الملخص
Abstract Livestock is a fundamental part of the agriculture industry in Pakistan and contributes more than 11.53% to GDP. Among livestock species, the buffaloes are regarded as the black gold of Pakistan. Being the highest milk producers globally, Nili-Ravi buffaloes are the most famous ones. Buffaloes are affected by many endemic diseases, and "Hemorrhagic septicemia" (HS) is one of them. This study was designed to ascertain the effects of experimental exposure ofP. multocida B:2 (oral) and its immunogens, i.e., LPS (oral and intravenous) and OMP (oral and subcutaneous) on reproductive hormonal profiles in Nili-Ravi buffaloes. Repeated serum samples were collected from the jugular vein of experimental animals for 21 days (0, 02, 04, 08, 12, 16, 20, 24, 36, 48, 72, 120, 168, 216, 264, 360, 456 and 504 hours). Hormonal assays to determine the serum concentrations of Gonadotropin-releasing hormone (GnRH), Follicle-stimulating hormone (FSH), Luteinizing hormone (LH), Estrogen (E2) and progesterone (P4) were performed using (MyBioSource) commercial Elisa kits. The hormonal profile of all treatment groups of the buffalo heifers exhibited significant (P<0.05) variations as compared to the control group (G-1). These results indicate suppression in Nili-Ravi buffaloes' reproductive hormonal profile on exposure to P. multocida B:2 and its immunogens. This influence warrants that exposure to H.S may be a possible reason for delayed puberty and poor reproduction performance in Nili-Ravi buffaloes.
Resumo A pecuária é uma parte fundamental da indústria agrícola no Paquistão e contribui com 11,53% do PIB nacional. Entre as espécies de gado, os búfalos são considerados o ouro negro do Paquistão. Sendo os maiores produtores de leite em todo o mundo, os búfalos Nili-Ravi são os mais famosos. Os búfalos são afetados por muitas doenças endêmicas, entre as quais a "septicemia hemorrágica" (SH). Este estudo busca verificar os efeitos da exposição experimental de P. multocida B:2 (oral) e seus imunógenos, ou seja, LPS (oral e intravenoso) e OMP (oral e subcutâneo), nos perfis hormonais reprodutivos em búfalos Nili-Ravi. Amostras de soro repetidas foram coletadas da veia jugular de animais experimentais por 21 dias (0, 2, 4, 8, 12, 16, 20, 24, 36, 48, 72, 120, 168, 216, 264, 360, 456 e 504 horas). Os ensaios hormonais para determinar as concentrações séricas do hormônio liberador de gonadotrofina (GnRH), hormônio foliculoestimulante (FSH), hormônio luteinizante (LH), estrogênio (E2) e progesterona (P4) foram realizados usando kits comerciais Elisa (MyBioSource). O perfil hormonal de todos os grupos de tratamento das novilhas bubalinas apresentou variações significativas (P < 0,05) em relação ao grupo controle (G-1). Esses resultados indicam supressão no perfil hormonal reprodutivo de búfalos Nili-Ravi na exposição a P. multocida B:2 e seus imunógenos. Essa influência garante que a exposição à SH possa ser uma possível razão para o atraso da puberdade e o baixo desempenho reprodutivo em búfalos Nili-Ravi.
الموضوعات
Animals , Female , Pasteurella Infections/veterinary , Reproduction , Gonadal Steroid Hormones/blood , Buffaloes , Progesterone , Cattle , Lipopolysaccharides , Gonadotropin-Releasing Hormone , Pasteurella multocidaالملخص
Abstract Livestock is a fundamental part of the agriculture industry in Pakistan and contributes more than 11.53% to GDP. Among livestock species, the buffaloes are regarded as the black gold of Pakistan. Being the highest milk producers globally, Nili-Ravi buffaloes are the most famous ones. Buffaloes are affected by many endemic diseases, and "Hemorrhagic septicemia" (HS) is one of them. This study was designed to ascertain the effects of experimental exposure ofP. multocida B:2 (oral) and its immunogens, i.e., LPS (oral and intravenous) and OMP (oral and subcutaneous) on reproductive hormonal profiles in Nili-Ravi buffaloes. Repeated serum samples were collected from the jugular vein of experimental animals for 21 days (0, 02, 04, 08, 12, 16, 20, 24, 36, 48, 72, 120, 168, 216, 264, 360, 456 and 504 hours). Hormonal assays to determine the serum concentrations of Gonadotropin-releasing hormone (GnRH), Follicle-stimulating hormone (FSH), Luteinizing hormone (LH), Estrogen (E2) and progesterone (P4) were performed using (MyBioSource) commercial Elisa kits. The hormonal profile of all treatment groups of the buffalo heifers exhibited significant (P 0.05) variations as compared to the control group (G-1). These results indicate suppression in Nili-Ravi buffaloes' reproductive hormonal profile on exposure to P. multocida B:2 and its immunogens. This influence warrants that exposure to H.S may be a possible reason for delayed puberty and poor reproduction performance in Nili-Ravi buffaloes.
Resumo A pecuária é uma parte fundamental da indústria agrícola no Paquistão e contribui com 11,53% do PIB nacional. Entre as espécies de gado, os búfalos são considerados o ouro negro do Paquistão. Sendo os maiores produtores de leite em todo o mundo, os búfalos Nili-Ravi são os mais famosos. Os búfalos são afetados por muitas doenças endêmicas, entre as quais a septicemia hemorrágica (SH). Este estudo busca verificar os efeitos da exposição experimental de P. multocida B:2 (oral) e seus imunógenos, ou seja, LPS (oral e intravenoso) e OMP (oral e subcutâneo), nos perfis hormonais reprodutivos em búfalos Nili-Ravi. Amostras de soro repetidas foram coletadas da veia jugular de animais experimentais por 21 dias (0, 2, 4, 8, 12, 16, 20, 24, 36, 48, 72, 120, 168, 216, 264, 360, 456 e 504 horas). Os ensaios hormonais para determinar as concentrações séricas do hormônio liberador de gonadotrofina (GnRH), hormônio foliculoestimulante (FSH), hormônio luteinizante (LH), estrogênio (E2) e progesterona (P4) foram realizados usando kits comerciais Elisa (MyBioSource). O perfil hormonal de todos os grupos de tratamento das novilhas bubalinas apresentou variações significativas (P 0,05) em relação ao grupo controle (G-1). Esses resultados indicam supressão no perfil hormonal reprodutivo de búfalos Nili-Ravi na exposição a P. multocida B:2 e seus imunógenos. Essa influência garante que a exposição à SH possa ser uma possível razão para o atraso da puberdade e o baixo desempenho reprodutivo em búfalos Nili-Ravi.
الملخص
Background & objectives: Limited data are available on the typing of Chlamydia trachomatis in India. Serovars D to K of C. trachomatis are chiefly responsible for urogenital infections. Thus, this study was conducted to determine the distribution of C. trachomatis serovars in patients with urogenital infections and to characterize omp A gene of the detected C. trachomatis isolates by sequence analysis. Presence of other co-infections was also evaluated. Methods: Endocervical swabs were collected from 324 women and urethral swabs/urine were collected from 193 men attending the sexually transmitted diseases outpatient clinic. The samples were screened for C. trachomatis by cryptic plasmid PCR and omp A gene PCR. Genotyping was performed by PCR-restriction fragment length polymorphism (RFLP) and sequencing of the omp A gene. Samples were screened for genital mycoplasmas, Neisseria gonorrhoeae, Treponema pallidum and human immunodeficiency virus (HIV). Results: C. trachomatis was found in 15.0 per cent men and 10.8 per cent women. Serovar D was the most prevalent followed by serovars E, F, I and G. Twenty two C. trachomatis isolates were selected for omp A gene sequencing. No mixed infection was found. Variability in omp A sequences was seen in 31.8 per cent cases. Both PCR-RFLP and omp A gene sequencing showed concordant results. The presence of Ureaplasma spp. and Mycoplasma hominis was observed in 18.7 and 9.5 per cent patients, respectively. Co-infection of C. trachomatis was significantly associated with Ureaplasma urealyticum and HIV. Interpretation & conclusions: The high occurence of C. trachomatis infections warrants its screening in addition to other sexually transmitted infections namely U. urealyticum and HIV. Genotyping of the omp A gene may provide additional information for vaccine development.
الملخص
Objective To establish a real-time quantitative PCR (RT-PCR) preliminary screening method for rapid detection of Brucella.Methods Based on the nucleotide sequence encoding Brucella's outer membrane proteins omp10 and omp31,four primers and probes of omp10,omp10-1,omp31 and omp31-1 were designed.The primers and probes were used to detect 19 strains of 6 categories of Brucella DNA of known organisms,10 strains of Brucella DNA that were isolated from Yuxi of Yunnan.And 224 negative Brucella DNA,including 35 strains of Bartonella DNA,103 strains of the Lord Komori enterocolitis DNA (including 4 parts of O ∶ 3 and 9 parts of O ∶ 9) and 86 samples of hybrids bacteria DNA.Then the specificity of primers and probes were evaluated based on the test results.Results The DNA of 19 standard Brucella strains could be amplified by omp10 and omp10-1,and the peak time and amplification curve of omp10 is better than omp10-1,and the DNA of negative control strains could not be amplified by omp10.The DNA of 16 standard Brucella strains could be amplified by omp31-1.The DNA of standard Brucella strains could not be amplified by omp31.The average Ct values of 10 strains of Brucella DNA which were isolated from Yuxi of Yunnan that were detected by omp10,omp10-1 and omp31-1 respectively were 19.87,19.14 and 17.52.Conclusion Omp10 has strong specificity and only specific for Brucella,so it can be used for rapid detection of Brucella.
الملخص
Outer membrane proteins (OMPs) of Gram-negative bacteria constitute the first line of defense protecting cells against environmental stresses including chemical, biophysical, and biological attacks. Although the 43-kDa OMP (OMP43) is major porin protein among Bartonella henselae-derived OMPs, its function remains unreported. In this study, OMP43-deficient mutant B. henselae (Δomp43) was generated to investigate OMP43 function. Interestingly, Δomp43 exhibited weaker proliferative ability than that of wild-type (WT) B. henselae. To study the differences in proteomic expression between WT and Δomp43, two-dimensional gel electrophoresis-based proteomic analysis was performed. Based on Clusters of Orthologus Groups functional assignments, 12 proteins were associated with metabolism, 7 proteins associated with information storage and processing, and 3 proteins associated with cellular processing and signaling. By semi-quantitative reverse transcriptase polymerase chain reaction, increases in tldD, efp, ntrX, pdhA, purB, and ATPA mRNA expression and decreases in Rho and yfeA mRNA expression were confirmed in Δomp43. In conclusion, this is the first report showing that a loss of OMP43 expression in B. henselae leads to retarded proliferation. Furthermore, our proteomic data provide useful information for the further investigation of mechanisms related to the growth of B. henselae.
الموضوعات
Bartonella henselae , Bartonella , Gram-Negative Bacteria , Information Storage and Retrieval , Membrane Proteins , Membranes , Metabolism , Proteomics , Reverse Transcriptase Polymerase Chain Reaction , RNA, Messengerالملخص
Objective To screen B and T cell antigen epitopes on Acinetobacter baumannii outer membrane protein 33×103-36×103 (OMP33-36).Methods B and T cell epitopes on OMP33-36 of Acinetobacter baumannii were predicted by bioinformatics methods and synthesized.Recombinant expression plasmid pET-30a-OMP33-36 was cloned and used to express OMP33-36 in a prokaryotic expression system.The expressed OMP33-36 was used to immunize BALB/c mice after purification.Serum sample was collected from each mouse in immunization and negative control groups, and then analyzed by indirect ELISA with synthesized peptides to identify B cell epitopes.Splenocytes were separated from every mouse and then cultured with each of the synthesized peptides, respectively.Double sandwich ELISA was performed to detect IFN-γ secretion in the supernatant of cell cultures for screening of T cell epitopes.Results Candidates of B and T cell epitopes were constructed, which were PB1, PB2, PB3, PT1, PT2 and PT3.Results of the indirect ELISA showed that peptides PB1 and PB2 reacted with the serum samples collected from immunized mice and A450 values of the immunization group were significant higher than those of the negative control group.Compared with the negative control group, enhanced secretion of IFN-γ following peptide PT3 stimulation was observed in the immunization group as indicated by the double sandwich ELISA.Conclusion Two B cell epitopes PB1 and PB2, and one T cell epitope PT3 on the OMP33-36 of Acinetobacter baumannii were successfully constructed and screened out.
الملخص
PURPOSE: At present, there is no vaccine available for the prevention of human brucellosis. Brucella outer membrane protein 2b (Omp2b) is a 36 kD porin existed in common Brucella pathogens and it is considered as priority antigen for designing a new subunit vaccine. MATERIALS AND METHODS: In the current study, we aimed to predict and analyze the secondary and tertiary structures of the Brucella abortus Omp2b protein, and to predict T-cell and B-cell epitopes with the help of bioinformatics tools. Subsequently, cloning and expression of the short form of Omp2b (SOmp2b) was performed using pET28a expression vector and Escherichia coli BL21 host, respectively. The recombinant SOmp2b (rSOmp2b) was purified with Ni-NTA column. RESULTS: The recombinant protein was successfully expressed in E. coli host and purified under denaturation conditions. The yield of the purified rSOmp2b was estimated by Bradford method and found to be 220 microg/mL of the culture. CONCLUSION: Our results indicate that Omp2b protein has a potential to induce both B-cell- and T-cell-mediated immune responses and it can be evaluated as a new subunit vaccine candidate against brucellosis.
الموضوعات
Humans , Brucella abortus , Brucella , Brucellosis , Clone Cells , Cloning, Organism , Computational Biology , Computer Simulation , Epitopes, B-Lymphocyte , Escherichia coli , Membrane Proteins , T-Lymphocytesالملخص
We produced and identified the monoclonal antibodies against Brucella melitensis U‐lipoprotein OMP19 .A DNA fragment coding omp19 of Brucella melitensis was amplified by PCR ,and inserted into the vector of pET‐30a(+ ) ,the result‐ant recombinant plasmid ,which we designated as pET‐30a(+ )/omp19 .We then transformed the plasmid into BL21(DE3) competent cells for the expression of the OMP19 protein .After induction with different concentrations of IPTG ,the colleted cells were analyzed by SDS‐PAGE ,and then OMP19 monoclonal antibodies were prepared through hybridoma technology . These mAbs were tested to reactivity to rOMP19 and nature membrance proteins (NMP) of Brucella melitensis by Western blot and IEST .We successfully constructed an expression vector of pET 30a(+ )/omp19 .An IPTG‐induced expression of the OMP19 protein (19 kDa in molecular weight) was demonstrated by SDS‐PAGE .The fusion protein existed in the form of solu‐ble ,and the OMP19 protein of high purity could be obtained by Ni‐NTA .Western blot assay showed that the refolded protein could be recognized by the anti‐serum against Brucella melitensis .Twenty‐three mAbs to OMP19 was produced in which 91 .30% were IgG1 ,twenty‐two (95 .65% ) mAbs could recognize nature OMP19 protein ,and eighteen (78 .26% ) mAbs could recognize NMP ,four mAbs could react with Brucella melitensis .The protein maintained good immunogenicity and twenty‐three mAbs were obtained ,which we believe provides a good protein candidate for the immunological research .
الملخص
In this study ,we aimed to understand the sequence characteristics ,transmembrane structures ,line B cell epitopes present in the OMP18 from Campylobacter jejuni ,and provide candidate antigens for the antibody detection and vac-cine development .NCBI/Blast ,TMHMM Server V2 and DNA Star softwares were used for the OMP18 sequence analysis . Based on the ELISA ,the whole bacterial antibody IgG of Campylobacter jejuni was used for the identification of the predicted line B cell epitopes .The OMP18 gene was found conserved in different Campylobacter jejuni strains .The OMP18 was predic-ted to be located on the outer surface of the bacteria .And three line B cell epitopes were determined to be present in the OMP18 protein .As a conclusion ,the OMP18 protein was confirmed to be an important outer membrane protein ;three line B cell epitopes were identified in the OMP18 ,which could be further used for Campylobacter jejuni antibody detection and vaccine development .
الملخص
Background & objectives: Carbapenem-resistant Acinetobacter spp. have gained increasing significance as opportunistic pathogens in hospitalized patients. Carbapenem resistance is often associated with the loss and/or decrease in outer membrane proteins (OMP) and overexpression of multidrug efflux systems. However, carbapenem-hydrolysing β-lactamases of Ambler Class B (metallo-enzymes) and Ambler Class D (oxacillinases) have also been detected in Acinetobacter spp. In this study we have investigated the role of the iron regulated outer membrane protein (IROMPs) and the loss of a 29-kDa OMP in carbapenem resistance of Acinetobacter calcoaceticus. Methods: Carbapenem resistant clinical isolates (n=39) of Acinetobacter baumannii / calcoaceticus were used. Identification of Acinetobacter spp. at species level was done by amplified ribosomal DNA restriction analysis (ARDRA). MIC was evaluated using agar dilution method according to CLSI standards. Presence of outer membrane proteins were determined by SDS-PAGE. A representative strain of A. calcoaceticus, S26 with the loss of 29-kDa OMP was selected for further analysis as strain S26 had unique resistance mechanism, that is, the presence of IMP-4 metallo-β-lactamases. IROMPs were expressed under iron deficit conditions. Bands corresponding to IROMPs were excised from SDS-PAGE and used to immunize rabbits for the production of polyclonal antibodies. The antibodies raised against IROMPs were detected by an in-house ELISA and then used for bactericidal activity against carbapenem resistant A. baumannii / calcoaceticus. Results: All isolates were resistant to all antibiotics including imipenem and meropenem and had loss of a 29-kDa OMP. The polyclonal antibodies showed bactericidal effect against the organism tested and it specifically killed the bacteria grown in iron deficit medium. Interpretation & conclusions: In this study, a 29-kDa OMP has been identified to be the major outer membrane protein in A. baumannii / calcoaceticus and loss of this porin and overexpression of IROMPs have contributed to carbapenem resistance. Polyclonal antibodies raised against IROMPs may have a role in antimicrobial therapy in these isolates.
الموضوعات
Acinetobacter baumannii/drug effects , Acinetobacter baumannii/metabolism , Bacterial Outer Membrane Proteins/drug effects , Bacterial Outer Membrane Proteins/physiology , Carbapenems/pharmacology , Drug Resistance, Microbial , Electrophoresis, Polyacrylamide Gel , Humans , Iron/physiology , Malaysiaالملخص
P13-OMP (29.1). P13-OMP and OMP68 group challenged with P13 and P11 can be efectivly protected; P13-WCB group challenged with P13 and P11 can not be efectivly protected; the control group were died out. The P13-OMP and OMP68 of Bordetella bronchiseptica has good immunogenicity and protection, so the results of this study lay good theoretical foundation for OMP subunit vaccine.
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BACKGROUND: As the incidence of bovine brucellosis increases in Korea, the incidence of human brucellosis is also increasing since 2002. However, it is difficult to identify Brucella species by using the conventional methods. METHODS: Three strains of gram-negative coccobacilli were isolated from blood specimens of three patients with prolonged fever, which were not identified by using the conventional methods. After extracting total DNA from these isolates, PCR amplification of 16S rRNA and omp2 genes was performed. These sequences secured by PCR assay were compared with known sequences by using GenBank BLAST. RESULTS: DNA sequences were obtained from 3 isolates by using PCR amplification of 16S rRNA. These sequences had more than 99.9% similarities with Brucella species by using GenBank BLAST. In the second place, after comparing DNA sequences secured by PCR amplification of omp2a and omp2b by using GenBank BLAST, these isolates were confirmed as B. abortus. CONCLUSIONS: DNA sequence analysis is a rapid and accurate method for identification of uncommon microorganisms, such as Brucella species.
الموضوعات
Animals , Cattle , Humans , Base Sequence , Brucella abortus , Brucella , Brucellosis , Brucellosis, Bovine , Databases, Nucleic Acid , DNA , Fever , Incidence , Korea , Polymerase Chain Reaction , RNA, Ribosomal, 16S , Sequence Analysis, DNAالملخص
BACKGROUND: Despite increasing importance of Acinetobacter baumannii in nosocomial infections and rapid development of multi-antimicrobial resistance in this strain, the resistance mechanisms of beta-lactam antimicrobials in A. baumannii were not clearly defined. In order to observe the resistance mechanisms against beta-lactams and carbapenem, we characterized the production of beta-lactamases and outermembrane protein (OMP) profiles for the 44 clinical isolates of A. baumannii. METHODS: The MICs of antimicrobials were determined by agar dilution test. The secondary beta-lactamases were characterized by isoelectric focusing, polymerase chain reactions and nucleotide sequencing, and the production of chromosomal beta-lactamases was quantitated by spectrophotometric method. For two strains with an elevated MIC of carbapenem, outermembrane protein (OMP) profile was analyzed by ultracentrifugation of the sonicated bacteral cells and SDS-PAGE. RESULTS AND CONCLUSION: Twenty two or 4 of 44 strains produced TEM-1-like beta-lactamase or PER-1 extended-spectrum beta-lactamase, respectively. However, when we analyzed the MICs of several beta-lactams with the beta-lactamase production, the resistance level of beta-lactam was mainly determined by the production of chromosomal beta-lactamase, not by the secondary beta-lactamases in the clinical isolates of A. baumannii. In two strains with an elevated MIC of imipenem, a decrease or loss of about 35 kDa and 22 kDa proteins in OMP was observed, which suggested that the change of OMP played a role in carbapenem resistance.
الموضوعات
Humans , Acinetobacter/drug effects , Acinetobacter Infections/drug therapy , Lactams/pharmacology , Bacterial Outer Membrane Proteins/biosynthesis , Carbapenems/pharmacology , Cross Infection/drug therapy , Drug Resistance, Bacterial , beta-Lactamases/biosynthesisالملخص
BACKGROUND: Chlamydia trachomatis is currently classified into 15 serovars (A, B, Ba, C, D, E, F, G, H, I, J, K, L1, L2, L3) and a large number of serovariants. Typing of C. trachomatis serovars has generally been performed by the MOMP (major outer membrane protein) serotyping method, which requires a large panel of polyclonal and monoclonal antibodies. Recently the PCR was successfully applied to the genotyping of different C. trachomatis serovars by means of restriction fragment length polymorphism (RFLP) analysis and direct sequencing of amplified omp1 DNA. The objective of this study was to evaluate the serotyping of C. trachomatis by PCR-FLP and to determine the serovars of C. trachomatis urogenital isolates. METHODS: The 15 reference strains of C. trachomatis and 27 clinical isolates were analyzed by PCR-FLP. The C. trachomatis omp1 gene was amplified by PCR. For serotyping by RFLP analysis, the omp1 PCR products were digested with AluI and were electrophoresed through a 7% polyacrylamide gel with ethidium bromide staining. If necessary, the PCR products were analyzed with HinfI, a combination of EcoRI and DdeI, or CfoI. RESULTS: In serotyping of 15 reference strains of C. trachomatis, serovars A, B, Ba, E, F, G, K, and L2 were clearly identified after AluI digestion. However serovars C, H, I, J, L3 and serovars D, L1 were respectively identical patterns after AluI digestion. Serovars C and J and serovars D and L1 were further discriminated by a second step of enzyme digestion with HinfI. Serovar H, I, and L3 were distinguished by enzyme digestion with EcoRI and DdeI. In serotyping of C. trachomatis from 27 urogenital isolates, 25 isolates were clearly typed. Nine were typed as serovar E, 8 were typed as serovar D, 6 were typed as F, 2 were typed as serovar H. Two isloates showed unidentifiable RFLP pattern which was not in accordance with any of the existing C. trachomatis prototypes. CONCLUSIONS: PCR-FLP analysis is a rapid, simple, and powerful tool for differentiating serovars of C. trachomatis. Therefore, this approach is recommended for future epidemiological studies of C. trachomatis. And the serovar E, D, and F are the most prevalent types found in urogenital specimens, representing 92% of those investigated.
الموضوعات
Antibodies, Monoclonal , Chlamydia trachomatis , Chlamydia , Digestion , DNA , Ethidium , Membranes , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Serotypingالملخص
BACKGROUND: Chlamydia trachomatis is currently classified into 15 serovars (A, B, Ba, C, D, E, F, G, H, I, J, K, L1, L2, L3) and a large number of serovariants. Typing of C. trachomatis serovars has generally been performed by the MOMP (major outer membrane protein) serotyping method, which requires a large panel of polyclonal and monoclonal antibodies. Recently the PCR was successfully applied to the genotyping of different C. trachomatis serovars by means of restriction fragment length polymorphism (RFLP) analysis and direct sequencing of amplified omp1 DNA. The objective of this study was to evaluate the serotyping of C. trachomatis by PCR-FLP and to determine the serovars of C. trachomatis urogenital isolates. METHODS: The 15 reference strains of C. trachomatis and 27 clinical isolates were analyzed by PCR-FLP. The C. trachomatis omp1 gene was amplified by PCR. For serotyping by RFLP analysis, the omp1 PCR products were digested with AluI and were electrophoresed through a 7% polyacrylamide gel with ethidium bromide staining. If necessary, the PCR products were analyzed with HinfI, a combination of EcoRI and DdeI, or CfoI. RESULTS: In serotyping of 15 reference strains of C. trachomatis, serovars A, B, Ba, E, F, G, K, and L2 were clearly identified after AluI digestion. However serovars C, H, I, J, L3 and serovars D, L1 were respectively identical patterns after AluI digestion. Serovars C and J and serovars D and L1 were further discriminated by a second step of enzyme digestion with HinfI. Serovar H, I, and L3 were distinguished by enzyme digestion with EcoRI and DdeI. In serotyping of C. trachomatis from 27 urogenital isolates, 25 isolates were clearly typed. Nine were typed as serovar E, 8 were typed as serovar D, 6 were typed as F, 2 were typed as serovar H. Two isloates showed unidentifiable RFLP pattern which was not in accordance with any of the existing C. trachomatis prototypes. CONCLUSIONS: PCR-FLP analysis is a rapid, simple, and powerful tool for differentiating serovars of C. trachomatis. Therefore, this approach is recommended for future epidemiological studies of C. trachomatis. And the serovar E, D, and F are the most prevalent types found in urogenital specimens, representing 92% of those investigated.
الموضوعات
Antibodies, Monoclonal , Chlamydia trachomatis , Chlamydia , Digestion , DNA , Ethidium , Membranes , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Serotypingالملخص
BACKGROUND: Chlamydia pneumoniae has recently been established as an important cause of acute respiratory tract infections such as pneumonia and bronchitis in humans. The purpose of our study was to define the sequence of the C. pneumoniae omp1 gene. METHODS: The omp1 gene of C. pneumoniae was amplified by touchdown polymerase chain reaction (PCR) on sputum samples. The PCR product was cloned into pT7Blue T-Vector using the TA cloning technique. The nucleotide sequence of the cloned omp1 gene was determined with the Cy5TM AutoReaderTM Sequencing Kit. RESULTS: We designated the cloned PCR product as CpT-207. The sequence of CpT-207 DNA was 96%-100% identical to the omp1 gene of C. pneumoniae isolated from other countries. CONCLUSIONS: The sequence analysis of CpT-207 DNA was almost identical to the sequences of the omp1 gene of C. pneumoniae isolated from other countries. The CpT-207 can be used as a control or a probe for the molecular diagnosis of C. pneumoniae.