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Quality Control of Biotechnological Inputs DetectingMycoplasma

Abstract

The aim of this work was to study the Polymerase Chain Reaction (PCR) as a tool of quality control of bovine sera and cellular cultures used in the biotechnological industry. A total of 46 samples of bovine sera derived from two slaughterhouses and 33 samples of BHK21 cells derived from two biotechnological industries were evaluated using the primers GPO-3 (sense) and MGSO (antisense). The PCR technique sensibility analysis showed that 280 bp were amplified for the quantities of 50 ng to 0.006 ng of Micoplasma DNA. The primers specificity was confirmed in the test using Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Candida albicans; except by the positive control, none of the samples showed amplification. The presence of Mycoplasma in bovine sera and in the cultures of BHK21 cells showed that 56.5 and 15.2%, respectively, were contaminated. Thus, it was possible to conclude that PCR was a fast and confident technique to detect mycoplasma and that it could be used to control the quality of immunobiological products and inputs, such as sera and cultures of BHK21 cells.

PCR; bovine serum; BHK21 cells; mycoplasma; cellular culture


INTRODUCTION

Mycoplasma belongs to the class Mollicutes and it differs phenotypically of other bacteria due to its reduced size and absence of cellular wall (Razin et al. 1998Razin S, Yogev D, Naot Y. Molecular biology and pathogenicity of mycoplasmas. Microbiol Mol Biol. 1998; 62(4): 1094-1156.; Domingues et al. 2005Domingues D, Nogueira F, Tavira L, Exposto F. Micoplasmas: Que papel nas Infecções Humanas?. Acta Med Port. 2005; 18: 377-384.). Due to its small size, it can pass through the filters with porosity from 0.45 to 0.22 µm that commonly are used to sterilize the reagents in industries and laboratories (Hay et al. 1989Hay RJ, Macy ML, Chen TR. Mycoplasma infection of cultured cells. Nature. 1989; 339: 487-488.; Tully 1992Tully JG. Mollicutes. In: Encyclopedia of microbiology. New York: Academic Press; 1992. p. 181-191.). Cellular cultures contaminated with mycoplasma represent an artificial habitat for such microorganisms. Studies performed in many countries have demonstrated that from 10 to 87% of the cellular cultures were infected with mycoplasma (Kazemiha et al. 2009Kazemiha VM, Shokrgozar MA, Arabestani MR, Moghadam MS, Azari S, Maleki S, et al. PCR-based detection and eradication of mycoplasmal infections from various mammalian cell lines: a local experience. Cytotechnology. 2009; 61: 117-124.). The species M. orale, M. salivarium, M. hyorhinis and M. arginini are most found as contaminants (Miyaki et al. 1989Miyaki C, Pral MM, Gallina NMF, Rizzo E. Micoplasma como contaminante de culturas celulares mantidas em laboratórios de instituições particulares e oficiais. Rev Saúde Públ. 1989; 23(1): 39-44.; Hu et al. 1995Hu M, Buck C, Jacobs D, Paulino G, Khouri H. Application of PCR for detection and identification of Mycoplasma contamination in virus stocks. In vitro cellular and developmental biology. In Vitro Cell Dev Biol. 1995; 31(3): 710-715.; Timenetsky et al. 2006Timenetsky J, Santos LM, Buzinbani M, Mettifogo E. Detection of multiple Mycoplasma infection in cell cultures by PCR. Braz J Med Biol Res. 2006; 39(7): 907-914.).

Mycoplasma orale and M. salivarium inhabit the human oropharynx and generally infect the cell cultures due to inadequate aseptic techniques. M. arginini has bovine origin and it is found in the cultures from the contaminated bovine serum. M. hyorhinis has porcine origin and it can contaminate the bovine serum in slaughterhouses when they are also used to slaughter swine (Rottem and Barile 1993Rottem S, Barile MF. Beware of mycoplasmas. Trends Biotechnol. 1933; 11: 143-151.; Smith and Mowles 1996Smith A, Mowles J. Prevention and control of mycoplasma infection of cell cultures. Molecular and diagnostic procedures in mycoplasmology. San Diego: Academic Press; 1996. p. 445-451.; Razin et al. 1998). The contamination through mycoplasma in bovine cells and sera used to produce vaccine antigens or used in diagnosis can generate non-confident results and add potentially noxious effects to biotechnological products. Contaminations through mycoplasma cause alterations in the cellular metabolism, decreasing the cellular division rate through the interference in DNA, RNA and protein synthesis, chromosomal aberrations and death with the monolayer release (Timenetsky et al. 1992Timenetsky J, Miyaki C, Mendes IF, Rizzo E. Identificação de micoplasmas pela inibição de culturas celulares. Rev Saúde Públ. 1992; 26(1): 17-20.). Products, such as vaccines, medicines or monoclonal antibodies, manufactured based on such inputs should be disenabled, which causes prejudices to the pharmaceutical and biotechnological industries (Uphoff and Drexler 2002Uphoff CC, Drexler HG. Comparative PCR analysis for detection of Mycoplasma infections in continuous cells lines. In vitro cellular and developmental biology. In Vitro Cell Dev Biol - Animal. 2002; 38: 79-85.).

The tests to detect the contamination by mycoplasma are necessary to assure the confidence of the results and the quality of the biotechnological products (Uphoff and Drexler 2002Uphoff CC, Drexler HG. Comparative PCR analysis for detection of Mycoplasma infections in continuous cells lines. In vitro cellular and developmental biology. In Vitro Cell Dev Biol - Animal. 2002; 38: 79-85.; Timenetsky et al. 2006Timenetsky J, Santos LM, Buzinbani M, Mettifogo E. Detection of multiple Mycoplasma infection in cell cultures by PCR. Braz J Med Biol Res. 2006; 39(7): 907-914.). As this kind of contaminant does not generate visual signals, because they do not cause turbidity, just specific tests enable its detection (Lincoln and Lundin 1990Lincoln CK, Lundin DJ. Mycoplasma detection and control. FCC Tools & Resources U. S. Pharmacopeial Newsletter. 1990: 20:1-3.; Timenetsky et al. 1992Timenetsky J, Miyaki C, Mendes IF, Rizzo E. Identificação de micoplasmas pela inibição de culturas celulares. Rev Saúde Públ. 1992; 26(1): 17-20.; Hu et al. 1995Hu M, Buck C, Jacobs D, Paulino G, Khouri H. Application of PCR for detection and identification of Mycoplasma contamination in virus stocks. In vitro cellular and developmental biology. In Vitro Cell Dev Biol. 1995; 31(3): 710-715.; Ossewaarde et al. 1996Ossewaarde JM, Vries A, Bestebroer T, Angulo AF. Application of a Mycoplasma group-specific PCR for monitoring decontamination of Mycoplasma infected Chlamydia sp. strains. Appl Environ Microbiol. 1996; 62(2): 328-331.). Among the different techniques to detect the contamination through mycoplasma, the histochemical staining, immunosorbent assays (ELISA), indirect immunofluorescence (IIFT), biochemical assays and polymerase chain reaction (PCR) are used (Kong et al. 2001Kong F, James G, Gordon S, Zelynski A, Gilbert GL. Species specific PCR for identification of common contaminant mollicutes in cell culture. Appl Environ Microbiol. 2001; 67(7): 3195-3200.;Uphoff and Drexler 2002Uphoff CC, Drexler HG. Comparative PCR analysis for detection of Mycoplasma infections in continuous cells lines. In vitro cellular and developmental biology. In Vitro Cell Dev Biol - Animal. 2002; 38: 79-85.; Sung et al. 2006Sung H, Kang SH, Bae YJ, Hong JT, Chung YB, Lee CK, Song S. PCR-based detection of Mycoplasma species. J Microbiol Korea. 2006; 44(1): 42-49.; Gopalkrishna et al. 2007Gopalkrishna V, Verma H, Kumbhar NS, Tomar RS, Patil PR. Detection of Mycoplasma species in cell culture by PCR and RFLP based method: effect of BMcyclin to cure infection. Indian J of Med Microbiol. 2005; 25(4): 364-368.). PCR has been the chosen methodology to detect mycoplasma due to its high specificity, speed in the technique performance and the possibility of analyzing different samples at the same time (Rottem 2003Rottem S. Interaction of mycoplasmas with host cells. Physiol Rev. 2003; 83: 413-432.).

This work aimed to study the use of PCR to detect Mycoplasma sp for the quality control of cellular cultures and bovine serum in a biotechnological industry and validating it testing the contamination by Mycoplasmasp in inputs used in the preparation of vaccines.

MATERIAL AND METHODS

Microrganisms Cultivation

The strains of M. orale, M. salivarium, M. arginini andM. hyorhinis, donated by Jorge Timenetsky of University of São Paulo, were cultivated in 10 mL of SP4 broth at 37ºC (Nascimento et al. 2002Nascimento CMO, Figueiredo CA, Timenetsky J. Sensitivity of Rabbit Fibrochondrocytes to Mycoplasmas. Braz J Microbiol. 2002; 33: 243-246. ). The growth of the microrganisms was confirmed due to the acidification of the pH in the broth, absence of turbidity and production of colonies looking like 'fried eggs' in SP4 agar plates. Mycoplasma was frozen at -20ºC with 20% of glycerol (Sigma-Aldrich, St. Louis, MO and USA) till its use.

Polymerase Chain Reaction

In order to extract the DNA of the microorganisms, the phenol/chloroform technique was used (Bashiruddin 1998Bashiruddin JB. Extraction of DNA from Mycoplasmas In: MILES, R. & NICHOLAS, R. eds. Mycoplasma Protocols: a Laboratory Manual. New Jersey: Humana Press Inc; 1998.). The DNA concentration of each sample was measured through the absorbance at 260 nm (NanoDrop 2000, Thermo Scientific, Waltham, MA, USA). For PCR, the primers described by Van Kuppeveld et al. (1994)Van Kuppeveld FJM, Johansson KE, Galama JMD, Kissing J, Bölske G, Van Der Logt JTM, Melchers WJG. Detection of Mycoplasma contamination in cell cultures by mycoplasma group-specific PCR. Appl Environ Microbiol. 1994; 60: 149-152.were used. The primer sense GPO-3 5'- GGGAGCAAACAGGATTAGATACCCT-3' and the antisense MGSO 5'-TGCACCATCTGTCACTCTGTTAACCTC - 3' were obtained from Invitrogen. PCR reaction contained 2.0 µL of buffer 10X, 1.5 mM of MgCl2, 0.2 μM of each deoxyribonucleotide triphosphate (dNTP), 12.5 pmol of each primer (GPO3/MGSO), 1 U of Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA), 50 ng of DNA (maximum volume of 8.0 µL) and ultrapure water to a final volume of 20 μL. The amplification was made in thermocycler (model Biocycler MG96) which one cycle at 94ºC by 5 min, 35 cycles at 94ºC 30 sec, 55ºC during 30 sec, 72ºC 30 sec and a final extension at 72ºC during 5 min. A negative control (ultra-pure water) and a positive control (DNA of the references strains) were added to all amplifications.

In order to determine the sensibility of the PCR reaction, dilutions from 50 ng to 0.003 ng of the DNA of M. salivarium were cultivated in SP4 broth medium and extracted through the phenol/chloroform method were used. To determine the specificity of the PCR, DNAs extracted through phenol/chloroform methodology from strains of Staphylococcus aureus ATCC 6538,Escherichia coli ATCC 25922, Bacillus subtilisATCC 6633 and Candida albicans ATCC 10231 were used and the PCR was performed.

Mycoplasma contamination detection

Forty six samples of bovine sera derived from slaughterhouses and 33 cultures of BHK21 cells derived from biotechnological industries were submitted to DNA extraction through phenol/chloroform method (Bashiruddin 1998Bashiruddin JB. Extraction of DNA from Mycoplasmas In: MILES, R. & NICHOLAS, R. eds. Mycoplasma Protocols: a Laboratory Manual. New Jersey: Humana Press Inc; 1998.) and then to PCR to detect the presence or absence of contamination by mycoplasma.

RESULTS AND DISCUSSION

Mycoplasma orale, M. salivarium, M. arginini and M. hyorhinis grew in broth and in SP4 agar culture medium. All the species presented the expected product (280 bp) in the PCR (Fig. 1).

Figure 1
Agarose gel electrophoresis at 1.5% of PCR products of mycoplasma grown in SP4 culture media. MM: Molecular Weight Marker (100 bp); 1: Negative Control; 2: Mycoplasma orale; 3: M. salivarium; 4: M. arginini; 6: M. hyorhinis.

According to Uphoff and Drexler (2002)Uphoff CC, Drexler HG. Comparative PCR analysis for detection of Mycoplasma infections in continuous cells lines. In vitro cellular and developmental biology. In Vitro Cell Dev Biol - Animal. 2002; 38: 79-85., to detect the mycoplasma based on PCR, it is possible to mention two main variants: the detection of any infection by mycoplasma (specific genus PCR), and the detection of just one species of mycoplasma (specific specie PCR).

In the study, specific genus was used and PCR employed the primers GPO-3 and MGSO to detect the contaminations by any species of mycoplasma. In the PCR technique, analytical sensibility test, the amplification of a 280 bp fragment for quantities from 50 ng to 0.006 ng DNA of mycoplasma was observed (Fig. 2).

Figure 2
Electrophoresis in agarose gel (1.5%) of the PCR products of M. salivarium growth in SP4 broth and DNA extraction method using phenol/cloroform to determinate the PCR reaction sensibility. MM: Molecular Mass Marker (100 bp); 1 - 15 different DNA concentration: 1: 50 ng; 2: 25 ng; 3: 12.50 ng; 4: 6.250 ng; 5: 3.125 ng; 6: 1.562 ng; 7: 0.781 ng; 8: 0.391 ng; 9: 0.195 ng; 10: 0.098 ng; 11: 0.049 ng; 12: 0.024 ng; 13: 0.012 ng; 14: 0.006 ng; 15: 0.003 ng; 16: PCR negative control.

The specificity of the primers used for Mycoplasma was confirmed, because except the positive control of mycoplasma, no amplification was observed for the DNAs extracted through the phenol/chloroform method ofS. aureus ATCC 6538, E. coli ATCC 25922,B. subtilis ATCC 6633 and C. albicans ATCC 10231 strains (Fig. 3).

Figure 3
Electrophoresis in agarose gel 1.5% of the PCR products of Mycoplasma salivarium growth in SP4 broth and DNA extraction method using phenol/cloroform to determinate the PCR reaction specificity. MM: Molecular Mass Marker (100 pb); 1: PCR negative control; 2: Staphylococcus aureus ATCC 6538; 3: Escherichia coli ATCC 25922; 4: Bacillus subtilis ATCC 6633; 5: Candida albicans ATCC 10231; 6: PCR positive control (DNA M. salivarium).

The PCR technique standardized in the study was highly sensitive, because it was able to detect the presence of mycoplasma DNA at 0.06 nanograms and it was also highly specific, because it did not amplify the DNA of other microorganisms' species that were commonly used in laboratories. Such method was fast and confident to detect different species of mycoplasma that contaminated cell cultures, which enabled its usage to control the quality of immunobiological products and inputs, such as sera and cultures of BHK21 cells.

Aiming to validate the consistency of the PCR methodology established to be used in industries as quality control of inputs, 46 samples of bovine sera derived from two slaughterhouses and 33 samples of BHK21 cells derived from two biotechnological industries were evaluated to detect the possible contaminations by mycoplasma (Fig. 4). In most cases, the contamination was through mycoplasma derived from the animal sera, mainly from contaminated cattle and also from aerosols derived from the humans due to non-aseptic practices in the laboratory environments (Smith and Mowles 1996Smith A, Mowles J. Prevention and control of mycoplasma infection of cell cultures. Molecular and diagnostic procedures in mycoplasmology. San Diego: Academic Press; 1996. p. 445-451.; Rottem 2003Rottem S. Interaction of mycoplasmas with host cells. Physiol Rev. 2003; 83: 413-432.), justifying why such inputs were important to chose.

Figure 4
Agarose gel electrophoresis at 1.5% of PCR products of cultures of BHK21 cells and bovine sera. MM: Molecular Weight Marker (100 pb); 1: Negative Control; 2 and 3: Samples of cultures of BHK21 cells; 4, 5 e 6: Samples of bovine sera; 7: PCR Positive Control (DNA M. salivarium).

Results showed that 56.5% of bovine sera and 15.2% of the cellular strains were contaminated (Table 1). According toKazemiha et al. (2009)Kazemiha VM, Shokrgozar MA, Arabestani MR, Moghadam MS, Azari S, Maleki S, et al. PCR-based detection and eradication of mycoplasmal infections from various mammalian cell lines: a local experience. Cytotechnology. 2009; 61: 117-124., up to 87% of the cellular cultures could be contaminated by mycoplasma. The variation of the percentage of contamination found in the literature was related to the size of the studied samples population, contamination control practices and efficiency of the used detection assays (Rottem and Barile 1993Rottem S, Barile MF. Beware of mycoplasmas. Trends Biotechnol. 1933; 11: 143-151.; Kong et al. 2001Kong F, James G, Gordon S, Zelynski A, Gilbert GL. Species specific PCR for identification of common contaminant mollicutes in cell culture. Appl Environ Microbiol. 2001; 67(7): 3195-3200.).

Table 1
Percentage of samples of bovine sera and BHK21 cells contaminated by mycoplasma.

The test that detects the contamination by mycoplasma is necessary to assure the confidence of the results and biotechnological products quality control (Uphoff and Drexler 2002Uphoff CC, Drexler HG. Comparative PCR analysis for detection of Mycoplasma infections in continuous cells lines. In vitro cellular and developmental biology. In Vitro Cell Dev Biol - Animal. 2002; 38: 79-85.). PCR has been the chosen methodology due to the high specificity, sensibility and speed during the performance of the technique (Rawadi and Dussurget 1995Rawadi G, Dussurget O. Advances in PCR-based detection of mycoplasmas contaminating cell cultures. Genome Res. 1995; 4: 199-208.). The specificity of such methodology occurs due to the usage of oligonucletides primers that are connected to the maintained region of the DNA that codifies the region 16S of rRNA or the intergenic regions of the rDNA 16S-23-S of mycoplasma (Uphoff and Drexler 2002Uphoff CC, Drexler HG. Comparative PCR analysis for detection of Mycoplasma infections in continuous cells lines. In vitro cellular and developmental biology. In Vitro Cell Dev Biol - Animal. 2002; 38: 79-85.; Sung et al. 2006Sung H, Kang SH, Bae YJ, Hong JT, Chung YB, Lee CK, Song S. PCR-based detection of Mycoplasma species. J Microbiol Korea. 2006; 44(1): 42-49.; Timenetskt et al. 2006).

CONCLUSIONS

Thus, it could be concluded that the PCR technique was adequate for the quality control of bovine serum and cultures of BHK21 cells and that the importance of preventing the contamination through such microorganisms should be highlighted as well as the implantation of quality control in the productive process such as detecting mycoplasma through PCR.

ACKNOWLEDGMENTS

Special thanks are due to the veterinary industry Ourofino Saúde Animal, its manager Silvia Barioni and its director Dan Artioli and to Dr. Jorge Timenetsky from the University of São Paulo and to Dr. Celso Caricati from Butantan Institute.

REFERENCES

  • Bashiruddin JB. Extraction of DNA from Mycoplasmas In: MILES, R. & NICHOLAS, R. eds. Mycoplasma Protocols: a Laboratory Manual. New Jersey: Humana Press Inc; 1998.
  • Domingues D, Nogueira F, Tavira L, Exposto F. Micoplasmas: Que papel nas Infecções Humanas?. Acta Med Port. 2005; 18: 377-384.
  • Gopalkrishna V, Verma H, Kumbhar NS, Tomar RS, Patil PR. Detection of Mycoplasma species in cell culture by PCR and RFLP based method: effect of BMcyclin to cure infection. Indian J of Med Microbiol. 2005; 25(4): 364-368.
  • Hay RJ, Macy ML, Chen TR. Mycoplasma infection of cultured cells. Nature. 1989; 339: 487-488.
  • Hu M, Buck C, Jacobs D, Paulino G, Khouri H. Application of PCR for detection and identification of Mycoplasma contamination in virus stocks. In vitro cellular and developmental biology. In Vitro Cell Dev Biol. 1995; 31(3): 710-715.
  • Kazemiha VM, Shokrgozar MA, Arabestani MR, Moghadam MS, Azari S, Maleki S, et al. PCR-based detection and eradication of mycoplasmal infections from various mammalian cell lines: a local experience. Cytotechnology. 2009; 61: 117-124.
  • Kong F, James G, Gordon S, Zelynski A, Gilbert GL. Species specific PCR for identification of common contaminant mollicutes in cell culture. Appl Environ Microbiol. 2001; 67(7): 3195-3200.
  • Lincoln CK, Lundin DJ. Mycoplasma detection and control. FCC Tools & Resources U. S. Pharmacopeial Newsletter. 1990: 20:1-3.
  • Miyaki C, Pral MM, Gallina NMF, Rizzo E. Micoplasma como contaminante de culturas celulares mantidas em laboratórios de instituições particulares e oficiais. Rev Saúde Públ. 1989; 23(1): 39-44.
  • Nascimento CMO, Figueiredo CA, Timenetsky J. Sensitivity of Rabbit Fibrochondrocytes to Mycoplasmas. Braz J Microbiol. 2002; 33: 243-246.
  • Ossewaarde JM, Vries A, Bestebroer T, Angulo AF. Application of a Mycoplasma group-specific PCR for monitoring decontamination of Mycoplasma infected Chlamydia sp. strains. Appl Environ Microbiol. 1996; 62(2): 328-331.
  • Razin S, Yogev D, Naot Y. Molecular biology and pathogenicity of mycoplasmas. Microbiol Mol Biol. 1998; 62(4): 1094-1156.
  • Rawadi G, Dussurget O. Advances in PCR-based detection of mycoplasmas contaminating cell cultures. Genome Res. 1995; 4: 199-208.
  • Rottem S, Barile MF. Beware of mycoplasmas. Trends Biotechnol. 1933; 11: 143-151.
  • Rottem S. Interaction of mycoplasmas with host cells. Physiol Rev. 2003; 83: 413-432.
  • Smith A, Mowles J. Prevention and control of mycoplasma infection of cell cultures. Molecular and diagnostic procedures in mycoplasmology. San Diego: Academic Press; 1996. p. 445-451.
  • Sung H, Kang SH, Bae YJ, Hong JT, Chung YB, Lee CK, Song S. PCR-based detection of Mycoplasma species. J Microbiol Korea. 2006; 44(1): 42-49.
  • Timenetsky J, Miyaki C, Mendes IF, Rizzo E. Identificação de micoplasmas pela inibição de culturas celulares. Rev Saúde Públ. 1992; 26(1): 17-20.
  • Timenetsky J, Santos LM, Buzinbani M, Mettifogo E. Detection of multiple Mycoplasma infection in cell cultures by PCR. Braz J Med Biol Res. 2006; 39(7): 907-914.
  • Tully JG. Mollicutes. In: Encyclopedia of microbiology. New York: Academic Press; 1992. p. 181-191.
  • Uphoff CC, Drexler HG. Comparative PCR analysis for detection of Mycoplasma infections in continuous cells lines. In vitro cellular and developmental biology. In Vitro Cell Dev Biol - Animal. 2002; 38: 79-85.
  • Van Kuppeveld FJM, Johansson KE, Galama JMD, Kissing J, Bölske G, Van Der Logt JTM, Melchers WJG. Detection of Mycoplasma contamination in cell cultures by mycoplasma group-specific PCR. Appl Environ Microbiol. 1994; 60: 149-152.

Publication Dates

  • Publication in this collection
    11 Nov 2014
  • Date of issue
    Mar-Apr 2015

History

  • Received
    09 May 2014
  • Accepted
    18 Sept 2014
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