A newly developed consensus polymerase chain reaction to detect Mycoplasma species using 16S ribosomal RNA gene
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Abstract:
Mycoplasmas are highly fastidious bacteria, difficult to culture and slow growing. Infections with Mycoplasma species can cause a variety of problems in living organisms and in vitro cell cultures. In this study, we investigated the usefulness of a genus-specific consensus PCR analysis method to detect Mycoplasma species. The developed consensus primer pairs MycoF and MycoR were designed specifically to amplify the 16S ribosomal RNA gene (rRNA) of Mycoplasma species by the optimized PCR system. The developed consensus PCR system effectively amplified 215 bp of Mycoplasma genus-specific region of 16S rRNA. In conclusion, we recommend this consensus PCR for monitoring Mycoplasma species in animals, human and cell culture system.Keywords:
Fastidious organism
Primer (cosmetics)
23S ribosomal RNA
23S ribosomal RNA
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Since the late 1960s, there have been an increasing number of reports describing the isolation and identification of fastidious strains of Mycoplasma hyorhinis in cell cultures but not in conventional mycoplasma media, i.e., agar and broth. The application of those techniques normally used in studying viruses, i.e., infection of cell cultures coupled with the subsequent use of immunological and biological test procedures, has provided a reliable alternative procedure for M. hyorhinis detection. Major isolation surveys have revealed that as many as 60 to 80% of M. hyorhinis isolates from contaminated tissue cultures failed to grow in agar medium. Efforts to elucidate the mechanisms involved in failure of the cell culture-derived M. hyorhinis strain to grow in standard cell-free mycoplasma media are ongoing. Initial data indicated that extracts prepared from tissue cultures (BHK 21) and incorporated in Macpherson's broth and agar medium would permit growth of fastidious strains. Moreover, it appears that these strains are particularly sensitive to inhibition by yeast products often found in mycoplasma media. While M. hyorhinis appears to be unique with respect to its sensitivity to medium components, these fastidious strains are isolated with such frequency that the routine use of an indicator cell system is strongly recommended.
Fastidious organism
Isolation
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Mycoplasma spp. are extracellular bacteria that colonize on the respiratory epithelium of humans and animals. It is a causative agent of pneumonia commonly complicated by opportunistic infectious bacteria. Mycoplasma spp. infection cause relatively mild disease in the absence of environmental stressors, but when complicated by secondary bacterial invaders the resultant disease can cause obvious clinical disease and severe production losses in intensively reared pigs Mycoplasma spp. are highly fastidious bacteria, difficult to culture and slow growing. Many species of Mycoplasma spp. are important pathogens causing respiratory infection in animals and known to induce huge economic losses. The aims of the present study were to develop a rapid isolation and culture method of wild type Mycoplasma spp. in pigs. We used Mycoplasma spp. genus specific direct PCR without DNA extraction procedure using PhireⓇ Animal Tissue Direct PCR Kit from the lung tissues with pneumonia lesions. Therefore, we could save the time for tissue processing and increase the accuracy of Mycoplasma spp. inclusion prediction in lung tissues. Thereafter, we used the optimized media to isolate and culture Mycoplasma spp. As the results, Mycoplasma spp. could be isolated and cultured quickly and efficiently. These results could provide an efficient strategy and method for the rapid and accurate isolation and culture of wild type Mycoplasma spp. in pigs.
Fastidious organism
Isolation
Mycoplasma pneumonia
Mycoplasmataceae
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The ribonuclease alpha-sarcin exclusively cleaves the phosphodiester bond after G2661 in the 23S rRNA within 50S subunits, thus inactivating the ribosomes. The resulting alpha-fragment is 243 nucleotides long and contains the 3'-end of the 23S rRNA. The specificity is changed dramatically if isolated 23S rRNA is used as substrate. We have shown previously that 23S rRNA is digested completely except for two fragments, one of which is identical to the alpha-fragment. Here we show that the other fragment comprises the 5'-end of 23S rRNA and contains 385 nucleotides. A similar fragment was obtained when isolated 23S rRNA was digested with RNase A (specific for pyrimidines in single strands). It appears that the 5'-domain (equivalent to 5.8S rRNA of eukaryotic ribosomes) as well as the 3'-domain (equivalent to 4.5S rRNA of chloroplast ribosomes) have a compact and defined tertiary structure in isolated 23S rRNA in contrast to the rRNA region in between. Thus, alpha-sarcin is a convenient tool for detecting compact domains in isolated RNA.
23S ribosomal RNA
50S
Ribonuclease III
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The objective was to investigate the presence of Mycoplasma and Chlamydia in the synovial fluid of patients with rheumatoid arthritis (RA) and other chronic arthritides. Samples of synovial fluid (SF) were collected from all patients presenting with an articular effusion. Seventy SF samples were subjected to study for Mycoplasma and cultured on standard media for Mycoplasma. The 70 other SF samples were subjected to study for the presence of Chlamydia and cultivated on cell cultures specially MC coy cell lines. All standard cultures for Mycoplasma and Chlamydia remained negative, consistent with the fact that synovial fluid is sterile, despite of many investigations that have indicated DNA of some bacteria in the SF of patients with arthritis. However, as many other attempts to detect the presence of these fastidious organisms in the joints of patients with such arthritides have failed, the question of their possible roll in the pathogenesis of human rheumatic diseases remains controversial and needs to be re-examined.
Fastidious organism
Pathogenesis
Mycoplasma genitalium
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ABSTRACT For still unknown reasons, the 23S rRNA of many α-Proteobacteria shows a unique fragmentation pattern compared to other bacteria. The 23S rRNA processing involves RNase III and additional, yet unidentified enzymes. The α-proteobacterium Rhizobium leguminosarum ATCC 10004 T possesses two fragmentation sites in its 23S rRNA. The first one harbors an intervening sequence in helix 9 which is cleaved by RNase III. We demonstrate that the mature 5′ end of the resulting 2.6-kb rRNA fragment is generated by additional removal of helix 10. A fraction of the 2.6-kb rRNA is further processed in domain III, giving rise to two 1.3-kb rRNA fragments. We mapped the domain III fragmentation site and found it to be at a position which has only been reported for trypanosomatid protozoa. This fragmentation site is also unique in that it lacks an intervening sequence. We found that the simultaneous occurrence of 2.6-kb and 1.3-kb rRNA fragments is not due to interoperonal sequence differences but rather reflects slow processing. The different characteristics of the two fragmentation sites in the 23S rRNA suggest that they are processed by different mechanisms. Interestingly, the amount of 2.6-kb rRNA varies during culture growth. We observed a transient increase in the relative amount of 2.6-kb rRNA fragments during the first hours after inoculation, which points to changes in the ratio of rRNA synthesis rate to domain III processing rate during the growth of a culture.
23S ribosomal RNA
Fragmentation
Rhizobium leguminosarum
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Mycoplasma is fastidious, a slow-growing organism that lacks a cell wall and is often isolated from the genitourinary tract. The aim of the study determines the relationships between the fastidious microbial infection (Mycoplasma spp. and Ureaplasma spp.) and toll-like receptor 6 concentration for urinary-genital tract infections (UGTIs). The study includes a collection (of 223) samples (females’ urine and blood) from UGTIs patients, who arrived Hospital in Babylon (from January to June 2021). The isolates were identified according to appropriate culture media by urine culture, Biochemical analyses have been used to verify the authenticity. The results of the immunological examination of TLR-6 concentration were done using ELISA for measurement. The mean TLR-6 urine concentration in patients (2.08±0.3 pg/ml) was significant increase p< 0.05. compared1with the control (1.70±0.4 pg/ml) while showing the mean TLR-6 serum concentration in patients (2.21±1.4 pg/ml) was no significant difference compared with the control group (1.80±0.5 pg/ml). we concluded that the risk factors associated with Mycoplasma ssp. are numerous. Therefore, it is necessary to use accurate and rapid diagnostic methods. The present study indicates that the patients with UGTIs due to mycoplasmas had significantly higher Toll-like receptor-6 than the control group in urine only.
Fastidious organism
Ureaplasma
Ureaplasma urealyticum
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Ribonucleic acid (RNA) isolated by four distinct methods and from a variety of Salmonella typhimurium strains lacked intact 23S ribosomal RNA (rRNA). On sucrose gradients which minimize aggregation, the vast majority of S. typhimurium rRNA sedimented as a 16S peak with a 14S shoulder. RNA from this region of the gradient was resolved into three discrete bands by electrophoresis in formamide. Two very minor S. typhimurium RNA peaks were resolved at 21S and 10S on sucrose gradients, and each peak formed discrete bands in electrophoresis. It is concluded that if S. typhimurium does possess an intact 23S rRNA species, this species is extremely "labile." The absence of isolatable S. typhimurium 23S rRNA possibly reflected in vivo processing of the rRNA before isolation. Under certain conditions, S. typhimurium rRNA formed discrete aggregates which sedimented similarly to intact Escherichia coli 23S rRNA.
23S ribosomal RNA
50S
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Interactions between tRNA or its analogs and 23S rRNA in the large ribosomal subunit were analyzed by RNA footprinting and by modification–interference selection. In the E site, tRNA protected bases G2112, A2392, and C2394 of 23S rRNA. Truncated tRNA, lacking the anticodon stem-loop, protected A2392 and C2394, but not G2112, and tRNA derivatives with a shortened 3′ end protected only G2112, but not A2392 or C2394. Modification interference revealed C2394 as the only accessible nucleotide in 23S rRNA whose modification interferes with binding of tRNA in the large ribosomal subunit E site. The results suggest a direct contact between A76 of tRNA A76 and C2394 of 23S rRNA. Protections at G2112 may reflect interaction of this 23S rRNA region with the tRNA central fold.
23S ribosomal RNA
P-site
5.8S ribosomal RNA
Footprinting
50S
5S ribosomal RNA
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We have previously shown that the 23S rRNA of Salmonella strains is highly fragmented by specific enzyme cleavages. In this article, we report that 23S rRNA of Salmonella strains is rapidly degraded as the cells enter the stationary phase. More than 90% of the 23S rRNA is degraded when the cells reach the stationary phase. The rate of degradation of 23S rRNA correlated with its degree of fragmentation. This degradation is probably mediated by newly synthesized protein factor(s), since treatment with chloramphenicol or rifampin inhibits the rRNA degradation. We propose that degradation of 23S rRNA is a novel mechanism in the regulation of the bacterial 23S rRNA and ribosome concentration and that this additional regulatory mechanism provides some selective advantage to cells.
23S ribosomal RNA
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