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A New PCR-based Mycoplasma Detection Method


Reliable, PCR-based mycoplasma detection

Douglas McKenzie Heinz Miller Ian Colbert
Stratagene

Stratagene introduces the Mycoplasma PCR Primer Set Plus, a kit that uses a PCR-based method which specifically amplifies an 874-bp PCR product from a single copy gene. In addition, the kit contains an internal control template that serves as a positive control for PCR. The kit offers a rapid and sensitive means to determine information about mycoplasma (and related acholeplasma) species. The kit expands on Stratagenes commitment to provide reagents that detect mycoplasma infection in cell cultures.

Mycoplasmas (and acholeplasmas) are wall-less eubacteria that exist as parasites within eukaryotic cells. As the smallest, simplest, and most primitive forms of self-replicating prokaryotes, they consist of only three major componentsa plasma membrane, ribosomes, and a double-stranded genome of approximately 580 kb. Because of their limited synthetic capacity, these organisms depend on host cells as a source of nutrients.

Mycoplasmas pose a recurring problem for investigators who use cells grown in culture. When mycoplasmas infect cell cultures, they dramatically effect virtually every aspect of cellular metabolism, without any apparent influence on cellular morphology. Often, only an increased cell doubling time indicates culture contamination. To avoid these artifactual effects, cell lines should be periodically evaluated.

Mycoplasma-specific Procedure Is Developed

The first protocols developed to detect mycoplasma infection in cell lines required unwieldy probe technologies and time-consuming cell culture methods using indicator cells and suicide substrates.1 Subsequently, Stratagene introduced the first PCR-based method for mycoplasma detection.2 While other companies have since introduced PCR-based methods, these competing technologies call for amplifying ribosomal RNA subunits or the spacer regions between them.3

Inasmuch as these targets share a large degree of sequence homology, both within the mycoplasma genus and with other prokaryotes, a mycoplasma-specific procedure needed to be developed. Unfortunately, this required two sequential PCRs using nested primersa cumbersome and time-consuming procedure.

We sought to develop a PCR-based method to detect a single copy gene. In our approach, we identified a target gene whose evolution rate was sufficiently rapid to contain regions of sequence divergence that could be used to speciate mycoplasmas. Once the target gene was identified, we examined the outer portions for regions of sequence conservation within the mycoplasma genus that could serve as targets for consensus PCR primers. We also evaluated the potential priming sites for their low degree of homology with other prokaryotic sequences and their complete lack of homology with eukaryotic sequences.

These resulting primers yielded a single 874-bp PCR product when used to amplify DNA from the four mycoplasma species commonly associated with cell culture infections. Restriction analysis of the PCR product produced distinctive fingerprints to diagnose mycoplasma species. Consequently, primary analysis of the PCR product and secondary restriction analysis could be conducted in parallel on a single agarose gel.

PCR Primers Amplified

We initially evaluated how well the mycoplasma-consensus primers amplified an 874-bp target sequence from crude extracts of mycoplasma/acholeplasma samples obtained from the American Type Culture Collection (ATCC). We tested the comprised mycoplasma/acholeplasma species most commonly encountered in infected cultures. To approximate the conditions used in testing tissue culture supernatants, each sample was diluted with RPMI media supplemented with 10% fetal calf serum, L-glutamine, and antibiotics. All samples were heated to 99C for 5 minutes, treated with StrataClean resin (included in kit) to remove potentially inhibitory components, and then added to the PCR mixture. Each PCR received the equivalent of 100,000 colony-forming units of the target mycoplasma/acholeplasma. This amount of mycoplasma template is similar to levels present in typical cell culture infections.

Figure 1

As demonstrated in Figure 1, all four mycoplasma samples yielded a PCR product of the expected size, while the single acholeplasma sample produced a characteristic doublet. The media control yielded no PCR product.

The mycoplasma PCR primers generated information about the species present in the culture as follows: The PCR products from a standard amplification reaction were subjected to restriction analysis with Sau 3A and analyzed using agarose gel electrophoresis. The restriction patterns for each mycoplasma-derived PCR product were unique (Figure 1); hence, individual species could now be distinguished. These data show that the kits primers amplify the target gene from mycoplasmas and their close relative, the acholeplasma. Furthermore, the PCR products can be used to obtain information about the mycoplasma/acholeplasma species present in a sample.

PCR Primers Evaluated

We subsequently evaluated the performance of the primers with bona fide laboratory samples. The experiments were conducted blind, using 12 coded cell-free supernatants provided by the Cell Culture Testing Facility at the University of California at San Francisco (UCSF). The supernatants were prepared for PCR using the same procedure described above. PCR reactions were conducted either with or without a limiting amount of an internal control template (provided in the kit). When the internal control template was amplified, it generated a 1-kB PCR product that was easily resolved from any sample amplification product using agarose electrophoresis for analysis.

Figure 2

In the absence of the internal control template (Figure 2), we scored four samples as positive and eight samples as negative for infection. In the presence of the internal control template (Figure 3), we yielded the same results but demonstrated that one of the eight negative samples inhibited PCR amplification. For three of the contaminated samples, the PCR-amplified internal control was not inhibited to any degree, denoting a low infectious load. In the remaining infected sample, the amplified 874-bp PCR product interfered with the amplified internal control standard; this signified that high levels of mycoplasma DNA were present.

Figure 3

The PCR products amplified from the four contaminated culture supernatants were subsequently subjected to restriction analysis as described above. We were able to match the restriction patterns with the laboratory samples to those for known mycoplasmas (Table 1). Altogether, the scoring for both infection and species completely concurred with the results obtained using UCSFs established testing procedure.

Table 1
Mycoplasma Species Determined Using Different Testing Procedures

Sample No.

UCSF Cell Culture Facility

Mycoplasma PCR Primer Set Plus

1

M. orale

M. orale

2

M. hyorhinis

M. hyorhinis

3

M. hyorhinis

M. hyorhinis

4

M. hyorhinis

M. hyorhinis

Conclusions

We introduce Stratagenes Mycoplasma PCR Primer Set Plus, using a new PCR-based method that exposes mycoplasma infection in cell cultures. The rapid procedure yields a single PCR band (doublet bands for acholeplasma) that, following restriction enzyme analysis, identifies mycoplasma (and acholeplasma) species. Because it is specific to mycoplasma infection, investigators now have a powerful, reliable tool with which to maintain the integrity of their cell cultures.

REFERENCES
  1. Uphoff C.C. et al. (1992) J. Immunol. Methods 149(1): 43-53.

  2. McKenzie, D. and Kaderli, M. (1993) Strategies 6: 58-59.

  3. Harawasa, R. et al. (1993) Res. Microbiol. 144(6): 489-493.


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