Detection of mycoplasma contaminations by the polymerase chain reaction

Wirth, Manfred; Berthold, Evelyn; Grashoff, Martina; Pfützner, H; Schubert, Uli; Hauser, Hansjörg

doi:10.1007/bf00754609

Cited by 38 publications

(19 citation statements)

References 41 publications

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“…Similar sensitive methods have also been used to know the possible aetiology of Mycoplasma infections in certain chronic diseases. 11,12 However, drawback of the studies conducted is that none of the primers used is speciÞ c for the identiÞ cation of species-speciÞ c Mycoplasma (Mollicutes) present in cell culture, virus stocks or any other chronic disease. 3,12 Hence, detection and identiÞ cation of species-speciÞ c Mycoplasma associated in cell cultures or virus stocks by using highly sensitive molecular methods such as PCR and restriction fragment length polymorphism (RFLP) based assays is of great importance in cell culture and virology laboratories.…”

Section: Membersmentioning

confidence: 99%

Detection of <i>Mycoplasma </i>species in cell culture by PCR and RFLP based method: Effect of BM-cyclin to cure infections

Gopalkrishna

Verma

Kumbhar

et al. 2007

Indian J Med Microbiol

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Section: Membersmentioning

confidence: 99%

Detection of <i>Mycoplasma </i>species in cell culture by PCR and RFLP based method: Effect of BM-cyclin to cure infections

Gopalkrishna

Verma

Kumbhar

et al. 2007

Indian J Med Microbiol

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“…A review has been recently published [5]. Wirth et al have described a two-step, nested PCR procedure that can detect 1 fg Mycoplasma DNA, which is equivalent to 1-2 genome copies [15]. The sensitivity of our assay is similar but uses a simpler one step method.…”

Section: Methodsmentioning

confidence: 85%

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

et al. 1996

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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 [l], 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 ~1 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 ~1 (or 2 6.7 x 10' 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 0.0015-0.01 5 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.

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“…Most of the PCR methods are however based on the direct detection of the 16S rRNA genes, these are either specific for particular groups of mycoplasma (Van Kuppeveld et al, 1994;Pruckler and Ades, 1995;Nissen et al, 1996;Uphoff and Drexler, 2002) or are species-specific (Roulland-Dussoix et al, 1994). The recently developed PCR method Drexler, 2002, 2004) (Wirth et al, 1994).…”

mentioning

confidence: 99%

Detection of mycoplasma contamination in cell cultures and bovine sera

Dvořáková¹,

Valíček²,

Reichelova³

2005

Vet. Med. - Czech

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Contamination of cell cultures and sera used for animal virus propagation with mycoplasmas represents a serious problem, especially in virology. Therefore specific control measures must be used. To achieve this we introduced PCR for the detection of mycoplasma species in cell cultures and compared its results with ELISA and microbiological culture. Seven mycoplasma species which are the most common contaminants of cell lines (Mycoplasma arginini, M. fermentans, M. hyorhinis, M. bovis, M. orale, M. hominis, and Acholeplasma laidlawii) were used to verify the method. Then we assessed five selected cell lines and three bovine sera by the PCR, ELISA and culture methods and compared the results. PCR was positive for all of the mycoplasma species tested. ELISA kit used (Mycoplasma detection kit, Roche, Germany) allowed detection of only four species of contaminating mycoplasmas (Acholeplasma laidlawii, Mycoplasma arginini, M. hyorhinis, and M. orale). All the methods detected contamination of the VERO and RK13 cell lines. The agents of contamination were determined by the species-specific ELISA kit as Mycoplasma arginini and M. orale, respectively. Other cell lines and sera tested were not contaminated with mycoplasma. The results confirmed that the PCR method used in the present study is a sensitive, fast and specific detection method of mycoplasma contaminations and is suitable for routine mycoplasma detection in cell cultures and bovine sera.

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Detection of mycoplasma contaminations by the polymerase chain reaction

Cited by 38 publications

References 41 publications

Detection of <i>Mycoplasma </i>species in cell culture by PCR and RFLP based method: Effect of BM-cyclin to cure infections

Detection of <i>Mycoplasma </i>species in cell culture by PCR and RFLP based method: Effect of BM-cyclin to cure infections

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

Detection of mycoplasma contamination in cell cultures and bovine sera

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