Development of New PCR Assay with SYBR Green I for Detection of Mycoplasma, Acholeplasma, and Ureaplasma sp. in Cell Cultures

Abstract

Mycoplasma, Acholeplasma, and Ureaplasma sp. are atypical bacteria responsible for in vitro cell culture contaminations that can warp the results. These bacteria also cause human and animal infections and may lead to chronic diseases. In developed polymerase chain reaction (PCR) in this study a quantitative PCR with SYBR Green I fluorochrome was applied to facilitate the Mycoplasma, Acholeplasma, and Ureaplasma sp. DNA detection and identification. Screening Test-1 v.1 (triplex qPCR) allowed for the detection of 11 species. Test-1 v.2 (three single qPCRs) pre-identified three subgroups, allowing for the reduction of using single qPCRs in Test-2 for species identification. The range of both tests was consistent with pharmacopeial requirements for microbial quality control of mammal cells and included detection of M. arginini, M. orale, M. hyorhinis, M. fermentans, M. genitalium, M. hominis, M. pneumoniae, M. salivarium, M. pirum, A. laidlawii, and U. urealyticum. Limit of detection values varied between 125-300 and 50-100 number of copies per milliliter in Test-1 and Test-2, respectively. Test-1 and Test-2 showed fully concordant results, allowed for time-saving detection and/or identification of selected species from Mycoplasma, Acholeplasma, and Ureaplasma in tested cell cultures.

Keywords: Acholeplasma and Ureaplasma sp. detection; Mycoplasma; cell cultures; qPCR validation; quality control.

Figure 1

Comparative analysis results of multiple sequence alignment generated by BioEdit software for GenBank sequences distinguished by M1 (a), M2 (b), and M3 Fw, Rv primer pairs (c) for presented species. Full concordance is shown for conserved regions of GenBank sequences characteristic for selected species and distinguished by M1, M2, M3 forward, and reversed sequence primers (area marked by dark blue color). Full sequence compliance shown in the black area contrasts with the sequence polymorphism demonstrated in the white space with colorful letters meaning nucleotide changes.

Figure 1

Comparative analysis results of multiple sequence alignment generated by BioEdit software for GenBank sequences distinguished by M1 (a), M2 (b), and M3 Fw, Rv primer pairs (c) for presented species. Full concordance is shown for conserved regions of GenBank sequences characteristic for selected species and distinguished by M1, M2, M3 forward, and reversed sequence primers (area marked by dark blue color). Full sequence compliance shown in the black area contrasts with the sequence polymorphism demonstrated in the white space with colorful letters meaning nucleotide changes.

Scheme 1

Test-1 and Test-2 preparation and results interpretation: (a) screening Test-1 v.1, the triplex qPCR for simultaneous detection of selected Mycoplasma. Acholeplasma. and Ureaplasma sp. (MAU group); (b) screening Test-1 v.2 performed with three separate qPCRs for detection at least one of 11 species of MAU 1–3 subgroups; (c) species identification in Test-2 using single qPCRs according to Test-1 results; Fw (forward); Rv (reversed) M1, M2, M3 primers.

Figure 2

Specificity of Test-1 v.1 and Test-1 v.2 performed for qPCR with M1; M2; and M3 qPCRs, (a) amplification plots and (b) dissociation curves of qPCR products with maximum temperature melting (Tm) peaks. PC1 (triplex qPCR), PC1a (single qPCR)—positive control with 0.05 ng M. hominis; DNA standard and accordingly PC2, PC2a with M. fermentans; PC3 PC3a with M. pneumoniae; IC—internal control with ACTB Fw, Rv primers and 10.0 ng of human DNA; NIC—negative internal control with H₂O instead of DNA template; NC1-3—negative controls with M1, M2, and M2 Fw and Rv primers.

Figure 3

Test-1 v. 2. qPCR results obtained for M1, M2, M3. Fw and Rv primers used in three single qPCRs; (a) melting curves with maximum Tm values; (b) standard curves show RSq values and efficiency expressed in %.

Figure 4

Cell cultures cell culture under phase-contrast (PC) microscope (a) L929 (mouse fibroblast cell culture) 100×, (b) L929 400×; (e) MG63 (osteosarcoma cell culture) 100×, (f) MG63 400×; DAPI—stained cell images shown under DAPI—filter (c,g); DAPI—filter and visible light (VIS) (d,h); Arrows in subfigure h show a slightly stronger glare of the peripheral part of cells. In that case mycoplasma contamination is suspected. M. arginini colonies cultured on Mycoplasma Agar Base (MAB) supplemented with mycoplasma selective supplement-G directly examined after a week (i) and 2 weeks (k) by the camera with 20× zoom; M. arginini colonies and under inverted microscope at 400× magnification after a week (j) and 2 weeks (l).

Figure 5

The results of Mycoplasma. Acholeplasma. and Ureaplasma sp. detection in cell cultures found in Test-1.