Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran

Abstract

Mycoplasmas are the most important contaminants of cell cultures throughout the world. They are considered as a major problem in biological studies and biopharmaceutical economic issues. In this study, our aim was to find the best standard technique as a rapid method with high sensitivity, specificity and accuracy for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran. Thirty cell lines suspected to mycoplasma contamination were evaluated by five different techniques including microbial culture, indirect DNA DAPI staining, enzymatic mycoalert(®) assay, conventional PCR and real-time PCR. Five mycoplasma-contaminated cell lines were assigned as positive controls and five mycoplasma-free cell lines as negative controls. The enzymatic method was performed using the mycoalert(®) mycoplasma detection kit. Real-time PCR technique was conducted by PromoKine diagnostic kits. In the conventional PCR method, mycoplasma genus-specific primers were designed to analyze the sequences based on a fixed and common region on 16S ribosomal RNA with PCR product size of 425 bp. Mycoplasma contamination was observed in 60, 56.66, 53.33, 46.66 and 33.33 % of 30 different cell cultures by real-time PCR, PCR, enzymatic mycoalert(®), indirect DNA DAPI staining and microbial culture methods, respectively. The analysis of the results of the different methods showed that the real-time PCR assay was superior the other methods with the sensitivity, specificity, accuracy, predictive value of positive and negative results of 100 %. These values were 94.44, 100, 96.77, 100 and 92.85 % for the conventional PCR method, respectively. Therefore, this study showed that real-time PCR and PCR assays based on the common sequences in the 16S ribosomal RNA are reliable methods with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products.

Keywords: Cell culture; DAPI staining; Mycoplasma contamination; PCR; Real-time PCR.

Fig. 1

a Indirect DNA DAPI staining of COS7, NB4, MG63, EL4, Saos2 and Vero cell lines infected with mycoplasma in the left figure (positive controls) and B95.8, A549, Nalm6, CHO, Vero and NSO cell lines free of mycoplasma contamination in the right figure (negative controls). b PCR gel electrophoresis results for detection of mycoplasma contamination in five positive control cell lines, five negative control cell lines and Vero cell line of positive and negative control and other cell lines. Lane 1 DNA Size marker (100 bp DNA Ladder, Roche XIV), lane 2 negative control (DNA-free water), lane 3 A549 (negative control), lane 4 B95.8 (negative control), lane 5 NSO (negative control), lane 6 CHO (negative control), lane 7 Nalm6 (negative control), lane 8 Vero (negative control), lane 9 Densovirus free C6/36 (negative), lane 10 Vero (positive control), lane 11 Vero (negative control), lane 12 MG63 (positive control), lane 13 EL4 (positive control), lane 14 NB4 (positive control), lane 15 Saos2 (positive control), lane 16 DNA Size marker (100 bp DNA Ladder, Roche XIV), lane 17 COS7 (positive control), lane 18 NCCIT (positive), lane 19 Vero (positive control). c PCR gel electrophoresis results for detection of mycoplasma contamination in Vero (positive control), NSO (negative control) and other cell lines. Lane 1 DNA Size marker (100 bp DNA Ladder, Roche XIV), lane 2 NSO (negative control), lane 3 NB2-11 (negative), lane 4 BFA (positive), lane 5 F25 (negative), lane 6 YAC1 (negative), lane 7 OLN-93 (positive), lane 8 MCF-HGH (positive), lane 9 TF1 (negative), lane 10 C28/I2 (positive), lane 11 SH-SY5Y (positive), lane 12 SKLC6 (negative), lane 13 D17 (positive), lane 14 Vero (positive control). d Analysis real-time PCR results of positive and negative control cell lines based on the linear amplification plot and reporter dyes (FAM Target Probe and VIC Internal Control) and Quencher (none). 1-negative control (DNA-free water), 2-NSO (negative control), 3-B95.8 (negative control), 4-A549 (negative control), 5-CHO (negative control), 6-Nalm6 (negative control), 7-MG63 (positive control), 8-COS7 (positive control), 9-NB4 (positive control), 10-EL4 (positive control), 11-Saos2 (positive control), 12-Positive control kit

Fig. 2

PCR gel electrophoresis results for detection of mycoplasma contamination with three different mycoplasma species in Vero cell line as positive control with mycoplasma species-specific primers. Lane 1 DNA Size marker (100 bp DNA Ladder, Roche XIV), lane 2 M. hominis-specific primer (negative), lane 3 M. pirum-specific primer (negative), lane 4 M. pneumoniae-specific primer (negative), lane 5 A. laidlawii-specific primer (negative), lane 6 M. salivarium-specific primer (negative), lane 7 M. hyorhinis-specific primer (positive with Amplicone size 334 bp), lane 8 M. arginini-specific primer (positive with Amplicone size 326 bp), lane 9 M. fermentans-specific primer (positive with Amplicone size 324 bp), lane 10 M. orale-specific primer (negative), lane 11 M. genitalium-specific primer (negative), lane 12 U. urealyticum-specific primer (negative), lane 13 DNA-free water (negative control)

Fig. 3

a Determination of the limit of detection for the PCR assay. Gel electrophoresis results of optimized PCR assay performed with universal primers on different serial dilutions of DNA obtained from a culture of M. hyorhinis. Lane 1 DNA Size marker (100 bp DNA Ladder, Roche XIV), lane 2 DNA-free water (negative control), lane 3 3 ng (positive), lane 4 300 pg (positive), lane 5 30 pg (positive), lane 6 3 pg (negative), lane 7 300 fg (negative), lane 8 30 fg (negative), lane 9 3 fg (negative), lane 10 300 atg (negative), lane 11 Undiluted-DNA strain of M. hyorhinis as a positive control (30 ng). b The alignment of the sequence of PCR product obtained by the amplification of M. hyorhinis 16S rRNA with the corresponding reference sequence at the NCBI Gene Bank data base. This comparison confirmed that the universal primers used in the study specifically amplified the desired fragment of a conserved sequence in all mycoplsma species

Fig. 4

a The results of real-time PCR assay using PromoKine’s PCR Mycoplasma Test Kit I/RT performed with specific primers and probes (Promokine diagnostic kit) on different serial dilutions of DNA obtained from a culture of M. hyorhinis Amplification curve 1 negative control (DNA-free water), 2 Undiluted-DNA strain of M. hyorhinis (30 ng, positive), 3 3 ng (positive), 4 300 pg (positive), 5 30 pg (positive), 6 3 pg (positive), 7 300 fg (negative), 8 30 fg (negative), 9 3 fg (negative), 10 positive control kit. b Gel electrophoresis results of the real-time PCR assay products. Lane 1 DNA Size marker (100 bp DNA Ladder, Roche XIV), Lane 2 negative control (DNA-free water), lane 3 Undiluted-DNA strain of M. hyorhinis (30 ng, positive), lane 4 3 ng (positive), lane 5 300 pg (positive), lane 6 30 pg (positive), lane 7 3 pg (positive), lane 8 300 fg (negative), lane 9 30 fg (negative), lane 10 3 fg (negative), lane 11. The kit positive control. FAM Fluorescein amidite (6-carboxyfluoresceine), VIC a proprietary fluorescent dye produced by Applied Biosystems. Detection of target amplification was performed in FAM channel whereas monitoring of inhibition control amplification in VIC channel.

Fig. 5

Comparison of the statistical parameters related to each method. The specificity, sensitivity, accuracy, predictive value of positive and negative results for all five methods were calculated