Mycoplasma free Madin-Darby Canine Kidney Epithelial Cells (MDCK Line) and HeLa (human cervical carcinoma cells) were obtained from the Mycoplasma laboratory belonging to “Instituto de Ciências Biomédicas-II, Universidade de São Paulo”, Brazil. Mycoplasma free hybridoma cells, cell supernatant containing monoclonal antibodies (IgG isotype) and ascitic fluid were kindly supplied by the National Center for the Production of Laboratory Animals (CENPALAB), in Cuba. The absence of mycoplasma in the cell lines and biological products was confirmed by microbiological culture and PCR, following the quality standards of the Reference Laboratory for Mycoplasma Diagnosis (MYCOLAB) at the National Center for Animal and Plant Health, Cuba.

A Mycoplasma arginini culture (NCTC 10129) containing 3,9x10⁸CFU/mL was used as positive control for the assays. The strain was grown in modified Hayflick medium (1919. Hayflick L. Tissue cultures and mycoplasmas. Tex Rep Biol Med 1965; 23: 285-303). Broth culture was placed at 37°C until color change. Afterwards, 100 µL of grown culture were transferred to Hayflick agar plates and placed at 37°C for 48 h. Mycoplasma colonies were visualized and counted to calculate and adjust CFU/mL.

PCR sensitivity for the detection of mycoplasmas in cell cultures (MDCK and HeLa cell lines) and biological products derived from the manufacture of monoclonal antibodies (hybridoma cells, cell supernatant containing an IgG isotype monoclonal antibody and ascitic fluid) was evaluated after thermal shock treatment of the samples. One milliliter of each matrix and nuclease-free water was spiked with3.9x10⁸CFU/mL Mycoplasma arginini and tenfold diluted to 3.9 CFU/mL. Each dilution was processed by thermal shock (2020. Fernández C, Chavez I: Use of a Polimerase chain reaction for mycoplasmas detection in cell cultures. Rev Salud Anim. 1999;18(1):31-34.).

Briefly, 1ml of each dilution was centrifuged at 12 000 rpm for 10 minutes. Pellets were homogenized in 1mL of sterile phosphate-buffered saline (PBS), shaken and centrifuged again. Such pellets were then resuspended in 100 µL of nuclease-free water. After boiling for 10 minutes, samples were cooled on ice and stored at -20⁰C.

PCR reaction was carried out in a 25 μL volume containing 1,25 U of GoTaq^® DNA polymerase; 1X of Green GoTaq^® Flexi Buffer; 1,5 mM of MgCL₂ (GoTaq^®Flexi DNA Polymerase kit, Promega); 200 μM of each dNTP (Promega) and 0,5 μM of each primer GPO-3 (5'-GGGAGCAAACAGGATrAGATACCCT-3') and MGSO (5'-TGCACCATCTG TCACTCTGTTAACCTC-3') amplifying a 270 bp region of the mycoplasma 16S rRNA (2121. Van Kuppeveld FJ, Johansson KE, Galama JM, Kissing J, Bolske G, van der Logt JT, et al. Detection of mycoplasma contamination in cell cultures by a mycoplasma group-specific PCR. Appl Environ Microbiol. 1994;60:149-152.). Five microliters of the extracted DNA were added to the reaction. The cycling conditions for PCR were 10 minutes at 94°C, followed by 40 cycles of 30s at 94°C, 30s at 60°C and 35s at 72°C, followed by a final step of 5 min at 72°C. PCR products were visualized under UV light after electrophoresis on a 2 % agarose gel stained with ethidium bromide.

The effect of the matrices on mycoplasma DNA amplification was determined by comparing the PCR detection limit in each matrix with that obtained in nuclease-free water. Matrices with a reduced PCR detection limit or no DNA amplification were considered slightly or totally inhibitory, respectively.

The usefulness of two different DNA purification methods to reduce the inhibitory effect of the matrices tested on mycoplasma DNA amplification was evaluated. Serial ten-fold dilutions of Mycoplasma arginini culture, as described above, were performed in the matrices and in nuclease-free water. Each dilution was processed as follows: first, using a method based on DNA extraction using silica/guanidinium thiocyanate, with modifications (2222. Pérez-Castillo A, Duque-OrtízA,Burgher-Pulgarón Y, Agüero F,Solis MS, Lobo-Rivero E. Development of species-specific PCRs for the detection of mycoplasmas cell culture contaminants. Revista de Salud Animal. 2020;42(2). E-ISSN: 2224-4700.). Briefly, 500 µL of lysis buffer and 40 µL of silica were added to 1ml of each dilution. The mix was shaken vigorously for 20s and kept at room temperature for 10 min. The pellet was obtained by centrifugation at 14000 rpm for 4 min, subsequently; it was washed twice with 500 µL of wash buffer, homogenized for 30s and centrifuged under the same conditions. It was then diluted in 500 µL of cold 70 % ethanol, homogenized for 30s and centrifuged. The latter was repeated but using acetone instead of 70 % ethanol. It was also homogenized with acetone, oven dried, eluted in 120 µL of nuclease-free water and placed for 10 minutes at 56°C. Finally, tubes were centrifuged and the supernatant was recovered and stored at -20⁰C until use.

In addition, DNA extraction and purification from the dilutions were performed using Wizard^® Genomic DNA Purification Kit (Promega), according to the manufacturer’s instructions. PCR reactions were performed as described above. The reduction of the inhibitory effect of the matrices was evaluated by comparing the PCR detection limit obtained after using both purification methods and that one obtained in nuclease free water.

A PCR with internal amplification control (PCR-IAC) was optimized to detect inhibitory samples from cell cultures and biological products previously processed by thermal shock during the detection of mycoplasma DNA. The plasmid pTtPV was obtained from the Animal Virology Laboratory at the National Center for Animal and Plant Health in Cuba, and it was used as internal amplification control. This plasmid was constructed by inserting a fragment of the L1 gene of the dolphin papillomavirus into the pGEM-T-easy vector (Promega), and it was preserved in recombinant Escherichia coli JM 109 recombinant cells. Plasmid DNA was extracted and purified using Wizard ^® Plus Midipreps DNA Purification System (Promega), following the manufacturer's instructions. DNA concentration was measured on a NanoDrop 3300 (ThermoScientific).

Different annealing temperatures (55ºC, 60ºC and 65ºC), MgCl₂ concentrations (from 0,5 mM to 3,5 mM) and IAC primer concentrations (from 0,1 μM to 0,6 μM) were evaluated. All PCR reactions were performed by simultaneously amplifying IAC plasmid and mycoplasma genomic DNA. For the amplification of mycoplasma DNA, 0,5 μM of primers GPO-3 and MGSO was used (2121. Van Kuppeveld FJ, Johansson KE, Galama JM, Kissing J, Bolske G, van der Logt JT, et al. Detection of mycoplasma contamination in cell cultures by a mycoplasma group-specific PCR. Appl Environ Microbiol. 1994;60:149-152.). Five microliters of genomic DNA extracted from a Mycoplasma arginini culture containing 39 CFU/mL (the lowest amount of Mycoplasma arginini detected by the PCR previously described) were added to the reaction mix. Mycoplasma genomic DNA was extracted by thermal shock. Primers M13 uni (-21) (5`TGTAAAACGACGCCAGT3`) and M13 rev (-29) (5`CAGGAAACAGCTATGACC3`) were used for the amplification of IAC plasmid. Those primers amplified a 654 bp fragment of the pTtPV plasmid. To determine IAC concentration to be added as a template during the optimization reactions, plasmid DNA was tenfold pre-diluted in nuclease-free water. The lowest IAC concentration that remained amplified by PCR was determined. IAC amplification during the optimization study was performed by adding 56.2 fg/µL of the plasmid pTtPV, being tenfold more concentrated than the lowest IAC concentration still amplified by PCR, as recommended (1717. Hoorfar J, Abdulmawjood A, Cook N, Wagner M, Fach P. Practical Considerations in Design of Internal Amplification Controls for Diagnostic PCR Assays. Journal of Clinical Microbiology 2004;42(5):1863-1868.). Primer specificity for simultaneous amplification of mycoplasma DNA and IAC was previously evaluated in silico and in vitro (data not shown).

To evaluate the analytical sensitivity of the assay to amplify the target DNA (mycoplasma DNA); 56,2 fg/µL of IAC were simultaneously amplified with decreasing concentrations of Mycoplasma arginini DNA, (50 ng/μlL, 25 ng/μL, 10 ng/μL, 1 ng/μL, 100 pg/μL, 75 pg/μL, and 50 pg/μL). Analytical specificity was evaluated using 100 ng of purified DNA from Lactobacillus spp, Streptococcus suis and Clostridium sporogenes, as recommended by the European Pharmacopoeia, 2014 (11. European Pharmacopoeia 8.0, 2014; section 2.6.7: Mycoplasmas.). Purified DNA from these bacteria was kindly provided by the Animal Bacteriology Laboratory at the National Center for Animal and Plant Health, Cuba. All PCR reactions were carried out using 1.5 U of GoTaq^® DNA polymerase, annealing temperature of 60ºC, 2 mM of MgCl₂ (GoTaq^®Flexi DNA Polymerase kit, Promega) and 0,4 µM of primers M13 uni (-21) and M 13 rev (-29), as previously optimized.

A DNA extraction method, based on the combination of silica and guanidinium thiocyanate (2222. Pérez-Castillo A, Duque-OrtízA,Burgher-Pulgarón Y, Agüero F,Solis MS, Lobo-Rivero E. Development of species-specific PCRs for the detection of mycoplasmas cell culture contaminants. Revista de Salud Animal. 2020;42(2). E-ISSN: 2224-4700.) and a commercial DNA purification kit (Wizard^®Genomic DNA Purification Kit, Promega), was used to remove inhibitors from the matrices analyzed to increase PCR sensitivity for mycoplasma detection.

PCR sensitivity to amplify mycoplasma DNA or IAC could have varied depending on the type of matrix (1818. OIE Terrestrial Manual, 2019. Chapter 1.1.5. Principios y métodos de validación de las pruebas de diagnóstico de las enfermedades infecciosas.). Therefore, an optimal concentration of plasmid DNA could be used as IAC whatever the matrix was (MDCK and HeLa cell lines, hybridoma cells, cell supernatant containing monoclonal antibodies, or ascitic fluid), without decreasing PCR sensitivity to amplify mycoplasma DNA in the contaminated samples.

Matrices were spiked with the lowest concentration of Mycoplasma arginini DNA still amplified in each of them and they were then processed by thermal shock. Five microliters of the DNA extracted in PCR reactions were used with different IAC amounts (5,62 fg/µL, 14,0 fg/µL and 28,0 fg/µL). Those IAC amounts approached the detection limit of the plasmid obtained in nuclease free water (5,62 fg/µL), as determined above. PCR was carried out as described above for IAC simultaneous amplification and mycoplasma DNA on the matrices. The optimal IAC concentration to be added during sample analysis was defined as the plasmid concentration at which two amplification bands (IAC and mycoplasma DNA) were observed in all matrices after visualization in 2 % agarose gel electrophoresis.

Seventy-five samples were tested, including three MDCK and three HeLa cell lines, 24 hybridoma cells, 32 cell supernatant samples with monoclonal antibodies (IgG isotype), and 16 ascitic fluid samples. They were previously analyzed by culture and PCR without internal amplification control by the Reference Laboratory for Mycoplasmas Diagnosis (MYCOLAB) at the National Center for Animal and Plant Health, Cuba. All samples were processed by thermal shock (2020. Fernández C, Chavez I: Use of a Polimerase chain reaction for mycoplasmas detection in cell cultures. Rev Salud Anim. 1999;18(1):31-34.). Five microliters of each sample and 28.0 fg/µL of IAC (IAC optimal concentration previously determined for matrices) were added to the PCR reaction. The PCR assay for IAC simultaneous amplification and mycoplasma DNA was performed as described above.

All matrices processed by thermal shock showed an inhibitory effect on mycoplasma DNA amplification. PCR detection limit in the matrices studied was reduced compared to that obtained in nuclease free water. Ascitic fluid was the least inhibitory matrix, whereas hybridoma cells were the most inhibitory, showing a strong reduction in PCR sensitivity for mycoplasma DNA amplification (Figure 1).

PCR inhibitors act through direct interaction with DNA or by affecting the enzymatic activity of the polymerase (1414. Bessetti J. An Introduction to PCR Inhibitors. Profiles in DNA, 2007.http://www.promega.com ), reducing or even blocking the amplification of target DNA in biological samples with respect to pure nucleic acid solutions (2323. Lantz PG, Abu Al-Soud W, Knutsson R, Hahn-Hägerdal B, Rådström P. Biotechnical use of the polymerase chain reaction for microbiological analysis of biological samples. Biotechnol Annu ver. 2000;5:87-130.).

IgG immunoglobulins are among the strongest inhibitors for PCR assays, probably through complex formation with the single-stranded DNA (DNAss). This effect is higher at temperatures above 95°C (2424. Abu Al-Soud W, Jönsson L, Radström P. Identification and characterization of inmunoglobulin G in blood as a major inhibitor of diagnostic PCR. J Clin Microbiol. 2000;38:345-350.). It is important to notice that IgG is in high concentration in hybridoma cells, ascitic fluid and the cell supernatants analyzed here. Old or high cell density cell cultures may also reduce PCR sensitivity due to inhibitor accumulation in the culture media (HD Biosciences Co. (2525. Minerva Biolabs. Validation of the Mycoplasma-PCR-Detection Kit VenorGeM®. 2001.).

As shown in Figure 1, PCR detection limit increased in MDCK and HeLa cells, in cell supernatant containing monoclonal antibodies and in ascitic fluid after silica/guanidinium thiocyanate treatment. However, not all inhibitors were removed from MDCK and HeLa cells by this method, since the sensitivity of the assay was lower than that obtained with nuclease free water. The treatment with silica/guanidinium thiocyanate failed to reduce PCR inhibition in hybridoma cells. Wizard® Genomic DNA Purification Kit was useful in removing all inhibitors in all matrices, including hybridoma cells, because the detection limit reached the same limit obtained in nuclease free water.

The commercial DNA purification kit employed here can guarantee an efficient nucleic acid extraction, providing high-quality DNA with minimum inhibitors (2626. Shutler GG, Gagnon P, Verret G, Kalyn H, Korkosh S, Johnston E, et al. Removal of a PCR inhibitor and resolution of DNA STR types in mixed human canine stains from a five year old case. J Forensic Sci. 1999;44(3):623-626.). However, it may not be the first choice for laboratories routinely analyzing large numbers of samples due to increased costs. The extraction of mycoplasma DNA by a thermal shock-based method is fast and inexpensive, but it does not allow DNA purification and nucleic acid remains together with cellular detritus, proteins and other inhibitors after cell membrane rupture (1616. Harada K, Uchiyama M, Hoshi T, Takahashi T. Comparison of three DNA extraction methods for detection of Erysipelothrix rhusiopathiae in chicken blood by polymerase chain reaction. J Vet Diagn Invest. 2009;21:354-358.). Therefore, when using this method, it is important to include an IAC to detect inhibitory samples.

IAC primers amplify a 654 bp fragment from the plasmid pTtPV. This fragment size prevents the competence between the target DNA (a 270 bp fragment from mycoplasma genome) and IAC. This is because shorter DNA fragments are preferentially amplified with regard to larger fragments in simultaneous amplification (1717. Hoorfar J, Abdulmawjood A, Cook N, Wagner M, Fach P. Practical Considerations in Design of Internal Amplification Controls for Diagnostic PCR Assays. Journal of Clinical Microbiology 2004;42(5):1863-1868.).

IAC plasmid concentration in PCR reaction is very important. To optimize critical PCR-IAC parameters, the plasmid was added at a concentration of 56.2 fg/µL, as the PCR detection limit for IAC amplification was 5.62 fg/µL (data not shown). This agrees with the recommendation that IAC be added in the reaction at a concentration close to its own PCR detection limit (2727. OIE Terrestrial Manual, 2019. Chapter 1.1.5. Validation and quality control of polymerase chain reaction methods used for the diagnosis of infectious diseases.), not only to limit competence with the target DNA but also to demonstrate PCR sensitivity (88. Faison T, Wang J, Johnson S, Brown M, Chiang MJ, Dolan S, et al. Bioprocess: Robustness with Respect to Mycoplasma Species. PDA J Pharm Sci Technol. 2020;74(2):201-212. doi: 10.5731/pdajpst.2018.009613. Epub 2019 Sep 13. PMID: 31519782.).

IAC primer concentration is also a critical point. In PCR-IAC assays based on a non-competitive amplification system, the plasmid and target DNA are amplified using a different primer pair. IAC amplification should be limited by keeping the concentration of its primers at a suboptimal level to reduce competition between IAC and target DNA for reaction components (1717. Hoorfar J, Abdulmawjood A, Cook N, Wagner M, Fach P. Practical Considerations in Design of Internal Amplification Controls for Diagnostic PCR Assays. Journal of Clinical Microbiology 2004;42(5):1863-1868.). In this research, better results were obtained in the simultaneous amplification of IAC and mycoplasma DNA with 0.4 µM IAC primers at annealing temperature of 55°C - 60°C with 2.0 mM MgCl2, (data not shown).

PCR-IAC sensitivity for mycoplasma DNA amplification in nuclease free water was 39 CFU/ml, the same detection limit of PCR without IAC. This demonstrates that the inclusion of IAC at the selected concentration (56.2 fg/µL) did not affect the efficiency of mycoplasma DNA amplification in nuclease free water. The assay was specific for Mycoplasma arginini DNA, as no cross-reactivity was observed with DNA from phylogenetically closed bacteria (Lactobacillus spp., Streptococcus suis and Clostridium sporogenes).

Inhibitory substances can reduce the analytical sensitivity of PCR-IAC in biological samples (1818. OIE Terrestrial Manual, 2019. Chapter 1.1.5. Principios y métodos de validación de las pruebas de diagnóstico de las enfermedades infecciosas.,2828. Moalic PY, Gesbert F, Kempf I. Utility of an internal control for evaluation of a Mycoplasma meleagridis PCR test. Vet Microbiol. 1998;61:41-49.). Therefore, the optimal quantity of IAC in the PCR reaction was carefully determined for each matrix, before testing the samples.

Although in this experiment, IAC detection limit in nuclease free water was 5.62 fg/µL, a higher concentration of the plasmid was needed to amplify IAC in hybridoma, MDCK and HeLa cells. At 28.0 fg/µL of IAC plasmid, mycoplasma DNA and IAC were simultaneously amplified in almost all the matrices tested. This strategy agrees with that of Moalic et al. (2828. Moalic PY, Gesbert F, Kempf I. Utility of an internal control for evaluation of a Mycoplasma meleagridis PCR test. Vet Microbiol. 1998;61:41-49.), who increased IAC concentration to guarantee its amplification during the detection of Mycoplasma meleagridis in clinical samples from chickens. In the case of hybridoma cells, the matrix was also processed using Wizard^® Plus Midipreps DNA Purification System (Promega), due to the strong inhibition for DNA amplification observed in latter after thermal shock treatment (Table 1).

IAC should be added to the reaction at a concentration that does not inhibit the amplification of the target DNA (2929. Majidzadeh K, Mohseni A, Soleimani M. Construction and Evaluation of a Novel Internal Positive Control (IPC) for Detection of Coxiella burnetiiby PCR. Jundishapur J Microbiol. 2014;7(1):8849.). At very high concentrations, IAC competes with the target DNA, reducing the analytical sensitivity of the assay and leading to false negative results (1717. Hoorfar J, Abdulmawjood A, Cook N, Wagner M, Fach P. Practical Considerations in Design of Internal Amplification Controls for Diagnostic PCR Assays. Journal of Clinical Microbiology 2004;42(5):1863-1868.). It is also important that IAC concentration is not too low, as this would limit its own amplification (3030. Ballagi-Pordany A, Belak S. The use of mimics as internal standards to avoid false negatives in diagnostic PCR. Mol Cell Probes. 1996;10(3):159-64.).

Using 28.0 fg/µL of the IAC, 51 inhibitory samples were detected (65 % of all samples of the matrices analyzed in this study). Some samples from the same matrix were inhibitory, while others were not. The 75 % (18/24) of hybridoma cell samples, 94 % (15/16) of ascitic fluid samples and 56 % (18/32) of cell supernatant samples containing monoclonal antibodies showed inhibition of DNA amplification in this assay. In the case of MDCK and HeLa cells, no sample (0/3) was inhibited, as mycoplasma DNA and IAC were always amplified. However, in previous experiments, those matrices showed inhibition for mycoplasma DNA amplification. These results suggest that different samples from the same matrix are not homogeneous, and may affect PCR sensitivity differently. Other authors have reported PCR inhibition in cell cultures (88. Faison T, Wang J, Johnson S, Brown M, Chiang MJ, Dolan S, et al. Bioprocess: Robustness with Respect to Mycoplasma Species. PDA J Pharm Sci Technol. 2020;74(2):201-212. doi: 10.5731/pdajpst.2018.009613. Epub 2019 Sep 13. PMID: 31519782., 2424. Abu Al-Soud W, Jönsson L, Radström P. Identification and characterization of inmunoglobulin G in blood as a major inhibitor of diagnostic PCR. J Clin Microbiol. 2000;38:345-350.).

The inhibitory samples identified in this study showed no mycoplasma DNA or IAC plasmid amplification band; whereas the non-inhibitory samples showed a mycoplasma DNA and/or IAC plasmid amplification band (Figure 2).

Despite the absence of IAC signal, some samples were considered non-inhibitory in the present experiment, since mycoplasma DNA amplification band was visible (Figure 2). In this case, a high amount of mycoplasma DNA in the sample could suppress IAC, which was found in the reaction mix at a much lower concentration (88. Faison T, Wang J, Johnson S, Brown M, Chiang MJ, Dolan S, et al. Bioprocess: Robustness with Respect to Mycoplasma Species. PDA J Pharm Sci Technol. 2020;74(2):201-212. doi: 10.5731/pdajpst.2018.009613. Epub 2019 Sep 13. PMID: 31519782.). Some authors state other causes of failure regarding IAC amplification. IAC DNA may be degraded or attached to the plastic tube, reducing the plasmid concentration in the PCR. IAC concentration in the reaction should be guaranteed by adequate plasmid conservation or by preparing and using new plasmid dilutions if necessary (77. Russell BJ, Horiuchi K, Velez JO, Goodman CH, Johnson BW. Mycoplasma detection in a historical arbovirus repository: Commercial kit comparison and implications for improved repository management. J Virol Methods. 2020. doi: 10.1016/j.jviromet.2019.113769. Epub 2019, 88. Faison T, Wang J, Johnson S, Brown M, Chiang MJ, Dolan S, et al. Bioprocess: Robustness with Respect to Mycoplasma Species. PDA J Pharm Sci Technol. 2020;74(2):201-212. doi: 10.5731/pdajpst.2018.009613. Epub 2019 Sep 13. PMID: 31519782., 1010. Uphoff CC, DrexlerHG.Comparative PCR analysis for detection of mycoplasma infections in continuous cell lines. In vitroCell Dev Biol Anim. 2002b;38(2):79-85.).