Validation of nested PCR and a selective biochemical method as alternatives for mycoplasma detection.

Direct culture is the most common way to reliably detect mycoplasma, but it is not practical for the qualitative control of cell therapeutics because of the elaborate culture medium, the prolonged incubation time, and the large sample volumes. Here, we chose two alternative methods using commercial detection kits, the PCR mycoplasma detection kit with nested PCR and the selective biochemical method, MycoAlert(®), and validated them with the direct culture method as a reference. We tested eight mycoplasma species and five validation parameters: specificity, detection limit, robustness, repeatability, and ruggedness, based on the regulatory guidelines in the US Pharmacopoeia. All experiments were performed using fibroblasts spiked with mycoplasma. Specificity tests for both methods included all mycoplasma species, except Mycoplasma pneumonia and M. genitalium for the nested PCR and Ureaplasma urealyticum for the MycoAlert(®) assay. Regarding the detection limit, the nested PCR proved to be as sensitive as the direct culture method and more sensitive than the MycoAlert(®) assay. The predicted median for probit = 0.9 was 54 (44-76) CFU/ml for M. hyorhinis and 16 (13-23) CFU/ml for M. hominis by the nested PCR, but 431 (346-593) CFU/ml and 105 (87-142) CFU/ml, respectively, with MycoAlert(®). Changes in the concentration of reagents, reagent lot, or individual analysts did not influence the results of the examined methods. The results of this study support nested PCR as a valuable alternative for mycoplasma detection.