Introduction to Mycoplasma Detection at Microbe Inotech Labs

Mycoplasma is a term used to denote any species included in the class Mollicutes. They are the smallest (0.2 - 2.0 m in diameter) and simplest free-living parasitic organisms known (1). Mycoplasma lack a peptidoglycan cell wall and possess the smallest recorded genome in living cells (580 - 2,220 kilobases) (1, 2). Mycoplasma are parasites of many animal species and typically exhibit host and tissue specificity. In humans, Mycoplasma (M.) pneumoniae is the respiratory pathogen responsible for atypical pneumonia and mycoplasma are frequently isolated from patients with immunodeficiencies associated with disease states (1, 2).

Mycoplasma are common contaminants of eukaryotic cell cultures and are known to alter the phenotypic characteristics of host cell lines (1, 3, 4). The published incidence of mycoplasma infected cell cultures has ranged from 4 - 92% (3). Of the 18 most common species recognized as culture contaminants, M. orale, M. hyorhinis, M. arginini, M. fermentans, and Acholeplasma (A.) laidlawii are the most frequently isolated, representing 80 - 90% of isolates (5). The small size of mycoplasma allows them to pass through 0.45 m sterilization filters. Mycoplasma are resistant to common antibiotics such as penicillin and streptomycin. Mycoplasma contamination typically does not produce visible changes in cell culture medium despite the fact that they can reach titers of 10⁸/mL. Sources of mycoplasma contamination include laboratory personnel, reagents, and mycoplasma-contaminated cell lines.

Mycoplasma contamination is detected by a number of methods. Microbiological culture is generally considered the most sensitive method for mycoplasma screening and is commonly used as the reference method for the evaluation of new mycoplasma detection techniques. However, culturing mycoplasma can take two to four weeks and cannot detect fastidious mycoplasma (6). Culturing mycoplasma requires special growth conditions and is generally restricted to specialized laboratories. Microscopic visualization of mycoplasma attached to host cells using fluorescent DNA staining displays extracellular fluorescence in addition to extranuclear fluorescence (7). This method is efficient for mycoplasma screening and particularly for detection of M. hyorhinis strains that cannot be cultivated on microbiological media. However, fluorescent staining cannot detect mycoplasma species that cyto-absorb poorly, and this method requires expertise for accurate interpretation of results. Enzyme-linked immunosorbent assays measuring mycoplasma-specific cell surface antigens have been described (8, 9), but typically lack sensitivity. A number of polymerase chain reaction (PCR)-based methods have been described for detection of mycoplasma (9 - 16) that achieve high sensitivity and may be amenable to species identification.

The MiL, Inc. uses two methods and has both methods as Standard Operaturing Procedures (SOPs) for GLP studies.

The first is a rapid and highly sensitive PCR procedure. Our Mycoplasma PCR protocol utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell culture derived biological. Detection requires less than 2 mycoplasma genomes per microliter of sample. Both our methods are not suitable for clinical diagnostic use. The assay will not detect clinically relevant species such as M. pneumoniae and U. urealyticum. This is our highest sensitivity assay, the second assay we recommend for cell cultures and will be described in more detail with typical data.

The second is a 4.5 hour colorimetric microplate assay designed for routine screening of mycoplasma contamination in cultured cells. This assay detects Mycoplasma 16S ribosomal RNA (rRNA) using a colorimetric signal amplification system with sensitivity comparable to PCR. The assay detects the most common mycoplasma cell culture contaminants, including M. hyorhinis, M. arginini, M. fermentans, M. orale, M. Pirum, M. hominis, M. salivarium, and A. laidlawii. These eight species account for approximately 95% of all mycoplasma contaminations. The assay can be used to detect mycoplasma contamination using either cell culture supernates or cultured cell pellets. Samples are prepared by dilution in a lysis buffer. Passage of cultured cells in antibiotic-free media is not required. The strip-well microplate format used is amenable to a small number of samples as well as applications requiring high sample throughput. The positive control included in this assay is a synthetic DNA oligonucleotide.

PRINCIPLE OF THE Elisa-like ASSAY

Cell culture supernatant or cultured cell pellet samples are lysed. Samples are hybridized in a microplate with biotin-labeled capture oligonucleotide probes and digoxigenin-labeled detection probes that are targeted to the 16S ribosomal RNA (rRNA) of the eight most common mycoplasma contaminants. The hybridization solution is then transferred to a streptavidin-coated microplate and the rRNA/probe hybrid is captured. Following a wash to remove unbound material, an anti-digoxigenin alkaline phosphatase conjugate is added. After washing away unbound conjugate, a substrate solution is added. An amplifier solution is then added and color develops in proportion to the amount of mycoplasma rRNA in the original sample. Color development is stopped and the intensity of the color is measured using a standard colorimetric plate reader.

MYCOPLASMA SCREENING GUIDELINES

Routine screening of cultured cell lines for mycoplasma contamination is considered a prudent laboratory practice. It is recommended that screening for mycoplasma contamination be done at least quarterly, and more frequently if there is a history of mycoplasma infection or if a large number of cultures from outside laboratories are handled (4). Cell lines introduced to a cell culture facility, even from reputable sources, should be quarantined until tested for mycoplasma contamination. Recently thawed cell lines should be tested for mycoplasma contamination if not tested prior to freezing.

LIMITATIONS OF THE PROCEDURE

• FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.

• We use ribonuclease-free (RNase-free) reagents and supplies when running this assay.

• This assay detects the following species: M. hyorhinis, M. arginini, M. fermentans,

• M. pirum, M. hominis, M. salivarium, and A. laidlawii

• A negative result does not indicate that other species of mycoplasma are absent. Additionally, mycoplasma may be present at levels below the detectable limits of this assay.

• Cell cultures that are visibly contaminated (i.e. turbidity and yellow media) are probably due to E. coli or fungal infection and should not be tested. Visibly contaminated cell cultures should be discarded and fresh cultures should be started from frozen stock.

• This assay cannot be used for mycoplasma species identification.

SAMPLE PREPARATION

Cell culture supernates (15 L/well) or cell pellets (4,700 to 19,000 cells/well) are needed as samples in this assay. Passage of cultured cells in the absence of antibiotics (i.e. penicillin and streptomycin) is not needed.

Cell cultures that are visibly contaminated (i.e. turbidity and/or yellow media) are probably due to E. coli or fungal infection and should not be tested. Visibly contaminated cell cultures should be discarded and fresh cultures should be started from frozen stock. Mycoplasma contamination of cell cultures is typically not visible, even at high concentrations of mycoplasma (10⁷ - 10⁸ mycoplasma/mL).

A. CELL LYSATE SAMPLES

Cell pellet samples should be stored on ice until lysed with Cell Lysis Diluent (1X) or stored at ♂ -20° C for use at a later time. We prepare cell lysate samples using our own procedure.

B. CELL CULTURE SUPERNATE SAMPLES

Cell culture supernate samples should be stored on ice prior to use or stored at ♂ -20° C for use at a later time. We prepare cell culture supernate samples using our own procedure.

TYPICAL DATA

SENSITIVITY

This assay detects the following mycoplasma species at the levels shown below. To determine the minimal amount detectable, each species was grown in pure culture, serially diluted, and tested. The first five species listed account for 80 - 85% of mycoplasma contamination in cultured animal cells.

CFU = Colony Forming Units

CROSS-REACTIVITY

Assay cross-reactivity was tested using 1 x 10⁴ CFU/well of microbes and 1.2 x 10⁵ cells/well of mammalian cell lines listed below. This assay recognizes the eight mycoplasma species listed in the sensitivity table and two closely related prokaryote species (based on 16S rRNA homology), Ureaplasma (U.) urealyticum and Lactobacillus (L.) casei. U. urealyticum is a mycoplasma associated with human urogenital diseases and is not found as a cell culture contaminant. U. urealyticum was detectable using as few as 2.7 x 10³ CFU/well. L. casei is a lactic acid fermenting bacteria that is not found as a cell culture contaminant. No significant cross-reactivity was observed for other microbes in the panel. Mammalian cells did not show cross-reactivity or interference when tested using the recommended concentration range.

Pricing: determined upon assay type and volume of samples.

REFERENCES

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