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1.
Lehmusvuori A Juntunen E Tapio AH Rantakokko-Jalava K Soukka T Lövgren T 《Journal of microbiological methods》2010,83(3):302-306
Chlamydia trachomatis infection is the most common bacterial sexually transmitted disease and a major public health problem worldwide. Fast and sensitive point-of-care diagnostics including non-invasive sample collection would be of value for the prevention of C. trachomatis transmission. The aim of this study was to develop a fast, reliable, non-invasive and easy-to-use homogenous PCR assay for the detection of C. trachomatis. Bacteria were concentrated from urine by a simple and fast centrifugation-based urine pretreatment method. Novel automated GenomEra technology was utilized for the rapid closed-tube PCR including time-resolved fluorometric detection of the target using lanthanide chelate labeled probes. We have developed a rapid C. trachomatis assay which provides qualitative results in 1 h with diagnostic sensitivity and specificity of 98.7% and 97.3%, respectively. The novel assay can be performed with minimal laboratory expertise and without sophisticated DNA-extraction devices and has performance comparable to current gold standard assays. 相似文献
2.
Chlamydia trachomatis is a widespread bacterium that causes trachoma and genital tract infections in humans. The fact that the growth of this pathogen
does not normally occur outside living cells poses a challenge in its diagnosis. The present study aimed to compare the efficacies
of different molecular and cultural methods in the detection of C. trachomatis in urine samples collected from patients with urinary tract infections. Examined detection methods involved the Gen-Probe
C. trachomatis (GP-CT) assay, direct antigen detection by enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR)
method. The efficacies of these methods were compared to that of the cell culture technique depending on sensitivity, specificity,
and accuracy. C. trachomatis was detected in 25 out of 50 (50%) of examined urine samples using the cell culture method. Compared with this standard technique,
the GP-CT assay was the most sensitive procedure, being able to detect the pathogen in all positive samples, followed by PCR
and ELISA, which showed 60% and 40% sensitivities, respectively. PCR and ELISA displayed the highest level of specificity
(100%) compared to the cell culture method with the GP-CT assay showing 40% specificity. The rate of accuracy was comparable
between the GP-CT, PCR, and ELISA methods ranging from 70–80% of the accuracy of the cell culture method. The above results
suggest that C. trachomatis is a frequent pathogen associated with upper and lower urinary tract infections. Both the GP-CT assay and PCR method can
be recommended as reliable detection methods for C. trachomatis, and the GP-CT can be used as a screening tool. 相似文献
3.
The sensitivity and specificity of the polymerase chain reaction (PCR) test kit, AMPLICOR Chlamydia trachomatis, were examined by the use of purified elementary bodies (EBs), cells having inclusions containing reticulate bodies alone and 20 clinical isolates. The numbers of EB and inclusion of C. trachomatis at the detection limit were determined to be approximately 2 to 4 EBs and one inclusion per assay, respectively. No reaction occurred for C. psittaci and C. pneumoniae. All clinical isolates were positively reacted in the PCR assay. 相似文献
4.
Human papillomaviruses and Chlamydia trachomatis are two of the most commonly found sexually transmitted infections in cervical Pap smears. They are often asymptomatic and if left untreated can progress to cause serious complications such as pre‐cancerous lesions and tubal factor infertility, respectively. The aim of this study was to develop a rapid multiplex PCR for the simultaneous detection of HPV and C. trachomatis in ThinPrep® liquid cytology samples. Two multiplexes were optimized. (A) For the detection of C. trachomatis using primers for the cryptic plasmid and for a chromosomal gene (Hsp60); (B) for the simultaneous detection of HPV and C. trachomatis using consensus primers for HPV and plasmid primers for C. trachomatis. Both multiplexes included a set of primers for a human housekeeping gene‐β‐globin. DNA from 34 ThinPrep® cervical samples was extracted using the QiAmp DNA Mini Kit (Qiagen Ltd, UK). All 34 samples were previously confirmed positive for C. trachomatis using another nucleic‐acid based test. Using multiplex A.for the detection of C. trachomatis, 33 of 34 samples were positive for C. trachomatis by either the plasmid or chromosomal gene primer set. All samples were positive for β‐globin. Ten of the 34 C. trachomatis positive samples were known positives for HPV. Using the combined HPV and C. trachomatis multiplex 10 of 10 were positive for both HPV and C. trachomatis. These simple multiplexes are cost‐effective, rapid and have potential for rapid screening of cervical ThinPrep samples simultaneously for both HPV and C. trachomatis. 相似文献
5.
Gharsallah H Frikha-Gargouri O Besbes F Sellami H Znazen A Hammami A 《Journal of applied microbiology》2012,113(4):846-855
Aim
To develop and evaluate an in‐house reverse hybridization technique for Chlamydia trachomatis genotype identification.Methods and Results
The evaluation of the developed and optimized reverse hybridization method on reference strains showed the specific detection of all genotypes. This technique showed its ability to type one inclusion‐forming unit of C. trachomatis genotype E and equivalent sensitivity to the Cobas TaqMan assay. It was also able to detect mixed infections in vitro. Application of the reverse hybridization method on 38 isolated C. trachomatis strains and their respective swabs allowed the detection of six urogenital genotypes D, E, F, G, H and K and one trachoma genotype B. Genotype E was the most prevalent, detected in 73% of the swab samples. Mixed infections were detected in 26% of swab cases.Conclusion
The reverse hybridization technique is simple and does not require specialized instruments. It is powerful in the diagnosis of mixed infections and is suitable for use in epidemiological studies.Significance and Impact of the Study
This technique allowed rapid C. trachomatis genotype identification. 相似文献6.
We have studied in vivothe phenotypes of 23S rRNA mutations G2582A, G2582U, G2583C, and U2584C, which are located at the A site of Escherichia coli50S ribosomal subunit. All mutant rRNAs incorporated into 50S ribosomal subunits. Upon sucrose gradient fractionation of cell lysates, 23S rRNAs mutated at G2582 to A and G2583 to C accumulated in the 50S and 70S fractions and were underrepresented in the polysome fraction. Induction of 23S rRNAs mutated at G2582 and G2583 lead to a drastic reduction in cell growth. In addition, mutations G2582A and G2583C reduced to one-third the total protein synthesis but not the RNA synthesis. Finally, we show that 23S rRNA mutations G2582A, G2582U, and G2583C cause a significant increase in peptidyl-tRNA drop-off from ribosomes, thereby reducing translational processivity. The results clearly show that tRNA–23S rRNA interaction has an essential role in maintaining the processivity of translation. 相似文献
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A novel approach to determine the tetracycline susceptibility of Chlamydia trachomatis directly from specimens without cell culture propagation and adaptation has been explored. Out of a total of 1290 genital specimens from a sexually transmitted disease clinic, 211 (16.4%) were positive for C. trachomatis. A tetracycline concentration of 0.032 g/ml completely inhibited the appearance of inclusions in all of the 211 positive specimens. Of the positive specimens, 120 (56.9%) and 18 (8.5%) respectively showed the presence of 1 to 9 and 100 or more inclusions per microtiter well in antibiotic free medium. Other antibiotics are being tested in the same manner.A part of this paper was presented at the Canadian Public Health Association, STD meeting in Ottawa, Canada, 28–29 October 1985 相似文献
9.
Chukwuemeka Louis Ajonuma Lam Kin Fok Sze Lok Ho Sheung Kay Paul Chan Pak H. Chow Ling Lai Tsang Yan Hau Connie Wong Jie Chen Shen Li Kenneth Dewi Rowlands Wa Yiu Chung Chang Hsiao Chan 《Cell biology international》2010,34(6):593-600
Chlamydia trachomatis is an obligate intracellular Gram‐negative pathogen affecting over 600 million people worldwide with 92 million new cases occurring globally each year. C. trachomatis enter the cells and replicate to infect different tissues/organs, giving rise to a spectrum of pathological conditions; however, the exact mechanism or receptor(s) for their entry is not well understood. Here we report that CFTR (cystic fibrosis transmembrane conductance regulator), an apical epithelial anion channel, is required for cellular entry and internalization of C. trachomatis. Human epithelial cell lines expressing functional CFTR internalized more C. trachomatis than the cells expressing mutant Δ508 CFTR. The in vitro cellular uptake of C. trachomatis can be blocked by CFTR inhibitors or antibody, and the in vivo cellular uptake of C. trachomatis in CFTR mutant (CFTR?/?) mice was significantly less compared with that in the wild‐type. Direct interaction between CFTR and C. trachomatis LPS (lipopolysaccharide) is demonstrated by their immune‐co‐localization and co‐immunoprecipitation. Despite an increase in CFTR expression observed upon C. trachomatis LPS challenge, a reduction in its ion channel activity is observed, consistent with the notion that CFTR functions as a receptor for cellular entry and internationization of C. trachomatis, with compromised ion‐channel function. These findings, for the first time, demonstrate that CFTR functions as a cell‐surface receptor for epithelial cell entry, and internalization of C. trachomatis and these findings may lead to the development of new treatment strategies to curtail the spread of chlamydial infections. 相似文献
10.
Xue Li Shaoya Zhang Qingfeng Liang Mei Wang Ailian Hu Xiuyuan Li Benshan Yang Mingxin Zhang Ningli Wang Xinxin Lu 《中国科学:生命科学英文版》2016,59(6):561-570
To study the molecular characteristics of Chlamydia trachomatis, the major outer membrane protein gene(omp A) of C. trachomatis from primary school students with trachoma residing in the Qinghai Tibetan area was sequenced and compared with the same serotype in Gen Bank. In Jianshetang Primary School and Galeng Central Primary School in the Galeng Tibetan Township of Qinghai Haidong Sala Autonomous County, scraped samples were collected from the upper tarsal conjunctiva and lower conjunctival sac of both eyes of 45 students with trachoma, stored at 4°C, and transported to Beijing Tongren Hospital by air within 24 h. The samples were screened for C. trachomatis by real-time PCR. The omp A gene from the C. trachomatis-positive samples was amplified by nested PCR. The serotype was confirmed by National Center for Biotechnology Information(NCBI) BLAST search and homology analysis. The entire omp A gene sequence was compared with the corresponding gene sequences of serotype B strains available in Gen Bank. Of the 45 students aged 6–13 years with trachoma, 26 C. trachomatis-positive students were identified by the initial real-time PCR screening(average age,(9.09±1.63) years; sex ratio, 1.0), accounting for 57.78%(26/45). The cycle threshold values for real-time PCR were 16.79–37.77. Half(13/26) of C. trachomatis-positive students had a bacterial copy number of 105. The compliance rate of the omp A gene sequences with the C. trachomatis serotype B strains in Gen Bank was up to 99%. Two novel genetic mutations were found when the omp A gene was compared with those of the 11 serotype B strains in Gen Bank. The two non-synonymous mutations were located at(i) position 271 in the second constant domain, an adenine(A) to guanine(G) substitution(ACT?GCT), changing the amino acid at position 91 from threonine to alanine(Thr?Ala) in all 26 strains; and(ii) position 887 in the fourth variable domain, a cytosine(C) to thymine(T) substitution(GCA?GTA), changing the amino acid at residue 296 from alanine to valine(Ala?Val) in four of the 26 strains. Six mutations were identified relative to ATCC VR-573. The strains could be divided into two gene clusters according to the mutation at nucleotide position 887: CQZ-1(China Qinghai Tibetan-1) and CQZ-2(China Qinghai Tibetan-2). We thus detected two novel serotype B mutant strains of C. trachomatis among study subjects with trachoma. 相似文献
11.
Brinkworth AJ Malcolm DS Pedrosa AT Roguska K Shahbazian S Graham JE Hayward RD Carabeo RA 《Molecular microbiology》2011,82(1):131-144
Bacterial type III secretion system (T3SS) chaperones pilot substrates to the export apparatus in a secretion‐competent state, and are consequently central to the translocation of effectors into target cells. Chlamydia trachomatis is a genetically intractable obligate intracellular pathogen that utilizes T3SS effectors to trigger its entry into mammalian cells. The only well‐characterized T3SS effector is TARP (translocated actin recruitment protein), but its chaperone is unknown. Here we exploited a known structural signature to screen for putative type III secretion chaperones encoded within the C. trachomatis genome. Using bacterial two‐hybrid, co‐precipitation, cross‐linking and size exclusion chromatography we show that Slc1 (SycE‐like chaperone 1; CT043) specifically interacts with a 200‐amino‐acid residue N‐terminal region of TARP (TARP1–200). Slc1 formed homodimers in vitro, as shown in cross‐linking and gel filtration experiments. Biochemical analysis of an isolated Slc1–TARP1–200 complex was consistent with a characteristic 2:1 chaperone–effector stoichiometry. Furthermore, Slc1 was co‐immunoprecipitated with TARP from C. trachomatis elementary bodies. Also, coexpression of Slc1 specifically enhanced host cell translocation of TARP by a heterologous Yersinia enterocolitica T3SS. Taken together, we propose Slc1 as a chaperone of the C. trachomatis T3SS effector TARP. 相似文献
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13.
Paolo Maltese Emanuele Canestrari Linda Palma Annamaria Ruzzo Francesco Corini Michele Menotta Francesca Andreoni Anna Latiano Vito Annese Mauro Magnani 《The Journal of steroid biochemistry and molecular biology》2009,113(3-5):269-274
Polymorphisms in the glucocorticoid receptor (GR) gene have been associated with altered sensitivity to glucocorticoids. We designed a high-resolution melting (HRM) assay to detect, simultaneously, the three most intriguing GR polymorphisms, selected on the bases of clinical relevance and frequencies in caucasian population as described in literature. HRM enables the detection of ER22/23EK and N363S genotypes but fails to discriminate homozygous mutant for the BclI polymorphism from wild-type samples, however a simple spike experiment leads to a clear discrimination between these genotypes. The analyses were performed on a cohort of 70 healthy Caucasian subjects. The method was validated by restriction fragment length polymorphisms; HRM results were found to be in 100% concordance with those observed with the restriction enzymes. We also employed this method on a population of 40 Crohn Disease patients; the analysis demonstrated a significantly higher frequency of the BclI polymorphism in patients than in healthy volunteers.This is, at now, the less expensive and time-and work-saving method to detect GR mutations, providing precision, fast screening and high throughput capabilities. 相似文献
14.
Mycoplasma bovis isolates from dairy calves in Japan have less susceptibility than a reference strain to all approved macrolides associated with a point mutation (G748A) combined with multiple species‐specific nucleotide alterations in 23S rRNA 下载免费PDF全文
Toyotaka Sato Hidetoshi Higuchi Shin‐ichi Yokota Yutaka Tamura 《Microbiology and immunology》2017,61(6):215-224
15.
Denise Wohlmeister Débora Renz Barreto Vianna Virgínia Etges Helfer Fabrícia Gimenes Marcia Edilaine Lopes Consolaro Regina Bones Barcellos Maria Lucia Rossetti Luciane Noal Calil Andréia Buffon Diogo André Pilger 《Memórias do Instituto Oswaldo Cruz》2016,111(2):106-113
The influence of different infectious agents and their association with human
papillomavirus (HPV) in cervical carcinogenesis have not been completely elucidated.
This study describes the association between cytological changes in cervical
epithelium and the detection of the most relevant aetiological agents of sexually
transmitted diseases. Samples collected from 169 patients were evaluated by
conventional cytology followed by molecular analysis to detect HPV DNA,
Chlamydia trachomatis, herpes simplex virus 1 and
2,Neisseria gonorrhoeae, Mycoplasma genitalium,
Trichomonas vaginalis, andTreponema pallidum,
besides genotyping for most common high-risk HPV. An association between cytological
lesions and different behavioural habits such as smoking and sedentariness was
observed. Intraepithelial lesions were also associated with HPV and C.
trachomatis detection. An association was also found between both simple
and multiple genotype infection and cytological changes. The investigation of HPV and
C. trachomatisproved its importance and may be considered in the
future for including in screening programs, since these factors are linked to the
early diagnosis of patients with precursor lesions of cervical cancer. 相似文献
16.
O. Frikha‐Gargouri R. Gdoura A. Znazen J. Gargouri A. Rebai A. Hammami 《Journal of applied microbiology》2009,107(6):1875-1882
Aim: To study the performance of the CT694 protein in relation to the microimmunofluorescence (MIF) and the pELISA tests for the serodiagnosis of Chlamydia trachomatis infections. Methods and Results: The CT694 protein was produced as recombinant protein and was used as antigen in ELISA test for the detection of C. trachomatis IgG antibodies. The performance of the developed ELISA test was compared to the MIF test at two cut‐off values of 16 and 64, and to the specific pELISA test using a panel of 342 sera. These sera were from children MIF C. trachomatis and Chlamydophila pneumoniae negative, patients MIF C. pneumoniae positive, patients MIF C. trachomatis positive, patients suspected to have chlamydial infections diagnosed by the Cobas Amplicor test, healthy blood donors and prostitutes. Our results indicate that the developed ELISA test has performed better compared with the MIF and the pELISA tests. The highest performance was obtained when comparing the developed ELISA test in relation to the pELISA, yielding an overall sensitivity and specificity of 85% and 87% respectively. Conclusions: The CT694 ELISA showed the best performance when compared to the species‐specific pELISA test and may be used for the serodiagnosis of C. trachomatis infections. Significance and Impact of the Study: The CT694 ELISA test responds to the criteria of both sensitivity and specificity according to the MIF and pELISA tests and may be used for serodiagnosis of C. trachomatis infections. 相似文献
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18.
Bošnjak Z Džijan S Pavlinić D Perić M Ružman N Križan IR Lauc G Antolović-Požgain A Burazin J Vuković D 《Current microbiology》2012,64(6):552-560
The purpose of this study was to determine prevalence of Chlamydia trachomatis (Ct) urogenital infection and its serotype distribution from clinical samples in north-eastern Croatia. During a 3-year period,
2,379 urogenital samples were analyzed by real-time polymerase chain reaction (A group), while 4,846 genital swabs were analyzed
by direct fluorescent antibody test (B group). 132 Ct positive specimens were genotyped by omp1 gene sequencing. The prevalence rate of Ct was 3.2 % in A and 1 % in B group. The most prevalent chlamydial genotype was
E (44 %), followed by F (33 %), K (11.5 %), G (8 %), J/UW (5.3 %), D-IC (4.4 %), D-B120 (1.8 %), and B/IU, J/IU, Ia/IU (0.9
% each) serotypes. Single-nucleotide polymorphisms (SNPs) of omp1 gene were detected in E, K, and G serotypes. Some of these SNPs (C/T at position 272 and G/A at position 813 in E strain;
C/T at position 884 in D strain) might represent novel omp1 variants. 相似文献
19.
Chunxue Lu Lei Lei Bo Peng Lingli Tang Honglei Ding Siqi Gong Zhongyu Li Yimou Wu Guangming Zhong 《PloS one》2013,8(7)
Glycogen has been localized both inside and outside Chlamydia trachomatis organisms. We now report that C. trachomatis glycogen synthase (GlgA) was detected in both chlamydial organism-associated and -free forms. The organism-free GlgA molecules were localized both in the lumen of chlamydial inclusions and in the cytosol of host cells. The cytosolic GlgA displayed a distribution pattern similar to that of a known C. trachomatis-secreted protease, CPAF. The detection of GlgA was specific since the anti-GlgA antibody labeling was only removed by preabsorption with GlgA but not CPAF fusion proteins. GlgA was detectable at 12h and its localization into host cell cytosol only became apparent at 24h after infection. The cytosolic localization of GlgA was conserved among all C. trachomatis serovars. However, the significance of the GlgA secretion into host cell cytoplasm remains unclear since, while expression of chlamydial GlgA in HeLa cells increased glycogen stores, it did not affect a subsequent infection with C. trachomatis. Similar to several other C. trachomatis-secreted proteins, GlgA is immunogenic in women urogenitally infected with C. trachomatis, suggesting that GlgA is expressed and may be secreted into host cell cytosol during C. trachomatis infection in humans. These findings have provided important information for further understanding C. trachomatis pathogenic mechanisms. 相似文献
20.
Min Lin Ji-Wei Jiao Xiu-Hui Zhan Xiao-Fen Zhan Mei-Chen Pan Jun-Li Wang Chun-Fang Wang Tian-Yu Zhong Qin Zhang Xia Yu Jiao-Ren Wu Hui-Tian Yang Fen Lin Xin Tong Hui Yang Guang-Cai Zha Qian Wang Lei Zheng Ying-Fang Wen Li-Ye Yang 《PloS one》2014,9(8)
β-thalassemia is a common inherited disorder worldwide including southern China, and at least 45 distinct β-thalassemia mutations have been identified in China. High-resolution melting (HRM) assay was recently introduced as a rapid, inexpensive and effective method for genotyping. However, there was no systemic study on the diagnostic capability of HRM to identify β-thalassemia. Here, we used an improved HRM method to screen and type 12 common β-thalassemia mutations in Chinese, and the rapidity and reliability of this method was investigated. The whole PCR and HRM procedure could be completed in 40 min. The heterozygous mutations and 4 kinds of homozygous mutations could be readily differentiated from the melting curve except c.-78A>G heterozygote and c.-79A>G heterozygote. The diagnostic reliability of this HRM assay was evaluated on 756 pre-typed genomic DNA samples and 50 cases of blood spots on filter paper, which were collected from seven high prevalent provinces in southern China. If c.-78A>G heterozygote and c.-79A>G heterozygote were classified into the same group (c.-78&79 A>G heterozygote), the HRM method was in complete concordance with the reference method (reverse dot blot/DNA-sequencing). In a conclusion, the HRM method appears to be an accurate and sensitive method for the rapid screening and identification of β-thalassemia mutations. In the future, we suggest this technology to be used in neonatal blood spot screening program. It could enlarge the coverage of β-thalassemia screening program in China. At the same time, its value should be confirmed in prospectively clinical and epidemiological studies. 相似文献