Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 25, Num. 4, 2007, pp. 364-368

Indian Journal of Medical Microbiology, Vol. 25, No. 4, October-December, 2007, pp. 364-368

Original Article

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

Gopalkrishna V, Verma H, Kumbhar NS, Tomar RS, Patil PR

National Institute of Virology, P.O. Box 11, Pune - 411 001

Correspondence Address: National Institute of Virology, P.O. Box 11, Pune - 411 001, India. Email: gopalvk58@hotmail.com.

Date of Submission: 20-Apr-2007
Date of Acceptance: 18-May-2007

Code Number: mb07100

Abstract

Keywords: BM-cyclin, cell cultures, Mycoplasma infections, polymerase chain reaction, restriction fragment length polymorphism

Mycoplasma contaminations in cell cultures have been known since the beginning of cell culture technology. Mycoplasmas , which belong to the class Mollicutes ( Mycoplasma and Acholeplasma ), are one of the most frequent contaminants found in cell cultures and also in virus stocks. ^[1] Among 100 Mycoplasma species that have been identified, at least 20 Mycoplasma and Acholeplasma species have been found as cell culture/virus stock contaminants. ^[2],[3] The frequency of Mycoplasma -infected cultures ranges from 10 to 50%. ^[4] Majority of them identified in cell cultures were as either M. arginini , M. Pirum , M. orale , M. hominis , M. fermentans , M. salivarium or Acholeplasma laidlawii . ^[5],[6] Mycoplasmas , generally, grow in culture supernatant and attach to the cell membranes. They cannot be visualized under inverted microscope, neither they show turbidity of the culture fluid unlike other bacterial contamination, in addition they are resistant to the commonly used antibiotics. These contaminations often affect various aspects of culture growth, lead to unreliable experimental results and are also considered potential harmful biological products in tissue cultures.

Many methods for detecting Mycoplasmas such as microbial culture, DNA fluorochrome staining, enzyme-linked immunosorbent assay, immunofluorescence, biochemical assays and DNA hybridization methods using nucleic acid probes have been described earlier. ^[4],[7],[8] However, each has its own limitations, advantages and disadvantages. These methods are generally time-consuming, insensitive and might not detect all the Mycoplasma species in cell culture contaminants. Polymerase chain reaction (PCR) based methods have been developed for detection of Mycoplasma and Acholeplasma contaminants in cell culture. ^{[1],[3],[8],[9],[10]} These methods are relatively rapid, sensitive and can detect low copy number of Mycoplasmas present in cell cultures. 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 specific for the identification of species-specific Mycoplasma ( Mollicutes ) present in cell culture, virus stocks or any other chronic disease. ^[3],[12] Hence, detection and identification of species-specific 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. This approach is certainly likely to replace the serological and culture methods.

A two-stage nested PCR that amplifies 16S-23S rRNA spacer region sequences from Mycoplasma and Acholeplasma in combination with RFLP has been described, which could detect and identify all species-specific Mycoplasma contaminants associated in cell culture/virus stocks. In addition, treatment of Mycoplasma -infected cultures with BM-cyclin for several passages could substantially eliminate Mycoplasmas . The single nested PCR-RFLP method is relatively time-consuming, highly sensitive, reproducible and identifies species-specific Mycoplasmas involved in cell culture and virus stocks.

Materials and Methods

Cell cultures

Cell lines such as HeLa, Hep-2, Vero, RD, MRC-5, BF-2, MDCK and virus stocks were grown in 25 cm ² culture flask in minimum essential medium supplemented with 10% foetal calf serum without antibiotics.

Nucleic acid extraction

Extraction of nucleic acid from monolayer cell cultures was carried out according to the methods described earlier. ^[3] Monolayer cell cultures were washed once with antibiotic free medium. Cells were scraped with cell scraper into 10 mL medium. One millilitre of the cell suspension was transferred into a 1.5 mL Eppendorf tube and centrifuged at 10,000 rpm for 10 minutes at 4 °C. Supernatant was discarded and pellet was suspended in 100 μL of lysis buffer (10 mm Tris-HCl (pH 8.3), 50 mm KCl, 2 mm MgCl ₂ , 0.001% gelatin, 0.5% NP-40, 0.5% Tween-20 and 1 μL of proteinase-K (10 mg/mL stock, Bangalore Genei Pvt. Ltd, Bangalore, India). The cell suspension was lysed at 60 °C for 1 hour with intermittent shaking. Nucleic acid was precipitated with 600 μL of isopropanol and kept at -20 °C deep freezer for 30 minutes and centrifuged for 30 minutes at 4 °C. Pellet was washed once with 70% ethanol and air dried. Pellet was resuspended in 50 mL of 1x Tris-EDTA (10 mm Tris-1 mm EDTA pH 8.0) and directly used for PCR.

Primers used for Mycoplasma detection

A two-step nested PCR was carried out. A total of seven Mycoplasma primers were used. In the first-stage PCR, four sets of primer sequences were used as described earlier. ^[3],[13] In addition to this, three additional primers (M78, M89, R34) selected from the 16S and 23S rRNA region of A. laidlawii were used. In the second round, two sets of outer primer (F1, R1, M78, M89) and two sets of inner primers F2, R2 and R34 were used. The primer locations and sequences utilized in first- and second-stage PCR are described in [Table - 1].

PCR amplifications

PCR amplification was carried out as per the protocols described earlier, ^[14],[15] in 50 μL of master mix contains 5 μL of 10x Taq buffer (100 mm Tris, pH8.4, 500 mm KCl, 25 mm MgCl ₂ ), 1 μL of 10 mm dNTPs, 20 pmol of each primer, 0.4 μL of platinum Taq polymerase (5 U/mL, Invitrogen Life Technologies, Carlsbad, CA, USA) and the volume was made up to 50 μL by adding sterile distilled water. To the mix, 5 μL (200 ng conc.) of the extracted DNA from cell lines was used as a template. Nucleic acid extracted from M. pirum and A. laidlawii (ATCC, Manassas, VA, USA) was used as positive controls. Amplifications were conducted at initial denaturation at 94 °C for 2 min followed by 30 cycles of denaturation at 94 °C for 30 s, primer annealing at 55 °C, 30 s and extension at 72 °C, 60 s. A final primer elongation was carried out at 72 °C for 5 min. In the first stage, 5 μL of the template DNA was added to 45 μL of the mix as described earlier with two outer primers [Table - 1]. Second-step PCR was carried out by adding 5 μL of the first-stage PCR product to 45 μL of the master mix with three inner primers [Table - 1]. PCR products were electrophoresed on 1.2% agarose gels, stained with ethidium bromide (10 μg/mL, Sigma Chem. Co, St. Louis, USA) and visualized in a gel documentation system (Syngene Co, UK).

Identification of species-specific Mycoplasma in cell cultures by PCR-RFLP

Characterization of species-specific Mycoplasma was carried out by using PCR-RFLP method. PCR products (second-step PCR) of 10 μL in volume were digested with restriction enzymes, Vsp I, Cla I and Hin dIII (1 U/μL, Invitrogen Life Technologies, USA) along with 2 μL of 10x restriction enzyme buffer. The volume was made up to 20 μL by adding distilled water. The reaction mixture was incubated at 37 °C for 1 h. Enzyme-digested PCR products were electrophoresed on 2.5% agarose gels. Restriction-fragmented amplimers were compared and identified along with the positive controls ( M. Pirum and A. laidlawii ). Restriction fragment size location of commonly encountered Mycoplasma species were mentioned in [Table - 2].

Elimination of Mycoplasmas in cell cultures and treatment with BM-cyclin

Mycoplasma -infected cell cultures detected by PCR and RFLP methods as described earlier were treated with BM-cyclin antibiotics (BM-cyclin 1 and 2) at a final concentration of 5 μg/mL (Stock BM-cyclin 37.5 μg; Roche diagnostics GmbH, Mannheim, Germany). Cell cultures were passaged three more times with the same concentration of BM-cyclin. Finally, cultures were passaged once with out adding BM-cyclin and tested for Mycoplasma infections by PCR-RFLP method as described earlier.

Results

Among cell cultures screened for Mycoplasma infections by PCR-RFLP method, some of the cultures i.e, Hep-2, MDCK cell lines were shown to have slow deterioration of the cells and non-adherence of the cells. These cell lines were screened for Mycoplasma infections by a single-nested PCR method using a total of seven primers of both Mycoplasma and Acholeplasma contaminations. It was observed that one of the cell lines got infected with M. pirum by showing an amplified PCR product of 323 bp fragment in 1.2% agarose gels [Figure - 1] indicative of M. pirum . Other cell line (MDCK) was found to be infected with M. orale by showing a 290 bp amplimer size product in our nested PCR [Figure - 2]. Also co-infections of other Mycoplasma species were detected by showing multiple amplimers in nested PCR [Figure - 3]. Since we have used two sets of outer and inner primers, most of the co-infections can also be detected by this method. PCR products were further characterized and analysed for species identification by RFLP method using multiple restriction enzyme digestion method ( Vsp I, Cla I and Hin dIII). Restriction fragment analysis revealed that enzyme-digested PCR products of M. pirum (323 bp) showed fragments sizes of 169 and 154 bp with Vsp I, 285 and 38 bp fragments with Cla -I enzymes, respectively. Hind III digested PCR products did not show any cleavage, since Hind III does not have any restriction site of M. pirum DNA. Similarly, when PCR products of M. orale (290 bp) were digested with the same restriction enzymes, product digested with Vsp I enzyme could show fragments of 151 and 139 bp agarose gels, in contrast Cla I and Hin dIII enzymes. Mycoplasma -infected cell cultures were further treated with BM-cyclin at a final concentration of 5 mg/mL successively for three passages. Subsequently, cell lines were passaged further with out addition of BM-cyclin and sub-cultured. These cell lines were tested further for Mycoplasma infections by nested-PCR. Mycoplasma infections were cured in BM-cyclin-infected cells. This is further confirmed by non-amplification of PCR products in BM-cyclin-treated cells as compared to untreated cells [Figure - 3].

Discussion

Mycoplasma detection by PCR-RFLP method is relatively very simple, rapid and time saving. The sensitivity of the Mycoplasma detection by the method ranges from 20 to 180 CFU/mL among species of Mycoplasmas and Acholeplasmas . In a single-nested PCR method by using four outer primers and three inner primers, we are able to detect most of the Mycoplasma and Acholeplasma infections associated in cell cultures without addition of species-specific inner primers. The highly sensitive PCR method is especially useful for monitoring even low copy number of Mycoplasmas present in cell cultures, virus stocks and also useful while eliminating infections from infected cell cultures. The method is also useful for detecting if more than one Mycoplasma infections present in cell cultures. The lengths and sequences of the spacer region in the rRNA operon differ among Mycoplasma species and this heterogeneity can be used for species identification also. ^{[3],[13],[16]} With these primer sets used a single DNA amplicon ranging from 236 to more than 365 base-pair fragments were produced with all the commonly encountered Mycoplasma contaminants. However, since A. laidlawii has at least two rRNA operons with tRNA genes inserted in the 16S-23S spacer region, ^[17] a double-banded PCR product with a size of 426 and 219 bp was identified with the method. Further characterization of PCR products with the RFLP method identifies species-specific Mycoplasmas and A. laidlawii involved in cell culture contaminants. Mycoplasma species identification is particularly useful in monitoring the source of contamination of cell lines, since Mycoplasma species arise from different sources. The construction and application of probes for the detection of cell culture Mycoplasmas have been reported by several groups. ^{[18],[19],[20]} PCR-based methods have also been developed. ^[3],[16] However, most of the methods described earlier might be time-consuming, lack of sensitivity and might not detect all the Mycoplasma species involved in cell culture and virus stock contaminants.

In conclusion, two-stage nested PCR assay system amplifies the 16S-23S rRNA spacer region sequences from Mycoplasma and Acholeplasma in cell culture and virus stock contaminants. Using PCR-RFLP assay could also detect and identify the species-specific Mycoplasmas involved in cell cultures and sources of their contaminations. Furthermore, treatment of Mycoplasma -infected cell lines with BM-cyclin for three successive passages could substantially eliminate all Mycoplasma contaminants. These pure Mycoplasma -free cell lines and virus stocks could also be revived and propagated in cell cultures. This approach will certainly be useful and provides as a quality control approach of the biological materials and reagents used in cell culture and virology research laboratories. Using Mycoplasma -free cell cultures will certainly give good experimental results and can also be used as an utmost important biological tool in cell culture and vaccine research.

References

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