used to amplify the 5S-23S rRNA intergenic spacer DNA from the whole cellular DNA of isolated GXLD-4, 9, 18 and Chang 14, and then the amplified products were cloned into plasmid pGEM-T Easy and sequenced. It was found that the 5S-23S rRNA intergenic spacer DNA of the four isolates was 242?bp, revealing the nucleotide sequence identity of more than 99%. The four isolates had higher sequence identify with Borrelia valaisiana than with other genetic groups. These four isolates most likely belong to Borrelia valaisiana genomic group.展开更多
To establish a rapid identification method for common pathogenic bacteria on the basis of molecular biology and to construct a preliminary Polymerase Chain Reaction-Capillary Electrophoresis - Restriction Fragment Len...To establish a rapid identification method for common pathogenic bacteria on the basis of molecular biology and to construct a preliminary Polymerase Chain Reaction-Capillary Electrophoresis - Restriction Fragment Length Polymorphism (PCR-CE-RFLP) database of bacteria isolated from clinical specimens frequently, 183 strains collected from clinical samples belonging to 12 genera and 19 species whose biochemical characterizations corresponded to the typical ones were examined. The genomic DNAs were amplified by two pairs of fluorescence labeled primers aiming at 16S rRNA gene and 16S-23S rRNA spacer region gene respectively at the same time. PCR products were then digested by restriction endonuclease HaeⅢ incompletely before taking capillary electrophoresis. The results with the PCR-CE-RFLP patterns of 16S rRNA genes were just alike within some genera, but when it comes to 16S-23S rRNA spacer region genes, each bacterium showed a unique pattern, which can be distinguished from each other easily. It seems that PCR-CE-RFLP patterns of 16S rRNA gene could only be used to classify the bacteria into family level, whereas the data of 16S-23S rRNA spacer region gene could be utilized to identify the whole microorganisms as precisely as the species level. In spite of the data of the spacer region gene alone can be sufficiently to verify the whole bacteria, we insist that the 16S rRNA gene could be of some assistant in case that there should be lots of families of bacteria, in which some similar ones, with the same RFLP data of 16S-23S rRNA spacer region gene, may coexist. This study proves that the utility of PCR-CE-RFLP is a convenient, rapid method to identify pathogenic bacteria, and is also a quick diagnosis measure for application to clinical use.展开更多
Rapid differential identification of Mycobacterium species is essential for effective diagnosis and management of mycobacteriosis. The aim of this study was to develop a novel multiplex probe array based on the 16S-23...Rapid differential identification of Mycobacterium species is essential for effective diagnosis and management of mycobacteriosis. The aim of this study was to develop a novel multiplex probe array based on the 16S-23S rRNA gene internal transcribed spacer sequence for the genotyping of mycobacteria to the species level. A pair of primers and a set of genus- and species-specific probes were designed from the conserved and polymorphic regions of the 16S rRNA gene, internal transcribed spacer, and 23S rRNA gene sequences of mycobacteria. We used a novel multiplex probe array for identification of 266 clinical specimens obtained from patients with mycobaterial infection. The results showed that the overall specificity and sensitivity of our novel probe array were both 100% for the genus-specific probe and Mycobacterium tuberculosis complex- specific probe. There were 79.3 % （23/29） of nontuberculous mycobacteria which could be identified to the species level directly in the specimens from China. Some intraspecies heterogeneity in M. avium, M. intracellulare, M. chelonae and M. abscessus was observed. With the increase of sequences of internal transcribed spacer and numbers of whole microbial genomes, and further optimization of probes, the multiplex probe army will become a promising tool for the rapid and accurate identification of mycobacteria in ordinary clinical laboratories.展开更多
基金Project (Nos. 2003C13015 and 021103128) supported by Scienceand Technology Department of Zhejiang Province, China
文摘The aim of this study was to develop and validate an oligonucleotide suspension array for rapid identification of 15 bacterial species responsible for bacteremia, particularly prevalent in Chinese hospitals. The multiplexed array, based on the QIAGEN LiquiChip Workstation, included 15 oligonucleotide probes which were covalently bound to different bead sets. PCR amplicons of a variable region of the bacterial 23S rRNA genes were hybridized to the bead-bound probes. Thirty-eight strains belonging to 15 species were correctly identified on the basis of their corresponding species-specific hybridization profiles. The results show that the suspension array, in a single assay, can differentiate isolates over a wide range of strains and species, and suggest the potential utility of suspension array system to clinical laboratory diagnosis.
基金Supported by A grant from the Ministry of Science,Technology and Innovation,Malaysia
文摘AIM： To characterize the types of mutations present in the 23S rRNA genes of Malaysian isolates of clarithromycin-resistant Helicobacter pylori （H pylorl~. METHODS： Clarithromycin susceptibility of H pylori isolates was determined by E test. Analyses for point mutations in the domain V of 23S rRNA genes in clarithromycin-resistant and -sensitive strains were performed by sequence analysis of amplified polymerase chain reaction products. Restriction fragment length polymorphism was performed using Bsa I and MboI enzymes to detect restriction sites that correspond to the mutations in the clarithromycin- resistant strains. RESULTS： Of 187 isolates from 120 patients, four were resistant to clarithromycin, while 183 were sensitive. The MIC of the resistant strains ranged from 1.5 to 24 pg/mL. Two isolates had an A2142G mutation and another two had A2143G mutations. A T2182C mutation was detected in two out of four clarithromycin-resistant isolates and in 13 of 14 clarithromycin-sensitive isolates. Restriction enzyme analyses with Bsa I and Mbo I were able to detect the mutations. CONCLUSION： Clarithromycin resistance is an uncommon occurrence among Malaysian isolates of Hpylori strains and the mutations A2142G and A2143G detected were associated with low-level resistance.
基金funded by Islamic Azad University Karaj Branch (grant number: 1193)
文摘Objectives: To investigate possible sources of Stenotrophomonas maltophilia(S. maltophilia) in the clinical environment.Methods: Different samples were collected from Amol City of Iran. Steps for the identification of S. maltophilia included culturing, biochemical tests, polymerase chain reaction(PCR) of 16 S r RNA gene and 23 S r RNA gene. In addition, production of melanin pigment and patterns of motility of the bacteria, were also investigated.Results: In our study, 20 S. maltophilia strains were isolated from clinical sources,oxygen manometer apparatus of hospitals were 7/110(6.36%), blood was 1/777(0.13%),sputum was 4/40(4%), urine was 1/2 947(0.03%), tap water was 1/240(0.42%) and dental suction was 6/120(5%). The isolated bacteria showed production of melanin pigment with rates of strong, moderate, weak, and lack of pigment. Types of motilities were seen in isolates.Conclusions: The highest percentage of bacteria is isolated of oxygen manometer system and dental suction, yet has not been reported from oxygen manometer system. These bacteria have also been associated with patients who have respiratory problems, so it is essential for staffs of hospitals to draw attention to this source of bacteria.
文摘目的观察幽门螺杆菌(HP)相关萎缩性胃炎对常用抗生素的耐药性,并就克拉霉素、左氧氟沙星耐药菌株的耐药基突变点位进行分析,为临床治疗Hp相关萎缩性胃炎敏感抗生素的选择提供参考。方法选取2019年6月至2020年6月在成都市双流区中医医院消化内科经胃镜检查确诊的Hp相关萎缩性胃炎患者210例,分离菌株,用体外药敏试验观察其对常用抗生素的耐药性,采用RT-PCR法检测Hp23s rRNA基因V区A2142G、A2143G突变和gyrA基因N87K(C261A)、D91N(G271A)突变。结果对分离的200株菌株进行7种常用抗生素的药敏试验,甲硝唑耐药140株(70.0%),左氧氟沙星耐药77株(38.5%),克拉霉素耐药31株(15.5%),阿莫西林耐药20株(10.0%),呋喃唑酮、庆大霉素和四环素耐药各2株(1.0%);N87K(C261A)突变耐药菌株15株,敏感组0株,差异具有统计学意义(P<0.05);D91N(G271A)突变耐药菌株13株,敏感组0株,差异具有统计学意义(P<0.05);A2142G突变耐药菌株17株,敏感菌株2株,差异具有统计学意义(P<0.05);A2143G突变耐药菌株13株,敏感菌株1株,差异具有统计学意义(P<0.05);T2182C突变耐药菌株15株,敏感菌株4株,差异无统计学意义(P>0.05)。结论本地区分离的HP耐药菌株中,对克拉霉素耐药率最高的以23S r RNA基因V区中的A2143G位点突变为主,对左氧氟沙星耐药率最高的以gyrA基因C261A、G271A位点突变为主。
文摘To study the genetic characterization of four strains of Borrelia burgdorferi isolated in China. PCR technique was used to amplify the 5S-23S rRNA intergenic spacer DNA from the whole cellular DNA of isolated GXLD-4, 9, 18 and Chang 14, and then the amplified products were cloned into plasmid pGEM-T Easy and sequenced. It was found that the 5S-23S rRNA intergenic spacer DNA of the four isolates was 242?bp, revealing the nucleotide sequence identity of more than 99%. The four isolates had higher sequence identify with Borrelia valaisiana than with other genetic groups. These four isolates most likely belong to Borrelia valaisiana genomic group.
文摘To establish a rapid identification method for common pathogenic bacteria on the basis of molecular biology and to construct a preliminary Polymerase Chain Reaction-Capillary Electrophoresis - Restriction Fragment Length Polymorphism (PCR-CE-RFLP) database of bacteria isolated from clinical specimens frequently, 183 strains collected from clinical samples belonging to 12 genera and 19 species whose biochemical characterizations corresponded to the typical ones were examined. The genomic DNAs were amplified by two pairs of fluorescence labeled primers aiming at 16S rRNA gene and 16S-23S rRNA spacer region gene respectively at the same time. PCR products were then digested by restriction endonuclease HaeⅢ incompletely before taking capillary electrophoresis. The results with the PCR-CE-RFLP patterns of 16S rRNA genes were just alike within some genera, but when it comes to 16S-23S rRNA spacer region genes, each bacterium showed a unique pattern, which can be distinguished from each other easily. It seems that PCR-CE-RFLP patterns of 16S rRNA gene could only be used to classify the bacteria into family level, whereas the data of 16S-23S rRNA spacer region gene could be utilized to identify the whole microorganisms as precisely as the species level. In spite of the data of the spacer region gene alone can be sufficiently to verify the whole bacteria, we insist that the 16S rRNA gene could be of some assistant in case that there should be lots of families of bacteria, in which some similar ones, with the same RFLP data of 16S-23S rRNA spacer region gene, may coexist. This study proves that the utility of PCR-CE-RFLP is a convenient, rapid method to identify pathogenic bacteria, and is also a quick diagnosis measure for application to clinical use.
文摘Rapid differential identification of Mycobacterium species is essential for effective diagnosis and management of mycobacteriosis. The aim of this study was to develop a novel multiplex probe array based on the 16S-23S rRNA gene internal transcribed spacer sequence for the genotyping of mycobacteria to the species level. A pair of primers and a set of genus- and species-specific probes were designed from the conserved and polymorphic regions of the 16S rRNA gene, internal transcribed spacer, and 23S rRNA gene sequences of mycobacteria. We used a novel multiplex probe array for identification of 266 clinical specimens obtained from patients with mycobaterial infection. The results showed that the overall specificity and sensitivity of our novel probe array were both 100% for the genus-specific probe and Mycobacterium tuberculosis complex- specific probe. There were 79.3 % （23/29） of nontuberculous mycobacteria which could be identified to the species level directly in the specimens from China. Some intraspecies heterogeneity in M. avium, M. intracellulare, M. chelonae and M. abscessus was observed. With the increase of sequences of internal transcribed spacer and numbers of whole microbial genomes, and further optimization of probes, the multiplex probe army will become a promising tool for the rapid and accurate identification of mycobacteria in ordinary clinical laboratories.
