Table 2.

qPCR optimization results obtained in Test-2 with species specific DNA standards.

DNA Standard Used for Standard Curve	Optimal Primers Concentration Fw/Rv (nM)	Dynamic Range (ng)	Dynamic Range Number of Copies (c) 1*	Limit of Detection LOD (c/mL)	Limit of Quantitation LOQ (c/mL)	Cycle Threshold Range (Ct)	Efficiency (E) (%)	Linearity (R2)	Specificity Based on Amplicon’s Tm °C	SDTm (±)
A. laidlawii	Ala 100/100	1.0 × 10−7–2.5 × 10−10	6.8 × 102–1.7 × 100	50	50	19.37–27.77	98.7	0.996	76.75	0.32
M. hyorhinis	Mhy 100/100	2.5 × 10−7–2.5 × 10−10	2.1 × 103–2.1 × 100	100	1050	27.58–38.40	99.2	0.992	77.85	0.35
M. fermentans	Mfe 100/100	2.5 × 10−6–2.5 × 10−10	2.1 × 104–2.1 × 100	100	100	21.26–35.63	91.7	0.993	77.35	0.35
M. hominis	Mho 100/100	2.5 × 10−6–2.5 × 10−10	1.3 × 104–1.3 × 100	50	650	24.78–39.50	90.9	0.995	78.84	0.37
M. orale	Mor 100/100	7.5 × 10−5–7.5 × 10−10	5.0 × 105–5.0 × 100	100	250	17.71–34.70	98.2	0.991	79.21	0.34
M. salivarium	Msa 100/100	2.5 × 10−6–2.5 × 10−10	1.7 × 104–1.7 × 100	100	100	18.05–31.39	95.8	0.997	77.65	0.35
M. arginini	Mar 100/100	2.0 × 10−6–2.0 × 10−10	1.7 × 104–1.7 × 100	100	100	22.89–35.89	101.5	0.986	78.32	0.33
M. genitalium	Mge 100/100	2.5 × 10−6–2.5 × 10−10	2.2 × 104–2.2 × 100	100	1100	25.00–37.15	91.5	0.990	78.32	0.33
M. pneumoniae	Mpn 100/100	2.5 × 10−6–2.5 × 10−10	1.8 × 104–1.8 × 100	100	900	23.69–38.15	101.9	0.992	79.85	0.71
U. urealyticum	Uur 100/100	2.5 × 10−6–2.5 × 10−10	1.4 × 104–1.4 × 100	50	5200	26.22–33.86	97.8	0.986	78.54	0.65
H. sapiens (IC)	ACTB 300/300	3.9 × 102–0.7 × 10−1	2.2 × 1012–4.0 × 109	-	-	11.90–21.03	96.3	0.998	88.60	0.30

M. pirum qPCR optimization and validation is not performed because of no availability of DNA standard. ¹* DNA concentration expressed in ng was converted to number of copies by MxPro software. The molecular weight required for unit conversion was obtained using DNA number of copies calculator and presented on the website http://scienceprimer.com/nucleotide-molecular-weight-calculator (accessed on 12 May 2021).