-
In Vitro Cellular & Developmental... 2006We have developed a simple method for rapid detection of mycoplasma contamination in cell cultures using SYBR Green-based real-time polymerase chain reaction (PCR). To...
We have developed a simple method for rapid detection of mycoplasma contamination in cell cultures using SYBR Green-based real-time polymerase chain reaction (PCR). To detect eight common contaminant mollicutes, including Mycoplasma (M. arginini, M. fermentans, M. orale, M. hyorhinis, M. hominis, M. salivarium, M. pirum) and Acholeplasma laidlawii, four primers were prepared based on the 23S rRNA regions. Using these primers and a minimum of 100 fg of mycoplasma genomic DNA, the 23S rRNA regions of these eight mycoplasma species were consistently amplified by real-time PCR. In contrast, no specific amplification product was observed using DNA templates prepared from various mammalian cell lines. Frozen and cultured samples of several cell lines were tested for mycoplasma contamination to evaluate the utility of this method. Of 25 samples that tested positive for mycoplasma by Hoechst staining, which requires two passages of cell cultures started from frozen samples, mycoplasma was detected by real-time PCR in 24 samples of cell extracts prepared directly from frozen samples. When cultured samples were used for this assay, the accuracy of the diagnoses was further improved. Thus, this technique, which is simple, rapid, and sensitive enough for practical application, is suitable for handling many samples and for routine screening for mycoplasma contamination of cell cultures.
Topics: Animals; Benzothiazoles; Cell Culture Techniques; DNA, Bacterial; Diamines; Fluorescent Dyes; Humans; Mice; Mycoplasma; Mycoplasma Infections; Organic Chemicals; Polymerase Chain Reaction; Quinolines; Rats; Sensitivity and Specificity; Swine
PubMed: 16759150
DOI: 10.1290/0505035.1 -
Cytotherapy 2006Mycoplasma contamination is amongst the most frequently occurring problems associated with cell cultures. In order to meet the legal requirements (European Pharmacopoeia...
BACKGROUND
Mycoplasma contamination is amongst the most frequently occurring problems associated with cell cultures. In order to meet the legal requirements (European Pharmacopoeia and FDA) for Mycoplasma testing of cell lines and therapeutics, we have developed a PCR-based method to detect mycoplasms and introduce a validation concept.
METHODS
The PCR assay specifically amplifies a 280-bp DNA fragment of the gene coding for the 16S rDNA. Simultaneous amplification of an artificial oligonucleotide containing primer-binding sites allowed control of the efficacy of the PCR. The validation of the PCR assay was performed with two Mycoplasma reference strains, M. orale and M. pneumoniae. The validation concept included (i) cultivation of M. orale and M. pneumoniae in medium with an indicator for bacterial metabolism, (ii) determination of the color-changing units (CCU) in repeated dilution experiments and (iii) correlation of the PCR results with CCU values.
RESULTS
The detection range was found to include all Mycoplasma species most commonly found in cell cultures. The analytical sensitivity of the PCR was the CCU equivalent of 100 for M. orale and M. pneumoniae. Probit analysis revealed a detection probability of 9% for a mean concentration of 1222 (935-1844) CCU/mL for M. pneumoniae and 2547 (1584-10,352) CCU/mL for M. orale.
DISCUSSION
The validation of the Mycoplasma detection assay supported PCR as an attractive diagnostic tool that will help manage the important issue of Mycoplasma contamination of cell cultures.
Topics: DNA, Bacterial; Humans; Mycoplasma; Polymerase Chain Reaction; RNA, Ribosomal, 16S; Reproducibility of Results; Research Design; Self-Sustained Sequence Replication; Sensitivity and Specificity
PubMed: 16627346
DOI: 10.1080/14653240500518413 -
Journal of Microbiology (Seoul, Korea) Feb 2006In 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....
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.
Topics: Animals; Bacterial Typing Techniques; Base Sequence; DNA Primers; DNA, Bacterial; DNA, Ribosomal Spacer; Humans; Molecular Sequence Data; Mycoplasma; Polymerase Chain Reaction; RNA, Ribosomal, 16S; RNA, Ribosomal, 23S; Sensitivity and Specificity; Species Specificity
PubMed: 16554716
DOI: No ID Found -
Cancer Science Apr 2004We aimed to determine whether mycoplasmas are present in Korean chronic gastritis, and to understand their roles in gastric cancer tumorigenesis, because mycoplasmas... (Comparative Study)
Comparative Study
We aimed to determine whether mycoplasmas are present in Korean chronic gastritis, and to understand their roles in gastric cancer tumorigenesis, because mycoplasmas resemble Helicobacter pylori in terms of ammonia production and induction of inflammatory cytokines in immune and non-immune cells. The presence and identity of mycoplasmas were assessed by semi-nested PCR and sequencing, and the results were compared with pathologic data. Fifty-six samples collected from Korean chronic gastritis patients were used for this study. Twenty-three (41.1%) were positive for mycoplasmas. Eighteen sequenced samples contained a single human mycoplasma or two mycoplasmas, which were identified as Mycoplasma faucium (13/18), M. fermentans (3/18), M. orale (1/18), M. salivarium (2/18), and M. spermatophilum (1/18). Mycoplasma-infected chronic gastritis samples showed significantly more severe neutrophil infiltration than non-infected samples (P = 0.0135). Mycoplasma profiles in the oral cavity (M. salivarium is major) and stomach were different, and the presence of significant proinflammatory responses in mycoplasma-positive patients suggests that the mycoplasmas are not simply contaminants. Further studies are required to understand whether mycoplasmas play a role in gastric tumorigenesis.
Topics: Chronic Disease; DNA, Bacterial; Gastritis; Gastroscopy; Humans; Korea; Molecular Sequence Data; Mouth; Mycoplasma; Mycoplasma Infections; Mycoplasma fermentans; Mycoplasma salivarium; Organ Specificity; Pyloric Antrum; Sequence Analysis, DNA; Stomach; Stomach Neoplasms
PubMed: 15072588
DOI: 10.1111/j.1349-7006.2004.tb03208.x -
Applied and Environmental Microbiology Mar 2004We have developed a reverse line blot (RLB) hybridization assay to detect and identify the commonest mollicutes causing cell line contamination (Mycoplasma arginini,...
We have developed a reverse line blot (RLB) hybridization assay to detect and identify the commonest mollicutes causing cell line contamination (Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, and Acholeplasma laidlawii) and human infection (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum). We developed a nested PCR assay with "universal" primers targeting the mollicute 16S-23S rRNA intergenic spacer region. Amplified biotin-labeled PCR products were hybridized to membrane-bound species-specific oligonucleotide probes. The assay correctly identified reference strains of 10 mollicute species. Cell cultures submitted for detection of mollicute contamination, clinical specimens, and clinical isolates were initially tested by PCR assay targeting a presumed mollicute-specific sequence of the 16S rRNA gene. Any that were positive were assessed by the RLB assay, with species-specific PCR assay as the reference method. Initially, 100 clinical and 88 of 92 cell culture specimens gave concordant results, including 18 in which two or more mollicute species were detected by both methods. PCR and sequencing of the 16S-23S rRNA intergenic spacer region and subsequent retesting by species-specific PCR assay of the four cell culture specimens for which results were initially discrepant confirmed the original RLB results. Sequencing of amplicons from 12 cell culture specimens that were positive in the 16S rRNA PCR assay but negative by both the RLB and species-specific PCR assays failed to identify any mollicute species. The RLB hybridization assay is sensitive and specific and able to rapidly detect and identify mollicute species from clinical and cell line specimens.
Topics: Acholeplasma; Base Sequence; Cell Culture Techniques; DNA Primers; DNA, Ribosomal Spacer; Humans; Mycoplasma; Nucleic Acid Hybridization; Polymerase Chain Reaction; RNA, Bacterial; RNA, Ribosomal, 16S; RNA, Ribosomal, 23S; Sensitivity and Specificity; Tenericutes; Ureaplasma
PubMed: 15006769
DOI: 10.1128/AEM.70.3.1483-1486.2004 -
Journal of Oral and Maxillofacial... Oct 2003The objective of this study was to find any relation between the presence of specific bacterial species in the synovial fluid of the temporomandibular joint (TMJ) and...
PURPOSE
The objective of this study was to find any relation between the presence of specific bacterial species in the synovial fluid of the temporomandibular joint (TMJ) and clinical parameters.
PATIENTS AND METHODS
We studied 43 patients (male-to-female ratio, 1:1.69; average age, 34.37 +/- 14.55 years). Thirty-three patients had a displaced disc in the TMJ (DD group), and 10 patients did not have a displaced disc of the TMJ or any symptom related to TMJ disorders (NDD group). Clinical examinations were made to determine maximum mouth opening, joint sounds, previous trauma history, systemic disease, and TMJ pain. Six bacterial species that were reported in other studies were chosen to evaluate the presence of bacteria in the TMJ for this study.
RESULTS
Mycoplasma genitalium was most frequently detected in synovial fluid (86.0%). Staphylococcus aureus, Mycoplasma fermentans/orale, Actinobacillus actinomycetemcomitans, and Streptococcus mitis were detected in 51.2%, 37.2%, 25.6%, and 7.0% of samples, respectively. beta-Hemolytic Streptococcus was not detected. The prevalence of S aureus was significantly higher in the DD group than in the NDD group (P <.05). The patients who had M. fermentans/orale were 5.40 times more likely to be younger than 30 years than were those without M. fermentans/orale (P <.05). Those with M. genitalium were 5.81 times more likely to be female than were those without M. genitalium (P <.05).
CONCLUSION
The presence of S. aureus in TMJ synovial fluid was related to TMJ disorder symptoms and clinical parameters seemed to be influenced by bacterial presence in TMJ synovial fluid.
Topics: Adult; Aggregatibacter actinomycetemcomitans; Analysis of Variance; DNA, Bacterial; Facial Pain; Female; Humans; Logistic Models; Male; Mycoplasma; Odds Ratio; Range of Motion, Articular; Staphylococcus aureus; Streptococcus mitis; Synovial Fluid; Temporomandibular Joint; Temporomandibular Joint Disorders
PubMed: 14586850
DOI: 10.1016/s0278-2391(03)00674-8 -
Current Microbiology Apr 2003In a previous study, using a primer set designed from Mycoplasma fermentans, we amplified a PCR fragment from Mycoplasma orale similar to the 206-bp DNA fragment... (Comparative Study)
Comparative Study
In a previous study, using a primer set designed from Mycoplasma fermentans, we amplified a PCR fragment from Mycoplasma orale similar to the 206-bp DNA fragment amplified from M. fermentans insertion-sequence-like element (ISLE). The presence of this similar ISLE fragment has the potential to cause confusion in the PCR diagnosis of M. fermentans and M. orale, which have significantly different clinical scenarios. An ISLE from three different M. orale strains was amplified by using a primer set designed from sequence within the left and right terminal stem and loop (S&L) structures flanking the ISLE of M. fermentans. Sequence analysis showed that the M. orale ISLE is 93% identical to that of M. fermentans at the nucleotide level and codes for two open reading frames also found in the M. fermentans ISLE. This is the first finding that two different mycoplasma species harbor highly homologous IS elements. This finding has great significance in clinical diagnosis and suggests a possibility of horizontal transfer of an IS element between two different mycoplasma species.
Topics: Amino Acid Sequence; Cloning, Molecular; DNA Transposable Elements; Molecular Sequence Data; Mycoplasma; Mycoplasma fermentans; Open Reading Frames; Sequence Homology, Amino Acid
PubMed: 12732982
DOI: 10.1007/s00284-002-3848-9 -
The Bulletin of Tokyo Dental College Nov 2002It has been suggested that infection by some mycoplasma species can act as possible cofactors in the acceleration of immunodeficiency in HIV-infected patients. The...
It has been suggested that infection by some mycoplasma species can act as possible cofactors in the acceleration of immunodeficiency in HIV-infected patients. The present study was designed to examine infections by oral mycoplasma species in HIV-seropositive (HIV(+)) patients. Mycoplasma salivarium and Mycoplasma orale were isolated from 59.5% and 16.7% of 42 HIV(+) patients, respectively. Non-M. salivarium and non-M. orale species were isolated from 40.5% of saliva samples from the HIV(+) group and 20.8% of those from 24 HIV-seronegative (HIV(-)) subjects, respectively. Although the production of superantigen by human peripheral lymphocytes in the isolated mycoplasma species from HIV(+) and HIV(-) subjects was evaluated, none of the examined mycoplasma strains, including ATCC strains of M. salivarium, M. orale, Mycoplasma buccae and Mycoplasma penetrans, were found to produce superantigen. Production of heat shock proteins (HSPs) by isolated mycoplasma strains was examined by immunoblotting using monoclonal antibodies against Helicobacter pylori HSP60. It was found that all the strains of M. salivarium, M. orale, and unidentified mycoplasma species isolated from HIV(+) and HIV(-) groups produced heat shock proteins. HSP production by oral mycoplasma may play a role in the immunomodulation of HIV(+) patients.
Topics: AIDS-Related Opportunistic Infections; Adult; Antigens, Bacterial; Bacterial Typing Techniques; Case-Control Studies; Electrophoresis, Polyacrylamide Gel; Female; HIV Seropositivity; Heat-Shock Proteins; Humans; Immunoblotting; Lymphocytes; Male; Middle Aged; Mycoplasma; Mycoplasma Infections; Polymerase Chain Reaction; Saliva; Superantigens
PubMed: 12687728
DOI: 10.2209/tdcpublication.43.231 -
Disseminated Mycoplasma orale infection in a patient with common variable immunodeficiency syndrome.Diagnostic Microbiology and Infectious... Oct 2002Human infection with Mycoplasma species other than M. pneumoniae are infrequent, but may be encountered in patients with immunodeficiencies. We report a patient with...
Human infection with Mycoplasma species other than M. pneumoniae are infrequent, but may be encountered in patients with immunodeficiencies. We report a patient with combined variable immunodeficiency that developed multiple abscesses and destructive bone disease caused by M. orale, an organism generally considered to be non-pathogenic. Molecular laboratory methods, 16S rRNA sequence analysis, were used to detect the organism directly in the surgical specimen and confirmed following isolating of the organism. This case demonstrates the importance of molecular technology in the diagnosis of difficult infectious disease problems.
Topics: Administration, Oral; Adult; Bacteremia; Common Variable Immunodeficiency; Doxycycline; Follow-Up Studies; Humans; Magnetic Resonance Imaging; Male; Mycoplasma; Mycoplasma Infections; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Treatment Outcome
PubMed: 12458129
DOI: 10.1016/s0732-8893(02)00429-7 -
Comparative Medicine Aug 2002Mycoplasma haemomuris, a small pleomorphic bacterium parasitic of red blood cells, often causes chronic and subclinical infection of rodents. Mycoplasma haemomuris is...
BACKGROUND AND PURPOSE
Mycoplasma haemomuris, a small pleomorphic bacterium parasitic of red blood cells, often causes chronic and subclinical infection of rodents. Mycoplasma haemomuris is uncultivable, and a serologic testing method is not readily available. The purpose of the study reported here was to develop a sensitive and specific polymerase chain reaction (PCR) test for detection of M. haemomuris in blood samples.
METHODS
On the basis of the regions of the M. haemomuris 16S rRNA gene most divergent from corresponding regions of related bacteria, M. haemomuris-specific primers were designed so that these primers could selectively amplify M. haemomuris DNA. A PCR test was performed, using blood samples from BALB/c mice infected with M. haemomuris strains TR 8564, TR 8563, and TR 8556.
RESULTS
Use of the PCR test enabled detection of M. haemomuris DNA in a minimum of 0.0001 microl of infected mouse blood. The test also was specific for M. haemomuris and did not amplify closely related species, such as M. haemofelis, M. haemosuis, M. orale, or Anaplasma marginale.
CONCLUSION
This method is sensitive and specific for detection of M. haemomuris.
Topics: Animals; Erythrocytes; Male; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Mycoplasma; Mycoplasma Infections; Polymerase Chain Reaction; RNA, Ribosomal, 16S; Sensitivity and Specificity
PubMed: 12211273
DOI: No ID Found