TABLE 3.

Quantitative detection of M. agalactiae in raw sheep milk^a

Approximate M. agalactiae cells/25 ml	Approximate M. agalactiae genome equivalents/reactionb	Signal ratioc	CT value ± SEMd	Relative accuracye
2.5 × 107	1 × 105	9/9	24.20 ± 0.02	87.17
2.5 × 106	1 × 104	9/9	27.12 ± 0.06	115.14
2.5 × 105	1 × 103	9/9	30.50 ± 0.05	110.98
2.5 × 104	1 × 102	9/9	34.09 ± 0.06	91.81
2.5 × 103	10	9/9	37.33 ± 0.23	97.43
2.5 × 102	1	9/9	38.63 ± 0.32	NA
25	0.1	0/9	NAf	NA
3	0.01	0/9	NA	NA
0f	0	0/9	NA	NA

^aRaw milk was collected directly from a sheep farm. Data show results from three independent experiments with three PCR replicates used in each. The overall efficiency was 0.996, and the linearity (R²) was 0.9989. NA, not applicable.

^bEstimated numbers of M. agalactiae genome equivalents in each PCR run, assuming 100% DNA extraction efficiency, are shown. Each reaction mixture contained 2 μl of a DNA preparation taken from the 1:10 dilution of the initial 50 μl extracted from 25 ml of raw sheep milk.

^cNumber of positive results out of nine reactions.

^dMean C_T value ± standard error of the mean (SEM) at which fluorescence intensity was equal to a fixed threshold. The experimental results were statistically significant (P < 0.05) taking into account unavoidable errors associated with serial dilutions.

^eDegree of correspondence between results obtained with the standard plating technique (M. agalactiae CFU/25 ml) and those obtained with the p40 Q-PCR method (M. agalactiae genome equivalents/25 ml).

^fNoncontaminated milk.