PCR和分离培养同时检测儿童呼吸道感染的支原体

为了解广州市儿童呼吸道支原体感染情况,用一条共同的上游引物,二条特异性的下游引物建立的ＰＣＲ方法能同时扩增ＭＰ的６９１ｂｐ和ＭＧ的４３８ｂｐ粘附因子基因片段,但不会扩增其他支原体和细菌的ＤＮＡ。     

Ingested foreign (phage M13) DNA survives transiently in the gastrointestinal tract and enters the bloodstream of mice

Is the epithelial lining of the mammalian gastrointestinal (GI) tract a tight barrier against the uptake of ingested foreign DNA or can such foreign DNA penetrate into the organism? We approached this question by pipette-feeding circular or linearized double-stranded phage M13 DNA to mice or by adding M13 DNA to the food of mice whose fecal excretions had previously been shown to be devoid of this DNA. At various post-prandial times, the feces of the animals was tested for M 13 DNA sequences by Southern or dot blot hybridization or by the polymerase chain reaction (PCR). On Southern blot hybridization, the majority of M13 DNA fragments were found in the size range between < 200 and 400 by (base pairs). For the PCR analysis, synthetic oligodeoxyribonucleotide primers were spaced on the M13 DNA molecule such that the sizes of the persisting M13 DNA fragments could be determined. We also extracted DNA from whole blood or from sedimented blood cells of the animals at different times after feeding M t3 DNA and examined these DNA preparations for the presence of M13 DNA by dot blot hybridization or by PCR. M13 DNA fragments were found between 1 and 7 h postprandially in the feces of mice. By PCR analysis, fragments of 712, 976, and 1692 by in length were detected. In DNA from blood, M13 DNA fragments of up to 472 by were found by PCR between 2 and 6 h after feeding. Dot blot or Southern blot hybridization revealed M13 DNA at 2 and 4 h, but not at 1, 8 or 24 h after feeding. This DNA was shown to be DNase sensitive. M13 DNA was found both in blood cells and in the serum. A segment of about 400 by of the DNA amplified by PCR from feces or blood was analyzed for its nucleotide sequence which was found to be identical to that of authentic M13 DNA, except for a few deviations. M13 DNA could not be detected in the feces or in the blood of the animals prior to feeding or prior to 1 h and later than 7 h after feeding. These controls attest to the validity of the results and also argue against the possibility that the murine GI tract had been colonized by phage M13. Moreover, M13 DNA-positive bacterial colonies were never isolated from the feces of animals that had ingested M13 DNA. The results of reconstitution experiments suggested that 2 to 4% of the orally administered M13 DNA could be detected in the GI tract of mice. A proportion of about 0.01% to 0.1% of the M13 DNA fed could be retrieved from the blood.     

PCR-based detection of Mycoplasma species

In this study, we describe our newly-developed sensitive two-stage PCR procedure for the detection of 13 common mycoplasmal contaminants (M. arthritidis, M. bovis, M. fermentans, M. genitalium, M. hominis, M. hyorhinis, M. neurolyticum, M. orale, M. pirum, M. pneumoniae, M. pulmonis, M. salivarium, U. urealyticum). For primary amplification, the DNA regions encompassing the 16S and 23S rRNA genes of 13 species were targeted using general mycoplasma primers. The primary PCR products were then subjected to secondary nested PCR, using two different primer pair sets, designed via the multiple alignment of nucleotide sequences obtained from the 13 mycoplasmal species. The nested PCR, which generated DNA fragments of 165-353 bp, was found to be able to detect 1-2 copies of the target DNA, and evidenced no cross-reactivity with the genomic DNA of related microorganisms or of human cell lines, thereby confirming the sensitivity and specificity of the primers used. The identification of contaminated species was achieved via the performance of restriction fragment length polymorphism (RFLP) coupled with Sau3AI digestion. The results obtained in this study furnish evidence suggesting that the employed assay system constitutes an effective tool for the diagnosis of mycoplasmal contamination in cell culture systems.     

The first performance report for the Bio-Rad Dx CT/NG/MG assay for simultaneous detection of Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium in urogenital samples

We evaluated the clinical performance of the Bio-Rad Dx CT/NG/MG assay for the detection of Chlamydia trachomatis, Mycoplasma genitalium and Neisseria gonorrhoeae in urogenital samples in comparison with the Roche COBAS? TaqMan? CT assay for C. trachomatis and an in-house TaqMan PCR assay for M. genitalium. Swab specimens were cultured for N. gonorrhoeae. In this prospective study, urogenital samples were obtained from symptomatic and asymptomatic patients attending the sexually transmitted disease clinic of Bordeaux, France, from January to April 2010. A total of 658 clinical specimens (259 male and 180 female urines, 191 vaginal, 21 endocervical and 7 urethral swabs) from 453 patients were analyzed. The prevalence of C. trachomatis and M. genitalium infections was 8.1% (21/260) and 1.9% (5/260) in men and 10.4% (20/193) and 2.1% (4/193) in women, respectively. The Bio-Rad Dx CT/NG/MG clinical sensitivity was 100% for C. trachomatis and M. genitalium in men and women. In male urine, the clinical specificity was 99.6% for C. trachomatis and 100% for M. genitalium. In women, the specificity was 99.5% for swabs and 100% for urines for detecting C. trachomatis and M. genitalium. All seven N. gonorrhoeae PCR-positive samples were also positive by culture. Patients were co-infected in 5/57 cases (8.8%), with C. trachomatis and M. genitalium in three cases, and C. trachomatis and N. gonorrhoeae in two cases. In conclusion, the Bio-Rad Dx CT/NG/MG assay can be recommended for the simultaneous detection of C. trachomatis, M. genitalium and N. gonorrhoeae in urogenital specimens of symptomatic and asymptomatic individuals.     

Detection and identification of mycoplasmas by amplification of rDNA

Alignment of published 16S rRNA sequences allowed the definition of a pair of oligonucleotides suitable for polymerase chain reaction (PCR). Using this pair of PCR primers, several mycoplasmas including the four human parasites Mycoplasma genitalium, M. hominis, M. salivarium and M. orale were detected. This DNA amplification was restricted to species of the genus Mycoplasma while no cross-reaction was observed with DNA from other bacteria and eukaryotic cells. Subsequent analysis of amplified products by either specific oligonucleotide hybridization or dideoxy sequencing specified the identity of the detected mycoplasmas. This method offers a highly discriminating and sensitive assay for the direct detection and identification of these microorganisms without the need for prior cultivation.     

An unusual gene containing a dnaJ N-terminal box flanks the putative origin of replication of Mycoplasma genitalium.

Origins of replication are known to be highly conserved among widely divergent microbial species, with the gene order in those regions being dnaA-dnaN-recF-gyrB. On the basis of sequence identities to entries in GenBank, the gene order of a 6-kb fragment of Mycoplasma genitalium DNA was determined to be dnaN-orf311-gyrB-gyrA-serS, which is structurally similar to the ancestral origin of replication. We have directly linked the dnaN gene to the M. genitalium dnaA gene by PCR amplification. However, we found a novel open reading frame, designated orf311, in place of an expected sequence encoding recF. Orf311 contains a DnaJ box motif at its N terminus, but it has no overall homology to any other protein or sequence in the database. We are unable to detect any recF homolog in M. genitalium by hybridization or during a random sequencing survey of the genome.     

Identification of a small RNA within the pdh gene cluster of Mycoplasma pneumoniae and Mycoplasma genitalium

A highly abundant and heterogeneous small RNA about 205 to 210 bases long named MP200 RNA has been identified in Mycoplasma pneumoniae. It was localized on the genome within a 319-bp-long intergenic space of the pyruvate dehydrogenase (pdh) gene cluster. A database search at the DNA level revealed the highest similarity to a sequence located within the pdh gene cluster of Mycoplasma genitalium that was also shown to be transcribed into two abundant, but smaller RNAs than the ones in Mycoplasma pneumoniae. The RNAs from both M. pneumoniae and M. genitalium have the potential to code for cysteine-rich 29- and 23-amino-acid-long peptides, but so far, these peptides have not been identified experimentally in bacterial protein extracts.     

A survey of the Mycoplasma genitalium genome by using random sequencing.

A total of 508 random clones from five Mycoplasma genitalium genomic libraries were partially sequenced and analyzed. This resulted in the identification of 291 unique contigs. Sequence information from these clones (100,993 nucleotides), representing approximately 17% of this pathogen's genome, was analyzed by comparison to the DNA and protein sequence data bases. The frequency with which clones could be identified, by virtue of possessing homology to another data base entry, was 46%. Sequence analysis indicated the following. (i) The M. genitalium genome contains many genes involved in various metabolic processes. (ii) Repetitive DNA may comprise as much as 4% of this genome. (iii) The MgPa adhesin gene may be the result of horizontal transfer from an unknown origin. (iv) Not all dinucleotide pairs are present in this genome at the expected frequency. (v) This genome potentially encodes approximately 390 proteins and makes very efficient use of its limited amount of DNA. In addition, this study allowed us to estimate the number of genes involved with various cellular functions.     

Functional characterization of the RuvB homologs from Mycoplasma pneumoniae and Mycoplasma genitalium

Homologous recombination between repeated DNA elements in the genomes of Mycoplasma species has been hypothesized to be a crucial causal factor in sequence variation of antigenic proteins at the bacterial surface. To investigate this notion, studies were initiated to identify and characterize the proteins that form part of the homologous DNA recombination machinery in Mycoplasma pneumoniae as well as Mycoplasma genitalium. Among the most likely participants of this machinery are homologs of the Holliday junction migration motor protein RuvB. In both M. pneumoniae and M. genitalium, genes have been identified that have the capacity to encode RuvB homologs (MPN536 and MG359, respectively). Here, the characteristics of the MPN536- and MG359-encoded proteins (the RuvB proteins from M. pneumoniae strain FH [RuvB(FH)] and M. genitalium [RuvB(Mge)], respectively) are described. Both RuvB(FH) and RuvB(Mge) were found to have ATPase activity and to bind DNA. In addition, both proteins displayed divalent cation- and ATP-dependent DNA helicase activity on partially double-stranded DNA substrates. The helicase activity of RuvB(Mge), however, was significantly lower than that of RuvB(FH). Interestingly, we found RuvB(FH) to be expressed exclusively by subtype 2 strains of M. pneumoniae. In strains belonging to the other major subtype (subtype 1), a version of the protein is expressed (the RuvB protein from M. pneumoniae strain M129 [RuvB(M129)]) that differs from RuvB(FH) in a single amino acid residue (at position 140). In contrast to RuvB(FH), RuvB(M129) displayed only marginal levels of DNA-unwinding activity. These results demonstrate that M. pneumoniae strains (as well as closely related Mycoplasma spp.) can differ significantly in the function of components of their DNA recombination and repair machinery.     

DNA methylation and Mycoplasma genomes

DNA methylation is one of the many hypotheses proposed to explain the observed deficiency in CpG dinucleotides in a variety of genomes covering a wide taxonomic distribution. Recent studies challenged the methylation hypothesis on empirical grounds. First, it cannot explain why the Mycoplasma genitalium genome exhibits strong CpG deficiency without DNA methylation. Second, it cannot explain the great variation in CpG deficiency between M. genitalium and M. pneumoniae that also does not have CpG-specific methyltransferase genes. I analyzed the genomic sequences of these Mycoplasma species together with the recently sequenced genomes of M. pulmonis, Ureaplasma urealyticum, and Staphylococcus aureus, and found the results fully compatible with the methylation hypothesis. In particular, I present compelling empirical evidence to support the following scenario. The common ancestor of the three Mycoplasma species has CpG-specific methyltransferases, and has evolved strong CpG deficiency as a result of the specific DNA methylation. Subsequently, this ancestral genome diverged into M. pulmonis and the common ancestor of M. pneumoniae and M. genitalium. M. pulmonis has retained methyltransferases and exhibits the strongest CpG deficiency. The common ancestor lost the methyltransferase gene and then diverged into M. genitalium and M. pneumoniae. M. genitalium and M. pneumoniae, after losing methylation activities, began to regain CpG dinucleotides through random mutation. M. genitalium evolved more slowly than M. pneumoniae, gained relatively fewer CpG dinucleotides, and is more CpG-deficient.     

Electrophoretic and immunologic comparative analysis of Mycoplasma pneumoniae and Mycoplasma genitalium proteins

The Mycoplasma pneumoniae FH strain routinely used in our laboratory for over 25 years as antigen in serological tests, 2 reference M. pneumoniae strains from ATCC (29342 and M129) and 3 isolates of M. pneumoniae obtained in 1995 from pneumonia patients were compared by SDS-PAGE, complement fixation test (CFT) and by Western-immunoblotting against human and rabbit serum samples with high level of mycoplasmal antibodies. On SDS-PAGE all M. pneumoniae strains showed the same number of 23 polypeptides on the gel with identical molecular weights. The same strains on immunoblotting against human and rabbit serum samples showed six bands: 170, 89, 75, 55, 38 and 33 kDa with the strongest antibody staining in 170-(P1 protein) and 89-kDa bands. Because of its known antigenic relationships Mycoplasma genitalium was used for comparison. The pattern of M. genitalium proteins on SDS-PAGE was similar to pattern of M. pneumoniae but distinguishable. On immunoblotting six proteins of M. genitalium (135, 127, 110, 95, 75 and 45 kDa) reacted with human and rabbits immunoglobulins for M. pneumoniae antigens. Furthermore in complement fixation test both antigens, prepared from M. pneumoniae and M. genitalium, reacted as well with human and rabbit immunoglobulins for M. pneumoniae and with rabbit immunoglobulins for M. genitalium. These cross-reactions observed in serological techniques could give false positive results in routine diagnosis of M. pneumoniae infections. In such situations showing on immunoblott of presence in tested serum sample of antibodies to 170- and 89 kDa proteins could confirm M. pneumoniae infection.     

Estimating the accuracy of polymerase chain reaction-based tests using endpoint dilution

Polymerase chain reaction (PCR)-based tests for various microorganisms or target DNA sequences are generally acknowledged to be highly "sensitive," yet the concept of sensitivity is ill-defined in the literature on these tests. We propose that sensitivity should be expressed as a function of the number of target DNA molecules in the sample (or specificity, when the target number is 0). However, estimating this "sensitivity curve" is problematic, since it is difficult to construct samples with a fixed number of targets. Nonetheless, using serially diluted replicate aliquots of a known concentration of the target DNA sequence, we show that it is possible to disentangle random variations in the number of target DNA molecules from the underlying test sensitivity. We develop parametric, nonparametric, and semiparametric (spline-based) models for the sensitivity curve. The methods are compared on a new test for M. genitalium.     

The RuvA homologues from Mycoplasma genitalium and Mycoplasma pneumoniae exhibit unique functional characteristics

The DNA recombination and repair machineries of Mycoplasma genitalium and Mycoplasma pneumoniae differ considerably from those of gram-positive and gram-negative bacteria. Most notably, M. pneumoniae is unable to express a functional RecU Holliday junction (HJ) resolvase. In addition, the RuvB homologues from both M. pneumoniae and M. genitalium only exhibit DNA helicase activity but not HJ branch migration activity in vitro. To identify a putative role of the RuvA homologues of these mycoplasmas in DNA recombination, both proteins (RuvA(Mpn) and RuvA(Mge), respectively) were studied for their ability to bind DNA and to interact with RuvB and RecU. In spite of a high level of sequence conservation between RuvA(Mpn) and RuvA(Mge) (68.8% identity), substantial differences were found between these proteins in their activities. First, RuvA(Mge) was found to preferentially bind to HJs, whereas RuvA(Mpn) displayed similar affinities for both HJs and single-stranded DNA. Second, while RuvA(Mpn) is able to form two distinct complexes with HJs, RuvA(Mge) only produced a single HJ complex. Third, RuvA(Mge) stimulated the DNA helicase and ATPase activities of RuvB(Mge), whereas RuvA(Mpn) did not augment RuvB activity. Finally, while both RuvA(Mge) and RecU(Mge) efficiently bind to HJs, they did not compete with each other for HJ binding, but formed stable complexes with HJs over a wide protein concentration range. This interaction, however, resulted in inhibition of the HJ resolution activity of RecU(Mge).     

Target selection and deselection at the Berkeley Structural Genomics Center

At the Berkeley Structural Genomics Center (BSGC), our goal is to obtain a near-complete structural complement of proteins in the minimal organisms Mycoplasma genitalium and M. pneumoniae, two closely related pathogens. Current targets for structure determination have been selected in six major stages, starting with those predicted to be most tractable to high throughput study and likely to yield new structural information. We report on the process used to select these proteins, as well as our target deselection procedure. Target deselection reduces experimental effort by eliminating targets similar to those recently solved by the structural biology community or other centers. We measure the impact of the 69 structures solved at the BSGC as of July 2004 on structure prediction coverage of the M. pneumoniae and M. genitalium proteomes. The number of Mycoplasma proteins for which the fold could first be reliably assigned based on structures solved at the BSGC (24 M. pneumoniae and 21 M. genitalium) is approximately 25% of the total resulting from work at all structural genomics centers and the worldwide structural biology community (94 M. pneumoniae and 86 M. genitalium) during the same period. As the number of structures contributed by the BSGC during that period is less than 1% of the total worldwide output, the benefits of a focused target selection strategy are apparent. If the structures of all current targets were solved, the percentage of M. pneumoniae proteins for which folds could be reliably assigned would increase from approximately 57% (391 of 687) at present to around 80% (550 of 687), and the percentage of the proteome that could be accurately modeled would increase from around 37% (254 of 687) to about 64% (438 of 687). In M. genitalium, the percentage of the proteome that could be structurally annotated based on structures of our remaining targets would rise from 72% (348 of 486) to around 76% (371 of 486), with the percentage of accurately modeled proteins would rise from 50% (243 of 486) to 58% (283 of 486). Sequences and data on experimental progress on our targets are available in the public databases TargetDB and PEPCdb.     

Identification and semiquantitation of Mycobacterium avium using a competitive PCR method.

A competitive PCR method with standard DNA (MIMIC) was developed for the rapid detection and semiquantitation of Mycobacterium avium (M. avium) using primers specific for the alpha antigen sequence of the bacteria. DNA from both M. avium and Mycobacterium marinum was amplified by polymerase chain reaction (PCR), but only M. avium could be detected by subsequent blotting confirmation with a probe specific for the bacteria. With the PCR and subsequent dot blot hybridization, as little as 10 fg of the M. avium DNA could be detected, equivalent to about 2 cells of the mycobacteria. In addition, we could distinguish 10(5) CFUs of M. avium from 10(4) CFUs or less by competitive PCR using a MIMIC. The present competitive PCR test enabled rapid identification and semiquantitation of M. avium, and could be used clinically to monitor disease severity and response to treatment of human M. avium disease.     

Genetic variation in the complete MgPa operon and its repetitive chromosomal elements in clinical strains of Mycoplasma genitalium

Mycoplasma genitalium has been increasingly recognized as an important microbe not only because of its significant association with human genital tract diseases but also because of its utility as a model for studying the minimum set of genes necessary to sustain life. Despite its small genome, 4.7% of the total genome sequence is devoted to making the MgPa adhesin operon and its nine chromosomal repetitive elements (termed MgPars). The MgPa operon, along with 9 MgPars, is believed to play an important role in pathogenesis of M. genitalium infection and has also served as the main target for development of diagnostic tools. However, genetic variation in the complete MgPa operon and MgPars among clinical strains of M. genitalium has not been addressed. In this study we examined the genetic variation in the complete MgPa operon (approximately 8.5 kb) and full or partial MgPar sequences (0.4-2.6 kb) in 15 geographically diverse strains of M. genitalium. Extensive variation was present in four repeat regions of the MgPa operon (with homology to MgPars) among and within strains while the non-repeat regions (without homology to MgPars) showed low-level variation among strains and no variation within strains. MgPars showed significant variation among strains but were highly homogeneous within strains, supporting gene conversion as the likely recombination mechanism. When applying our sequence data to evaluate published MgPa operon-based diagnostic PCR assays and genotyping systems, we found that 11 of 19 primers contain up to 19 variable nucleotides and that the target for one of two typing systems is located in a hypervariable repeat region, suggesting the likelihood of false results with some of these assays. This study not only provides new insights into the role of the MgPa operon in the pathogenesis of M. genitalium infection but has important implications for the development of diagnostic tools.     

A latex agglutination test for the detection of Mycoplasma pneumoniae in respiratory exudates: a comparative study with a commercially available DNA-probe test.

We prepared polyclonal antibody specific to Mycoplasma pneumoniae. Using this antibody, we developed a latex agglutination test (LAT) for detecting the organism in respiratory exudates as rapid diagnosis of M. pneumoniae infection. Further, LAT was compared with DNA-probe test (DP) which was the only commercially available test for the rapid detection of the organism. In LAT, both M. pneumoniae and M. genitalium give positive agglutination, but the titer of M. genitalium was significantly lower than that of M. pneumoniae. The detection limit of LAT was 2 x 10(5) CFU/ml and that of DP was 5 x 10(4) CFU/ml in vitro. It was considered that target molecules in LAT were accumulated in the pharyngeal portion of the patients, because of their long half-life at 37 C. However, ribosomal RNA which was target molecule in DP was destroyed at 37 C much sooner, and the accumulation could not be expected. Actually, positive rate in LAT was higher than that in DP among clinical specimens in which M. pneumoniae was detected by culture method. The procedure of LAT is much easier and more rapid than that of DP in which radioactive isotope is required. LAT could be the choice of test for rapid diagnosis of M. pneumoniae infection.     

生殖支原体脂质相关膜蛋白激活核因子κB诱导人单核细胞表达前炎症细胞因子及凋亡

为了解生殖支原体(Mg)潜在的致病性及其脂质相关膜蛋白(LAMPs)诱导人单核细胞(THP-1)凋亡及表达前炎症细胞因子(CKs)的分子机制,用Mg提取的LAMPs刺激THP-1细胞,以ELISA法和RT-PCR方法分析CKs产生和其mRNA的表达。不同试实验组的细胞经AnnexinV联合PI染色后通过流式细胞仪检测细胞凋亡。采用EMSA方法检测LAMPs处理的THP-1细胞中核转录因子kappaB(NF-κB)的激活,并分析NF-κB抑制剂二硫代氨基甲酸吡咯烷(pyrrolidine dithiocoarbamate,PDTC)对LAMPs处理的THP-1细胞产生CKs的量和其mRNA表达及细胞凋亡的影响。LAMPs能以时间和剂量依赖方式刺激THP-1细胞产生TNF-α、IL-1β和IL-6,且能激活NF-κB诱导THP-1细胞表达CKs的mRNA及发生凋亡,PDTC能显著抑制CKs的mRNA表达水平和细胞凋亡。由于LAMPs能激活NF-κB诱导THP-1细胞表达CKs及产生细胞凋亡,因而可能是一个重要的致病因素。