2.8. Diagnostic Sensitivity, Specificity and Accuracy Evaluation Using PromoKine qPCR Test Kit I/RT, Variant C

Thirty DNA samples isolated from different cell lines were tested by commercial PromoKine qPCR Test Kit I/RT Variant C according to the manual manufacturers (PK-CA91-3025C). 500 μL of cell culture supernatant was transferred into sterile DNA-free reaction tube and incubated at 95 °C for 5 min, then centrifugated at 10,000× g for 25 s. Finally 2 μL of the extract was used as a template for qPCR that was performed using manual instructions and MxPro 3005P QPCR System with appropriate settings.

To obtain diagnostic sensitivity and specificity 30 DNA samples derived from selected cell cultures (Table 3) were additionally analyzed by both Test-1, Test-2 described in this study and commercial PromoKine qPCR Test Kit I/RT, Variant C. We compared qualitative results obtained by both tests PromoKine qPCR Test Kit I/RT and Test-1 for the diagnostic sensitivity and specificity evaluation according to Kralik et al. [30].

Comparison of Test-1 and Test-2 results obtained during detection of Mycoplasma/Ureaplasma/Acholeplasma presence in selected 12 different cell lines in 30 samples.

¹* Full name of cell line abbreviations are placed in Material and Method Section; ²* P—positive results; N—negative results; ³* All tests with other specific primers form Test-2 were negative for all tested cell lines.

The tests’ accuracy showed the level agreement of qualitative results obtained in both methods for the same tested cell samples. All obtained results achieved for M1, M2, and M3 tested primer pairs of Test-1 and obtained in Test-2 were compared with the results obtained with commercial PromoKine qPCR Test Kit I/RT, Variant C that was used in this study as a referenced method. Defining accuracy, we considered four primary categories applicable in binary classification tests (recommended for qualitative detection). We analyzed thirty samples by Test-1 and Test-2, calculating their concordance with the reference method according to the formula recommended by Kralik et al. [30].