Mycoplasmas In Cell Cultures Research Articles

Development and Evaluation of a New qPCR Assay for the Detection of Mycoplasma in Cell Cultures.

In recent years, cell culture has become an important tool not only in research laboratories, but also in diagnostic and biotechnological development laboratories. Mycoplasma contamination is present in up to 35% of cell cultures used in research and in cell therapies. This fact represents a significant problem since such contamination can cause disastrous effects on eukaryotic cells by altering their cellular parameters, which, in turn, can lead to unreliable experimental results. For this reason, it is mandatory to carry out continuous testing for the presence of Mycoplasma in cell culture and the development of appropriate methodologies for this purpose. An ideal detection methodology should be fast, sensitive, and reliable. In this study, we propose an alternative detection method based on real-time PCR in conjunction with a novel combination of primers and probes that have been improved to increase their efficiency. The new PCR method demonstrates 100% sensitivity and specificity results in the detection of common Mycoplasma species that contaminate cell cultures. Whilst 11 of 45 tested supernatants were positive for Mycoplasma (24.4%) using the new PCR method (corresponding to 5 of the 14 lines tested (35.71%)), only 10 of 45 supernatants showed positive results with the commercial Venor®GeM qEP and Plasmotest® kit. In addition, the new PCR method exhibits a high capacity to detect less-frequent Mycoplasma species, such as those related to the M. mycoides cluster. The use of an alternative Mycoplasma-detection method in cell culture labs can guarantee the detection of Mycoplasma contamination, especially in cases when dubious results are recorded.

Identification of cell culture contamination by an unusual species of Mycoplasma related to the M. mycoides cluster.

Mycoplasma contamination is a significant problem in cell culture replication and maintenance. From more than 200 known species, a limited number of Mycoplasma species have been detected in cell cultures, representing new species or variants that can escape detection systems. A qPCR commercial kit was used for Mycoplasma detection in cell cultures. Furthermore, an amplified Mycoplasma species was sequenced and summited for sequence assembly, clustering, and evolutionary analysis study. Our work has identified a new and unusual variant or species of Mycoplasma that possesses a high degree of homology with species related with M. mycoides cluster. This variant is usually associated with cattle but has been detected contaminating a cell culture. Mycoplasma testing (even for unusual species) in cell cultures is essential to ensure the validity and reproducibility of research that uses cell cultures and to ensure the quality of cell line deposits in biobanks. For this reason, it is necessary to perform continuous checks for the absence of Mycoplasma in cell cultures and engage in the continuous adaptation of relevant detection systems.

Apply Mycoflour kit for detection of Mycoplasma in cell lines at National Institute for Control of Vaccines and Biologicals

Control of Mycoplasma in cell culture used in testing is necessary to ensure the accuracy of quality control and vaccine release. Research on fluorescent dyeing process using Mycoplasma detection cell indicators to develop standard operating procedures before routinely used at National Institute for Control of Vaccines and Biologicals (NICVB). Outcome showed that the parameters of the Mycoplasma detection procedure by the Vero cell indicator method using the MycoFlourTM kit at NICVB were optimized for the concentration of Vero cells inoculated into coverslip 10 x104 TB/ml; Wash cells twice with PBS (-)1X and meet the requirements for specificity in cell quality assessment.

Aptamer Cocktail to Detect Multiple Species of Mycoplasma in Cell Culture.

Mycoplasma contamination of cell line cultures is a common, yet often undetected problem in research laboratories. Many of the existing techniques to detect mycoplasma contamination of cultured cells are time-consuming, expensive, and have significant drawbacks. Here, we describe a mycoplasma detection system that is useful for detecting multiple species of mycoplasma in infected cell lines. The system contains three dye-labeled detection aptamers that can specifically bind to mycoplasma-infected cells and a dye-labeled control aptamer that minimally binds to cells. With this system, mycoplasma-contaminated cells can be detected within 30 min by using a flow cytometer, fluorescence microscope, or microplate reader. Further, this system may be used to detect mycoplasma-contaminated culture medium. This study presents an novel mycoplasma detection model that is simple, rapid, inexpensive, and sensitive.

Molecular genetic techniques and oligonucleotides for mycoplasma identification – a review

Control of distribution of mycoplasmal infections in cattle herds is essential in the majority of countries world-wide. Various PCR procedures are available to detect mycoplasmas in cell cultures and bovine mycoplasma in different types of samples. We reviewed some common PCR techniques and specific primers targeted to different bacterial genetic regions of mycoplasma. Several researchers used the same PCR approach and Mycoplasma spp. as a target but their results could not be compared because different primer pairs were used. These methods and primers were first developed to identify mycoplasma species that contaminate animal cell cultures, and then were used by other researchers to differentiate mycoplasmas as a cow infecting agent. Our analysis of the specificity of these primer pairs to nucleotide sequences of five Mycoplasma spp. showed that oligonucleotides have less specificity to them. Numerous commercially available PCR kits are applicable to find mycoplasma contamination in cell cultures and fewer of them can be used in veterinary diagnostics. Although serological and culture techniques are still used, it is necessary to develop a new multiplex PCR technique with a more specific primer set especially in agrarian countries.

Testing for mycoplasma in cell culture by PCR

Testing for mycoplasma in cell culture by PCR

Detection of Mycoplasma in cell cultures

Mycoplasma is a prokaryotic organism that is a frequent and occult contaminant of cell cultures. This organism can modify many aspects of cell physiology, rendering experiments that are conducted with contaminated cells worthless. Because of their small size, Mycoplasmas can pass through filters used to prevent bacterial and fungal contamination and potentially spread to all the cultures in a laboratory. It is essential that all new cell cultures entering a laboratory and all cell banks are tested for the presence of Mycoplasma. It is recommended that two techniques be used, selected from a PCR-based method, indirect staining and an agar and broth culture. This protocol describes these three tests for detecting Mycoplasma, which take from 1 d to 3-4 weeks, and such tests should be an obligatory component of quality control in every tissue culture laboratory.

Rapid and sensitive detection of Mollicutes in cell culture by polymerase chain reaction

Infections with Mollicutes species (such as Mycoplasma, Acholeplasma, and Ureaplasma) can induce a variety of problems in living organisms and laboratory cell cultures. Therefore, it is necessary to establish a routine diagnostic protocol for Mycoplasma infection in order to ensure reliable research results, as well as the safety of commercial biological products. For that purpose a novel PCR-based procedure using specific designed primers complementary to 16S rRNA genome region of mollicute species was evaluated. PCR was optimized and sensitivity and specificity was evaluated by defined cell count concentrations (2-31250 CFU/ml) of different strains of Mycoplasma, Acholeplasma and Ureaplasma. Amplicon (272 bp) was cloned by PCR-cloning and sequenced by dideoxy chain termination. PCR, was found to be able to detect 10 copies of mollicute target DNA. No cross-reactivity with genomic DNA of non-mollicute bacteria or human cell lines was observed. Forty seven human and animal cell lines were evaluated for mollicute contamination. Twenty five cell lines (53%) were correctly identified as contaminated by this molecular approach. The results of this study demonstrated that this PCR-based method is not only fast and reproducible, but also highly sensitive and specific for detecting contaminant mycoplasmas in cell cultures.

Semi-automated relative quantification of cell culture contamination with mycoplasma by Photoshop-based image analysis on immunofluorescence preparations

Mycoplasma contamination in cell culture is a serious setback for the cell-culturist. The experiments undertaken using contaminated cell cultures are known to yield unreliable or false results due to various morphological, biochemical and genetic effects. Earlier surveys revealed incidences of mycoplasma contamination in cell cultures to range from 15 to 80%. Out of a vast array of methods for detecting mycoplasma in cell culture, the cytological methods directly demonstrate the contaminating organism present in association with the cultured cells. In this investigation, we report the adoption of a cytological immunofluorescence assay (IFA), in an attempt to obtain a semi-automated relative quantification of contamination by employing the user-friendly Photoshop-based image analysis. The study performed on 77 cell cultures randomly collected from various laboratories revealed mycoplasma contamination in 18 cell cultures simultaneously by IFA and Hoechst DNA fluorochrome staining methods. It was observed that the Photoshop-based image analysis on IFA stained slides was very valuable as a sensitive tool in providing quantitative assessment on the extent of contamination both per se and in comparison to cellularity of cell cultures. The technique could be useful in estimating the efficacy of anti-mycoplasma agents during decontaminating measures.

Tandem use of immunofluorescent and DNA staining assays to validate nested PCR detection of mycoplasma

Mycoplasma contamination in cell culture is a serious setback to cell culturists across the world with a very high rate of reported occurrence particularly because of difficult early detection. Out of a variety of detection methods known, the double-step nested polymerase chain reaction (PCR)-based detection of mycoplasma in cell culture has been critically viewed upon because of chances of producing reliable results. A nested PCR technique, described to detect a large range of cell-culture-contaminating mycoplasma species, with greater sensitivity to detect as low a contamination as a few organisms, was compared with the results from two cytological techniques employed in tandem. These are DNA staining using Hoechst, the gold standard, and an immunofluorescent assay using a highly specific monoclonal antibody. The study undertaken on randomly collected cell cultures revealed a false-negative and several false-positive results in comparison to the cytological methods employed. The observations were particularly more unambiguous with the immunofluorescent assay employed in the study while simultaneously employed Hoechst staining serving as an indicator of bacterial contamination. There is a general apprehension that genus-specific PCR approaches could be associated with inaccurate outcome and only species-specific PCRs may be satisfactory in routine screening for mycoplasma contamination in cell cultures. At this juncture, it may be suggested that caution must be exercised while adopting the two-step nested PCR-based detection approaches, and the simultaneous employment of cytological methods used in this investigation could prove to be practicable in the proper interpretation of results.

Detection of multiple mycoplasma infection in cell cultures by PCR

A total of 301 cell cultures from 15 laboratories were monitored for mycoplasma (Mollicutes) using PCR and culture methodology. The infection was detected in the cell culture collection of 12 laboratories. PCR for Mollicutes detected these bacteria in 93 (30.9%) samples. Although the infection was confirmed by culture for 69 (22.9%) samples, PCR with generic primers did not detect the infection in five (5.4%). Mycoplasma species were identified with specific primers in 91 (30.2%) of the 98 samples (32.6%) considered to be infected. Mycoplasma hyorhinis was detected in 63.3% of the infected samples, M. arginini in 59.2%, Acholeplasma laidlawii in 20.4%, M. fermentans in 14.3%, M. orale in 11.2%, and M. salivarium in 8.2%. Sixty (61.2%) samples were co-infected with more than one mycoplasma species. M. hyorhinis and M. arginini were the microorganisms most frequently found in combination, having been detected in 30 (30.6%) samples and other associations including up to four species were detected in 30 other samples. Failure of the treatments used to eliminate mycoplasmas from cell cultures might be explained by the occurrence of these multiple infections. The present results indicate that the sharing of non-certified cells among laboratories may disseminate mycoplasma in cell cultures.

Detecting Mycoplasma contamination in cell cultures by polymerase chain reaction.

The detection of mycoplasmas in human and animal cell cultures is mandatory for every cell culture laboratory, because these bacteria are common contaminants, persist unrecognized in cell cultures for many years, and affect research results as well as the purity of cell culture products. The reliability of the mycoplasma detection depends on the sensitivity and specificity of the method and should also be convenient to be included in the basic routine of cell culture quality assessment. Polymerase chain reaction (PCR) detection is one of the acknowledged methodologies to detect mycoplasmas in cell cultures and cell culture products. Although the PCR offers a fast and simple technique to detect mycoplasmas, the method is also susceptible to errors and can produce false positive as well as false-negative results. Thus, the establishment and the routine application of the PCR assay require optimization and the inclusion of the appropriate control reactions. The presented protocol describes sample preparation, DNA extraction, PCR run, the analysis of the PCR products, and speciation of the contaminant. It also provides detailed information on how to avoid artifacts produced by the method. Established properly, PCR is a reliable, fast, and sensitive method and should be applied regularly to monitor the contamination status of cell cultures.

Detection of mycoplasmas in cell cultures by cultural methods.

Detection of mycoplasmas in cell cultures by cultural methods.

Detection of mycoplasmas in cell cultures by fluorescence methods.

AbstractThe fluorescence stains used most often for mycoplasma detection are DNA binding fluorochromes (DNAFs) and fluoresceinated antibodies. Both permit direct visualization of individual mycoplasma organisms. DNAFs will bind to any appropriately conformed DNA that is present in a sample preparation and are, therefore, not specific for mycoplasmas. Conversely, fluoresceinated antibodies are highly specific for mycoplasmas, discriminating to species level. The two stains can be used individually or in combination, and they can be used either directly on the cells of interest or they may be used in a system involving mycoplasma-free indicator cells, which further enhances the diagnostic reliability of tests utilizing these fluorescent reagents.KeywordsCover SlipEvans BlueIndicator CellMycoplasma SpeciesMycoplasma HominisThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Quantitative detection of cell culture Mycoplasmas by a one step polymerase chain reaction method

A rapid, sensitive assay was developed that can detect the six species of Mycoplasmas that account for the vast majority of cell culture infections. This assay, a modification of the method published by Wong-Lee & Lovett [1], allows direct evaluation of culture medium by a single-step PCR method that utilizes primers complementary to conserved 16S rRNA sequences. Extensive testing of medium from uninfected cultures spiked with purified Mycoplasma DNAs showed that the method described in this report can detect the equivalent of one Mycoplasma in 15 μl culture medium; thus, evaluation of a single culture sample allows detection of Mycoplasmas in cultures infected with the equivalent of 10 or more Mycoplasmas per 15 μl (or ≥6.7×102 Mycoplasma equivalents/ml) with greater than 99.99% confidence. Comparison of results obtained with this PCR-based assay and a standard biological colony-forming assay revealed that the PCR assay is capable of detecting 0.0015-0.015 colony forming units, suggesting that the PCR assay may also be detecting nonviable Mycoplasmas. The high level of amplification achieved with this method allows direct detection of amplification products by ethidium bromide staining of agarose gels, and thus allows rapid screening of cell cultures.

Use of merocyanine 540 and Hoechst 33258 for the selective killing of contaminating mycoplasmas in cell cultures

Mycoplasma infection can substantially affect the biological properties of cells in vitro. We have devised a method for the selective killing of mycoplasmas, e.g., A. laidlawii, M. fermentans M. hyorhinis and M. arginini, from experimentally infected cell cultures. This approach is based on the differential binding of the lipophilic fluorescent probe Merocyanine 540 followed by illumination with visible light. The efficiency of the procedure depends on the Merocyanine 540 concentration, the intensity of illumination, and the presence of oxygen in the medium. When A. laidlawii contaminated correal endothelial cell cultures were treated simultaneously with Merocyanine 540 and DNA-binding fluorochrome Hoechst 33258 and then illuminated, a significant degree of eradication was observed, even after one cycle of treatment. This combined treatment is therefore recommended as an effective method of purging mycoplasmas from contaminated cultures.

Detection of mycoplasmas in cell cultures by PCR: a one year study

Over a one year period, 372 different cell cultures were received for the detection of mycoplasmas. PCR analysis of the 16SS rRNA gene was compared to DNA fluorescence staining by DAPI (4′,6-diamidine-2-phenylindole dihydrochloride). 278 samples (75%) were found to be negative using both methods, 86 samples (23%) were found positive with the PCR and the DAPI staining method, and 6 samples (2%) were found to be infected with other bacteria. The 86 positive samples were also subjected to specific amplification in order to identify the species. Only two of them (3.5%) could not be identified. In conclusion, PCR is a rapid and reliable method for detecting and identifying mycoplasmas in tissue cultures.

Detection of mycoplasma contamination in cell cultures by a mycoplasma group-specific PCR.

The suitability of a 16S rRNA-based mycoplasma group-specific PCR for the detection of mycoplasma contamination in cell cultures was investigated. A total of 104 cell cultures were tested by using microbiological culture, DNA fluorochrome staining, DNA-rRNA hybridization, and PCR techniques. A comparison of the results obtained with these techniques revealed agreement for 95 cell cultures. Discrepant results, which were interpreted as false negative or false positive on the basis of a comparison with the results obtained with other methods, were observed with nine cell cultures. The microbiological culture technique produced false-negative results for four cell cultures. The hybridization technique produced false-negative results for two cell cultures, and for one of these cell cultures the DNA staining technique also produced a false-negative result. The PCR may have produced false-positive results for one cell culture. Ambiguous results were obtained with the remaining two cell cultures. Furthermore, the presence of contaminating bacteria interfered with the interpretation of the DNA staining results for 16 cell cultures. For the same reason the hybridization signals of nine cell cultures could not be interpreted. Our results demonstrate the drawbacks of each of the detection methods and the suitability of the PCR for the detection of mycoplasmas in cell cultures.

Detection and tentative identification of dominant mycoplasma species in cell cultures by restriction analysis of the 16S-23S rRNA intergenic spacer regions

Mycoplasma (class Moliicutes) is one of the most common contaminants of cell cultures (McGarrity et aL, 1992). It is not always easy to find mycoplasma contamination in cell culturcs because the changes are not apparent. Viral s~ocks harvested from mycoplasma-contaminated celle cultures may be contaminated with a corresponding mycoplasma species; one recent case is that of the human immunodeficiency virus (HIV) type l which was grown in a lymphoblastoid cell line contaminated with Mycoplasma fermentans (Lemaitre et al., 1992). Live virus vaccines prepared from mycoplasmacontaminated cell cultures may become contaminated with mycoplasmas. Biopharmaceutical drugs produced usag animal cell cultures may also contain myceplasma cells. Mycoplasma contamination of virus vaccines and drugs may be a serious concern because mycoplasmas are currently suspected cofactors of AIDS (Lo, 1992). Another problem is that mycoplasmas can induce various cytokines including tumour necrosis factor, which may enhance HIV replication (Chowdhuri et aL, 1990). Most cytokineproducing cell lines are not only contaminated with mycoplasmas but also induced by mycoplasmas themselves (McGarrity et al., 1992). Thus it is imperative to examine mycoplasma contamination of cell cultures using reliable methods. Although various procedures are now available to detect mycoplasmas in cell cultures (Uphoff et al., 1992), serological and culture methods are still indispensable for the identification of mollicute species. Five mollicute species, M. fermentans, M. hyorhinis, M. orale, M. arginini, and Acholeplasma laidlawii, are known to account for more than 95 % of the mycoplasma contamination in cell cultures (McGarrity et aL, 1992). We developed a sent, tive and practical method to identify as v.c2! ,~ to detect these mycoplasma species which c o n taminate animal cell cultures.

Staining with an Acridine Orange Derivative for the Detection of Mycoplasmas in Cell Cultures

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