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The optimized PCR test using GPO-1 and MGSO primers. Column M: DNA ladder 100bp (Fermentas). lanes 1: positive control (DNA of mycoplasma arginini), 2: negative control: (sterile water) (Agarose 1.5% and TBE 0.5 × buffer)
Source publication
Contamination of cell lines and biological products is one of the major problems of cell culture techniques. Rapid detection of mycoplasma contamination in cell culture is an important part of quality control standards in related laboratories. The aim of this study was to evaluate the efficacy of PCR in detection of myroplasma as contaminants in ce...
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Citations
... Culture media was replaced every other day. Cells were regularly tested for mycoplasma contamination with PCR assay with primers for 16SrRNA of mycoplasma (F: 5'-ACACCATGGGAGCTGGTAAT-3', R: 5'-CTTCWTCGACTTYCAGACCCAAGGCAT-3') (Tabatabaei-Qomi et al, 2014) and used between passages 1 and 5. Tissue culture plates were coated with 0.1% gelatin (Sigma) for 30 min at 37°C before cell plating. Lipofectamine RNAimax reagent (Invitrogen) was used for gene knockdown. ...
Endothelial cells differ from other cell types responsible for the formation of the vascular wall in their unusual reliance on glycolysis for most energy needs, which results in extensive production of lactate. We find that endothelium-derived lactate is taken up by pericytes, and contributes substantially to pericyte metabolism including energy generation and amino acid biosynthesis. Endothelial-pericyte proximity is required to facilitate the transport of endothelium-derived lactate into pericytes. Inhibition of lactate production in the endothelium by deletion of the glucose transporter-1 (GLUT1) in mice results in loss of pericyte coverage in the retina and brain vasculatures, leading to the blood-brain barrier breakdown and increased permeability. These abnormalities can be largely restored by oral lactate administration. Our studies demonstrate an unexpected link between endothelial and pericyte metabolisms and the role of endothelial lactate production in the maintenance of the blood-brain barrier integrity. In addition, our observations indicate that lactate supplementation could be a useful therapeutic approach for GLUT1 deficiency metabolic syndrome patients.
... In addition to M.pneumoniae, they identified other bacteria of this genus. Bacteria including Mycoplasma arginini, Mycoplasma hyorinis, Mycoplasma orale, Mycoplasma synoviae, Mycoplasma gallinarum were also identified [24]. ...
Mycoplasma pneumoniae, which causes atypical pneumonia, is a well-established pathogen of the respiratory tract. This bacterium is intrinsically susceptible to fluoroquinolones. But Recently, drug-resistant forms of this bacterium have been reported. This study aims to determine the prevalence of this bacterium by ELISA and PCR and MIC to ciprofloxacin. The clinical samples (blood and nasopharyngeal swab) were collected from 100 patients, who were referred to selective hospitals in Tehran with respiratory complaints, were enrolled in 2017. Nasopharyngeal swab sample collections were cultured on PPLO broth and PPLO agar. After culturing and DNA extraction, PCR was performed by specific P1 genes primers. Ciprofloxacin's MIC of Mycoplasma pneumonia isolated was determined by the Micro-broth dilution method. The serum of IgG antibody titers was also measured by ELISA Mycoplasma pneumonia. In this study, out of 100 samples 12, bacteria were isolated on PPLO agar. Using specific primers, 7 samples of Mycoplasma speciesism-specific were positive for the presence of M.pneumoniae and 2 Ciprofloxacin resistant isolates were evaluated. ELISA results show that IgG titer antibody is existent in 19 samples and 5 samples are intermediate as well. IgG antibody titer average in the whole sample is 27/66 U/ml, but it is in Positive samples by P1 PCR is 45/75 U/ml. This study showed that PCR is a sensitive and reliable method for rapid detection of M. pneumoniae bacteria in respiratory infectious samples, but the results of this method are different from the ELISA method. Additionally, It seems that the Resistance to ciprofloxacin is relatively common among M. pneumoniae.
... MGSO; reverse primer: 5'-TGCACCATCTGTCACTCTGTTAACCTC-3') (Kong et al., 2001;Tabatabaei-Qomi et al., 2014). The PCR reaction consisted of 500 mg of template DNA, 10µl of master mix (Ampliqon), 1 μl of each one of forward (GPO1) and reverse (MGSO) primers, and 3μl of sterile deionized distilled water. ...
Infertility has recently become a growing social and economic world problem. Genital mycoplasmas, such as Mycoplasma hominis and M. genitalium, are most frequently associated with several adverse effects on men’s fertility. The present study aimed to determine the prevalence of M. hominis and M. genitalium in the semen samples in thenortheast of Iran. During thiscross-sectional study from February to May, 2018, 100 semen samples were collected from 100 infertile men in Mashhad, Khorasan Razavi province, northeast of Iran. The presence of M. hominis and M. genitalium was detected by cultivation, polymerase chain reaction (PCR), and Multiplex PCR assays. The colony of mycoplasma was confirmed by Diene’s stain; moreover, arginine hydrolysis, glucose, and urea utilization were evaluated. The following semen indices were analyzed according to World Health Organization guidelines for semen analysis: color, volume, appearance, liquefaction, viscosity, concentration, pH, leukocyte concentration, progressive motility, morphological normality, motile sperm concentration, functional sperm concentration, sperm motility index, and functional sperm. The gene of 16SrRNA (GPO1& MGSO primers) was used as the target gene of the Mycoplasma genus in PCR assay. Multiplex-PCR was performed with a specific primer for conserved regions in the 16SrRNA gene for M. hominis (RNAH1& RNAH2 primers) and the 140-kDa Adhesion Protein Gene for M. genitalium (MG1 & MG2 primers).According to the results,9 (9%) samples were PCR-positive for Mycoplasma spp , while there were 7 (7%) cases isolated by cultivation. M. hominis was detected in 8 (8%) samples by Multiplex PCR, while there was no evidence for M. genitalium. The mean age scores of all infertile and infected men were obtained at 31 and 30 years, respectively. The study could not show any statistical correlation between mycoplasma infection and abnormal semen parameters. The heterogeneity of mycoplasma prevalence in the reports can be ascribed to differences in geographic areas, the sensitivity of the identification method, condition of the group (fertile/infertile), sample size, and operator proficiency. Various results have been reported in numerous studies conducted on the relationship between mycoplasma infection and abnormal semen parameters.
... In addition to M.pneumoniae, they identi ed other bacteria of this genus. Bacteria including Mycoplasma arginini, Mycoplasma hyorinis, Mycoplasma orale, Mycoplasma synoviae, Mycoplasma gallinarum were also identi ed [24] . ...
Objective
Mycoplasma pneumoniae, which causes atypical pneumonia, is a well-established pathogen of the respiratory tract. This bacteria is intrinsically susceptible to fluoroquinolones. But Recently, drug-resistant forms of this bacteria have been reported. This study aims to determine the prevalence of this bacteria by ELISA and PCR and MIC to ciprofloxacin.
Methods
The clinical samples (blood and nasopharyngeal swab) were collected from 100 patients who were referred to selective hospitals in Tehran with respiratory complaints were enrolled in 2017.Nasopharyngeal swab sample collections were cultured on PPLO broth and PPLO agar. After culturing and DNA extraction, PCR was performed by specific P1 genes primers. Ciprofloxacin's MIC of Mycoplasma pneumonia isolated was determined by Micro-broth dilution method. Also, measure serum IgG antibody titers were measured by ELISA Mycoplasma pneumonia.
Results
In this study, out of 100 samples 12, bacteria were isolated on PPLO agar.Using specific primers,7 samples of Mycoplasma speciesism-specific were positive for the presence of M.pneumoniae. 2 Ciprofloxacin resistant isolates were evaluated. ELISA results show that the IgG titre antibody In 19 samples is existent. Also,5 samples are intermediate. IgG antibody titre average in the whole sample is 27/66 U/ml but it is in Positive samples by P1 PCR is 45/75 U/ml.
Conclusions
This study showed that PCR is a sensitive and reliable method for rapid detection of M. pneumoniae bacteria in respiratory infectious samples.but the results of this method are different from the ELISA method. also, It seems that the Resistance to ciprofloxacin is relatively common among M. pneumoniae.
... The main disadvantage of the compendial USP Ͻ63Ͼ reference culture method for Mycoplasma testing is the slow turnaround (28-day culture). Molecular testing for Mycoplasma has therefore become a favorable alternative in the industry, with several laboratory developed (45)(46)(47)(48)(49) and commercial assays available on the market, including My-coTOOL (Roche) (50,51), MycoSEQ (Applied Biosystems), and VenorGeM PCR (Minerva Biolabs). The availability of commercial assays has removed laborious assay validation requirements, as documented qualification points already covered by the kit manufacturer can replace qualification by the user (52). ...
Sterility testing of cellular therapy products along with the associated environmental monitoring requirements for aseptic facilities, including compounding pharmacies, continues to impact clinical microbiology laboratories as evidenced by the numerous discussions recurring on American Society for Microbiology DivC and ClinMicroNet listservs. This mini review provides an overview of this complex field of current good manufacturing practices (cGMP) based on biopharmaceutical industry standards, and will summarize the compendial and alternative rapid microbial test methods available for product sterility and Mycoplasma testing. In addition, this mini review highlights major overarching regulatory requirements governing any laboratory performing product testing as regulated by the United States Food and Drug Administration (FDA). These requirements are different to the clinical familiarities of Clinical Laboratory Improvement Act of 1988 (CLIA '88), the College of American Pathologists (CAP), and the Joint Commission on Accreditation of Healthcare Organizations (JCAHO) – all of which have no jurisdiction in this area. As the cellular therapy field continues to advance and an increasing number of medical centers participate in clinical trials of these novel therapies, it is critically important that laboratories have a sound understanding of the major regulations and cGMP practices governing microbiological testing in the biopharmaceutical industry.
... The DNA from mccp culture was extracted by boiling method as described elsewhere (Tabatabaei et al., 2014). The harvested Mccp culture was centriguged at 15000 x g for 5 min and the supernatant was discarded. ...
Contagious caprine pleuropneumonia (CCPP) caused by Mycoplasma capricolum subsp. capripneumoniae (Mccp) is a highly contagious and deadly disease of small ruminants, especially goats. It is an economically important disease, causing high morbidity and mortality in goats. More recently Mccp Pakistan strain has been identified and isolated by our laboratory. Alarmingly, antimicrobials in animals are inappropriately using and resultantly leading to antimicrobial resistance (AMR) including mycoplasmas, which are causing serious infections in ruminants. Since the growth curve of our local strain and its susceptibility to commonly using antimicrobials is unknown. Therefore, this study was aimed to evaluate the growth curve and susceptibility of Mccp against commercially available quinolones in Pakistan by broth microdilution method. Quinolones including moxifloxacin, levofloxacin, ciprofloxacin, and enrofloxacin were selected for in vitro susceptibility of Mccp Pakistan strain. The concentration for each drug was measured distantly depending on high concentration available in the market and was then used in various concentrations ranges from higher to lower i.e. moxifloxacin 80-0.156 µg/ml, levofloxacin 250-0.488 µg/ml, ciprofloxacin and enrofloxacin 50-0.097 µg/ml. The resultant effective antimicrobial by minimum inhibitory concentration (MIC) and minimum mycoplasmacidal concentration (MMC) value was ciprofloxacin having lowest MIC and MMC value (0.390 µg/ml and 0.781 µg/ml), respectively. It was concluded that ciprofloxacin had best
mycoplasmacidal and mycoplasmastatic activities among tested chemotherapeutics agents against Mccp. Ciprofloxacin could be more effective antimicrobial in field against Mccp infection in goat.
... PCR amplification of 16S rRNA gene by using forward primer, GPO-1 and a reverse primer, MGSO of Mycoplasma genus specific yielded approximately 700bp length. The PCR results were in agreement with Tabatabaei et al. (2014) shown in the Fig 1. The partial sequence of 16S rRNA gene of uncultured Mycoplasma species of chicken was submitted to GenBank and the accession number MH035883 was obtained. ...
In the present study, Mycoplasma gallinaceum was detected by PCR amplification of 16S rRNA gene from chronic respiratory disease in village chickens of Cauvery delta region of Tamil Nadu. Necropsy was performed to find out the etiological agent in desi birds mortality. At necropsy, airsacculitis with caseous exudate were found in the thoracic and abdominal cavity. Caseous material from airsacs was collected aseptically from dead birds for detection of Mycoplasma species. DNA was extracted from caseous material by using tissue DNA extraction kit. PCR was carried out using primers to amplify 16S rRNA gene belonging to Mycoplasma species. The amplified product yielded approximately 700-bp length (703 to 713 bp) of the 16S rRNA gene specific for Mycoplasma species. Further, it was subjected to sequence analysis and confirmed as Mycoplasma gallinaceum by NCBI blast analysis. In the present communication, detection of M. gallinaceum by PCR amplification of 16S rRNA gene provides a powerful tool for rapid diagnosis.
... The DNA from mccp culture was extracted by boiling method as described elsewhere (Tabatabaei et al., 2014). The harvested Mccp culture was centriguged at 15000 x g for 5 min and the supernatant was discarded. ...
... All cell lines were checked for mycoplasma contaminations by PCR assay [27,28]. Melanoma cells were grown in DMEM supplemented with 10% FBS (D10), 50 U/ml penicillin and 50µg/ml streptomycin. ...
Background
Melanoma is the most aggressive skin cancer, and BRAF (V600E) is the most frequent mutation that led to the development of BRAF inhibitors (BRAFi). However, patients treated with BRAFi usually present recidivism after 6-9 months. Curcumin is a turmeric substance, and it has been deeply investigated due to its anti-inflammatory and antitumoral effects. Still, the low bioavailability and biodisponibility encouraged the investigation of different analogs. DM-1 is a curcumin analog and has shown an antitumoral impact in previous studies.
Methods
Evaluate DM-1 stability and cytotoxic effects for BRAFi-sensitive and resistant melanomas, as well as the role in the metalloproteinases modulation.
Results
DM-1 showed growth inhibitory potential for melanoma cells, demonstrated by reduction of colony formation, migration and endothelial tube formation, and cell cycle arrest. Subtoxic doses were able to downregulate important metalloproteinases (MMPs) related to invasiveness, such as MMP-1, -2 and -9. Negative modulations of TIMP2 and MMP-14 reduced MMP-2 and -9 activity; however, the reverse effect is seen when increased TIMP-2 and MMP-14 resulted in raised MMP-2.
Conclusion
These findings provide essential details into the functional role of DM-1 in melanomas, encouraging further studies in the development of combinatorial treatments for melanomas.
... The DNA from mccp culture was extracted by boiling method as described elsewhere (Tabatabaei et al., 2014). The harvested Mccp culture was centriguged at 15000 x g for 5 min and the supernatant was discarded. ...
