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1.
Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention 总被引:11,自引:0,他引:11
The contamination of cell cultures by mycoplasmas remains a major problem in cell culture. Mycoplasmas can produce a virtually
unlimited variety of effects in the cultures they infect. These organisms are resistant to most antibiotics commonly employed
in cell cultures. Here we provide a concise overview of the current knowledge on: (1) the incidence and sources of mycoplasma
contamination in cell cultures, the mycoplasma species most commonly detected in cell cultures, and the effects of mycoplasmas
on the function and activities of infected cell cultures; (2) the various techniques available for the detection of mycoplasmas
with particular emphasis on the most reliable detection methods; (3) the various methods available for the elimination of
mycoplasmas highlighting antibiotic treatment; and (4) the recommended procedures and working protocols for the detection,
elimination and prevention of mycoplasma contamination. The availability of accurate, sensitive and reliable detection methods
and the application of robust and successful elimination methods provide powerful means for overcoming the problem of mycoplasma
contamination in cell cultures.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
2.
Ishikawa Y Kozakai T Morita H Saida K Oka S Masuo Y 《In vitro cellular & developmental biology. Animal》2006,42(3-4):63-69
Summary 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 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 evaluated 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, in suitable for handling many samples and for routine
screening for mycoplasma contamination of cell cultures. 相似文献
3.
Abstract Mycoplasma contamination of cell cultures is a menace to diagnostic and research procedures. Rapid and reliable detection methods are, therefore, sorely needed. After comparing 16S rRNA sequences from those mycoplasmas that contaminate cell cultures, three different oligonucleotide probes were constructed. Two of these probes were designed to be group-specific and one to be species-specific. The three oligonucleotide probes were designed to cover all mycoplasmas commonly isolated from cell cultures. Contaminated cell lines could easily be detected by a direct filter hybridization assay in which the probes were incubated jointly. The assay proved to be rapid and sensitive with the possibility to perform and evaluate the mycoplasma testing within one working day. 相似文献
4.
Transmission electron microscopy has been employed for the rapid detection of mycoplasma in sera and cell cultures. High speed centrifugation of sera or low speed centrifugation of cell debris, followed by negative staining of the resuspended pellet, detected mycoplasma contamination more frequently than a culture method followed by direct fluorescence (DAPI), which was used as a control procedure. The appearance of the mycoplasma cell border and content gives some information about particle viability. 相似文献
5.
A high-sensitive, easy and rapid proximate method has been developed to reveal mycoplasmas in the monolayer cell cultures using home fluorescent antibiotic olivomycin. This method has been used to screen many cell lines with its high effectiveness being shown. As based on this method the following control methods are worked out: indication of mycoplasmas in animal blood sera, used for the cultivation of cell lines detection of contaminant microorganisms of the cell cultures (fungi and bacteria) and human ureaplasmas. 相似文献
6.
Comparative PCR analysis for detection of mycoplasma infections in continuous cell lines 总被引:8,自引:0,他引:8
Mycoplasma contamination of cell lines is one of the major problems in cell culturing. About 15-35% of all cell lines are infected with a limited number of mycoplasma species of predominantly human, swine, or bovine origin. We examined the mycoplasma contamination status in 495 cell cultures by polymerase chain reaction (PCR) assay, microbiological culture method, and deoxyribonucleic acid-ribonucleic acid (DNA-RNA) hybridization, and in 103 cell cultures by PCR and DNA-RNA hybridization, in order to determine the sensitivity and specificity of the PCR assay in routine cell culture. For those two cohorts, results for the three or two assays were concordant in 92 and 91% of the cases, respectively. The sensitivity (detection of true positives) of this PCR detection assay was 86%, and the specificity (detection of true negatives) was 93%, with positive and negative predictive values (probability of correct results) of 73 and 97%, respectively. PCR defined the mycoplasma status with 92% accuracy (detection of true positives and true negatives). The mycoplasma contaminants were speciated by analyzing the PCR amplification fragment using several restriction enzymes. Most of the cultures (47%) were infected with Mycoplasma fermentans, followed by M. hyorhinis (19%), M. orale (10%), M. arginini (9%), Acholeplasma laidlawii (6%), and M. hominis (3%). To sum up, PCR represents a sensitive, specific, accurate, inexpensive, and quick mycoplasma detection assay that is suitable for the routine screening of cell cultures. 相似文献
7.
The contamination of cell lines with mycoplasmas is certainly one of the major problems occurring in cultured cells. Analyzing more than 460 human leukemia-lymphoma (LL) cell lines, we found that 28% of the cultures were mycoplasma-positive. Mycoplasmas can produce extensive changes, growth arrest and cell death in the infected cultures. While mycoplasma-infected cell lines can be truly cleansed from the contaminants, all the efforts would be in vain when the cells return to a mycoplasma-infested environment or are handled with unsuitable culture practices. Hence, the main focus of mycoplasma control should be on preventing cell culture contamination. Mycoplasmas can be introduced through several routes including culture reagents and laboratory personnel. Cross-contamination from infected cell cultures within one laboratory continues to be the major source for the spread of mycoplasma. Specific technical protocols and cell culturing guidelines may be followed in order to minimize the risk of mycoplasma contamination of cell lines. This "good culture practice" is of utmost importance as faulty cell culture techniques appear to be also the main reason for the high incidence of cross-contaminated LL cell lines which according to our experience using DNA fingerprinting of some 500 LL cell lines is about 15%. 相似文献
8.
Infection of cell cultures by mycoplasmas can be detected by hybridization of the DNA of suspected cell cultures with recombinant plasmids containing fragments of the mycoplasma DNA. The test is very sensitive and allows detection of as little as 1 ng of mycoplasmal DNA, roughly equivalent to the DNA amount of 10(6) mycoplasmas. This approach turns out to be effective for detection and identification of mycoplasmas in clinical material, plant and insect tissues. A set of DNA probes for detection of mycoplasmas infecting cell cultures by dot hybridization has been constructed. This set consists of specific DNA probes and universal DNA probe. Recombinant plasmids, pAl32, pMa13, pMh9, containing specific DNA fragments of Acholeplasma-laidlawii, Mycoplasma arginini, Mycoplasma hominis (the prevalent mycoplasma contaminants of home cell cultures) are species-specific DNA probes. Recombinant plasmid pMg16 containing rRNA genes of Mycoplasma gallisepticum is the universal DNA probe for detection of any mycoplasma (or any prokaryote) contaminations. These two classes of DNA probes may be considered as complementing each other. These 32P labeled probes do not hybridize with eukaryotic DNA. The set of DNA probes allows not only to detect infection of cell cultures by mycoplasmas but also to identify the species of mycoplasmas and to evaluate the multiplicity of mycoplasma infection. 相似文献
9.
In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain 总被引:97,自引:0,他引:97
T. R. Chen 《Experimental cell research》1977,104(2):255-262
A simple, fast, and easily reproducible routine laboratory technique for detecting mycoplasma contamination in cell cultures is reported. Cells grown on a coverslip are fixed directly with Carnoy's, air-dried, stained with DNA-specific fluorescent Hoechst 33258, and examined microscopically. All cultures that were infected with mycoplasmas had readily discernible, small, morphologically uniform, bright fluorescent bodies in the extranuclear and intercellular space in contrast to the non-contaminated control cultures in which the extra-nuclear background appeared uniformly dark. To probe the degree of sensitivity to detect mycoplasmas, control cultures were infected with aliquots from serially diluted cells or media collected from Mycoplasma hyorhinus infected cultures. The lowest infection rate (0.40% by sampling 1 000 cells in average per culture 4–24 h after infection) scored presently, however, can easily be lowered by increasing sample size since a cell infected with even one mycoplasma can be discerned. These mycoplasmas resisted centrifugation at 2 500 rpm for 30 min and easily filtered through 0.22 μm pore-size filter membrane. Amazingly infection rate of 0.63% scored from 24 h post-infection incubation attained 100% contamination with several hundreds of mycoplasmas per host cell within 120 h. 相似文献
10.
Molla Kazemiha V Azari S Amanzadeh A Bonakdar S Shojaei Moghadam M Habibi Anbouhi M Maleki S Ahmadi N Mousavi T Shokrgozar MA 《Cytotechnology》2011,63(6):609-620
Mycoplasma contamination is a deleterious event for cell culture laboratories. Plasmocin™ is used to prevent and eradicate
mycoplasma infections from cell. In this study, 80 different mammalian cell lines from various sources; human, monkey, mice,
hamster and rat were used to study and evaluate plasmocin™ efficiency and compare it to commonly used antibiotics such as
BM-cyclin, ciprofloxacin and mycoplasma removal agent (MRA). It was shown that mycoplasma infections were eradicated by plasmocin™,
BM-cyclin, ciprofloxacin and MRA in 65%, 66.25%, 20%, and 31.25%, respectively, of infected cell cultures. However, re-infection
with mycoplasmas after the period of 4 months occurred in 10–80% of the studied cell lines. Cell cytotoxicity and culture
death was observed in 25, 17.5 and 10% of the treated cells, for plasmocin™, BM-cyclin and MRA, respectively. In this study,
Plasmocin™ showed strong ability to eradicate mollicutes from our cell lines with minimal percentage of regrowth. However,
due to its high cell cytotoxicity it should be used with caution especially when dealing with expensive or hard-to-obtain
cell lines. Amongst the antibiotics tested, BM-cyclin was shown to remove mycoplasma with the highest efficiency. 相似文献
11.
Detection of mycoplasma contamination in cell cultures by a mycoplasma group-specific PCR. 总被引:6,自引:3,他引:3
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F J van Kuppeveld K E Johansson J M Galama J Kissing G Blske J T van der Logt W J Melchers 《Applied microbiology》1994,60(1):149-152
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. 相似文献
12.
George P. Studzinski John F. Gierthy Jolanta J. Cholon 《In vitro cellular & developmental biology. Plant》1973,8(6):466-472
Summary Autoradiographic detection of incorporation of tritiated thymidine into the cytoplasm of cultured mammalian cells has been
evaluated as a test of contamination of the cultures by cell-associated microorganisms, which usually are mycoplasmas. Criteria
which indicate the presence of cell-associated mycoplasmas have been established, and the reliability of the standardized
autoradiographic method has been assessed by testing the same cultures by two colony formation methods of mycoplasmal detection.
The autoradiographic method demonstrated cell-associated microorganisms in all cultures from which characteristic colonies
were grown on mycoplasma agar. The autoradiographic method did not produce false positive results, and the outcome of this
test was evident in 3 days as opposed to 7 to 14 days by agar culture methods. Some applications of the autoradiographic method
are shown, and it is suggested that this method be employed for routine surveillance for mycoplasmal contamination in laboratories
where facilities for frequent agar culture tests are not easily available.
This research was supported by U.S. Public Health Service Grants CA 12351-02 and CA 12334-01 from the National Cancer Institute. 相似文献
13.
Taylor-Robinson, David (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), Otakar Sobeslavsky, and Robert M. Chanock. Relationship of Mycoplasma pneumoniae to other human Mycoplasma species studied by gel diffusion. J. Bacteriol. 90:1432-1437. 1965.-Conditions are presented for the production of four lines of precipitate between Mycoplasma pneumoniae antigen and homologous hyperimmune rabbit serum in double diffusion in agar. The specificity of the reaction was shown by the fact that M. pneumoniae antigen did not react with antisera to the other human mycoplasma species, nor did M. pneumoniae antiserum produce lines with antigens prepared from the other human mycoplasmas. In addition, there was no reduction in the number or intensity of precipitation lines after absorption of M. pneumoniae antiserum with heterotypic mycoplasma antigens, or after absorption of heterotypic mycoplasma antisera with M. pneumoniae antigen. These findings indicate that, of the human mycoplasma species so far studied, M. pneumoniae is antigenically the most distinct. 相似文献
14.
Dmitriy V. Volokhov Hyesuk Kong Joseph George Christine Anderson Vladimir E. Chizhikov 《Applied microbiology》2008,74(17):5383-5391
In this study, we describe our results on the evaluation of the ability of different permissive mammalian cell lines to support the biological enrichment of mycoplasma species known to be bacterial contaminants of cell substrates. The study showed that this approach is able to significantly improve the efficiency of mycoplasma detection based on nucleic acid testing or biochemical technologies (e.g., MycoAlert mycoplasma detection). Of 10 different cell lines (Vero, MDBK, HEK-293, Hep-G2, CV-1, EBTr, WI-38, R9ab, MDCK, and High Five) used in the study, only MDCK cell culture was found to support the efficient growth of all the tested mycoplasmas (Mycoplasma arginini, M. bovis, M. fermentans, M. gallinaceum, M. gallisepticum, M. synoviae, M. hominis, M. hyorhinis, M. orale, M. salivarium, and Acholeplasma laidlawii) known to be most frequently associated with contamination of cell substrates and cell lines in research laboratories or manufacturing facilities. The infection of MDCK cells with serial dilutions of each mycoplasma species demonstrated that these common cell line contaminants can be detected reliably after 7-day enrichment in MDCK cell culture at contamination levels of 0.05 to 0.25 CFU/ml. The High Five insect cell line was also found to be able to support the efficient growth of most mycoplasma species tested, except for M. hyorhinis strain DBS1050. However, mycoplasma growth in insect cell culture was demonstrated to be temperature dependent, and the most efficient growth was observed when the incubation temperature was increased from 28°C to between 35 and 37°C. We believe that this type of mycoplasma enrichment is one of the most promising approaches for improving the purity and safety testing of cell substrates and other cell-derived biologics and pharmaceuticals. 相似文献
15.
Rapid Detection of Bovine Mycoplasma Antigens by Counterimmunoelectrophoresis 总被引:3,自引:1,他引:2
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Twelve reference strains of mycoplasma and acholeplasma previously reported to have been recovered from cattle were tested against hyperimmune rabbit serum by counterimmunoelectrophoresis. This technique detected antigen by the formation of precipitin lines with antibody within 1 h and promises to be a useful technique for detecting and identifying mycoplasma isolates in either pure or mixed cultures. 相似文献
16.
L Stipkovits L Bodon J Romváry L Varga 《Acta microbiologica Academiae Scientiarum Hungaricae》1975,22(1):45-51
Kidneys and sera of calves from various farms and primary kidney tissue cultures were tested for mycoplasma and acholeplasma contamination. Altogether 24 strains belonging to Mycoplasma arginini and Acholeplasma axanthum were isolated from tissue cultures, kidneys and sera. The same species were detected from lungs and peribronchial lymph nodes of calves, together with A. laidlawii, A. modicum and M. bovirhinis species. There was a close correlation between the mycoplasma content of tissue cultures, sera and kidneys and the clinical picture observed in the farm, as well as between the quality of tissue cultures and the mycoplasma content of organs, sera and tissue cultures. 相似文献
17.
Summary Mycoplasma contamination of established cell lines is a well-known but often poorly controlled artefactual problem in immunological studies of human tumor cell lines. We have evaluated four methods for detecting mycoplasmas in cell lines, namely direct culture, DNA staining, uridine phosphorylase assay, and a fourth technique based on our finding that the supernatant medium of mycoplasma-infected cell cultures inhibits thymidine uptake of mitogen-stimulated peripheral blood lymphocytes. In our hands the simplest, most reliable, and least expensive means of monitoring cell cultures for mycoplasma proved to be DNA staining. The uridine phosphorylase assay was unsuitable for use with melanoma cell lines, as six of eight lines that were negative with the other three techniques were positive with this assay.Of 14 contaminated cell lines injected to nude mice, eitht produced tumors, five of which were shown to be mycoplasma-free after one to five passages, confirming the usefulness of this approach for salvaging contaminated cell lines. 相似文献
18.
19.
Demonstration of cross-reactive antibodies to mycoplasmas in human sera by ELISA and immunoblotting 总被引:2,自引:0,他引:2
Varying levels of cross-reactivity to some mycoplasma species were observed in the sera of patients infected with Mycoplasma pneumoniae and even in normal human sera by enzyme-linked immunosorbent assay (ELISA). The absorption of the patients' sera with M. pneumoniae lysate showed the decrease in ELISA titers not only to M. pneumoniae, but also to other mycoplasma species. These results suggested the existence of cross-reactive antibodies to mycoplasmas in human sera. Cross-reactive antibodies to M. pneumoniae and other mycoplasmas in the patients' sera were also demonstrated by Western blotting technique. 相似文献
20.
Manfred Wirth Evelyn Berthold Martina Grashoff Horst Pfützner Uli Schubert Hansjörg Hauser 《Cytotechnology》1994,16(2):67-77
The polymerase chain reaction (PCR) has been used for the general detection ofMollicutes. 25Mycoplasma andAcholeplasma species were detected including important contaminants of cell cultures such asM. orale, M. arginini, M. hyorhinis, M. fermentans, A. laidlawii and additional human and animal mycoplasmas. PCR reactions were performed using a set of nested primers defined from conserved regions of the 16S rRNA gene. The detection limit was determined to be 1 fg mycoplasma DNA, which is equivalent to 1–2 genome copies of the 16S rRNA coding region. The identity of the amplification products was confirmed by agarose gel electrophoresis and restriction enzyme analysis. DNA from closely and distantly related micro-organisms did not give rise to specific amplification products. The method presented here offers a much more sensitive, specific and rapid assay for the detection of mycoplasmas than the existing ones. 相似文献