Table 5. Comparison between direct qPCR without any DNA extraction and qPCR with crude or purified DNA extraction.

Bacteria conc. (cfu/ml)		1.6 × 105	1.6 × 104	1.6 × 103	1.6 × 102	1.6 × 101	1.6 × 100
C. sakazakii ATCC29544	DNA*	22.1 ± 0.78¶	25.2 ± 0.42	28.6 ± 0.85	32.3 ± 0.99	36.1 ± 1.13	41.9 ± 1.56
	Cell†	21.6 ± 0.44	25.0 ± 0.26	28.9 ± 0.78	32.2 ± 0.75	35.6 ± 0.92	40.6 ± 1.27
	Clean-up‡	26.2 ± 0.63	29.5 ± 0.55	35.3 ± 1.03	ND × 2	ND × 2	ND × 2
	Boil‡	28.8 ± 0.69	32.3 ± 0.61	38.2 ± 1.14	ND × 2	ND × 2	ND × 2
C. muytjensii ATCC51329	DNA*	19.9 ± 0.71¶	23.4 ± 0.35	26.0 ± 0.85	30.7 ± 0.85	35.4 ± 1.13	40.2 ± 1.70
	Cell†	18.3 ± 0.52	21.8 ± 0.51	24.5 ± 0.69	29.4 ± 0.84	34.8 ± 0.86	39.6 ± 1.03
	Clean-up‡	23.3 ± 0.62	27.8 ± 0.54	31.6 ± 0.77	37.1 ± 1.15	ND × 2	ND × 2
	Boil‡	25.4 ± 0.71	28.9 ± 0.73	33.9 ± 0.89	ND × 2	ND × 2	ND × 2

^*DNA represents qPCR using serially diluted purified chromosomal DNA.

^†Cell represents direct qPCR using Cronobacter cells (all of the pellet obtained through centrifugation of 1-ml suspension) without DNA extraction. Through centrifugation of 1.6 × 10⁵ cfu/ml of bacterial suspension (1-ml), SPC counts revealed that 1.3 × 10⁵ cfu of Cronobacter cells was recovered in the pellet.

^‡Clean-up indicates DNA purification using FastPure DNA Kit (Takara Bio) for 1-ml suspension; subsequently, an aliquot of 5 μl of purified DNA solution (200 μl) was used for qPCR. Boil indicates the crude extraction of boiling 1-ml suspension for 10 min; subsequently, an aliquot of 5 μl of the supernatant obtained through centrifugation of the boiled suspension (1-ml) was used for qPCR.

^¶The estimated chromosomal DNA amounts in 1.3 × 10⁵ cfu of C. sakazakii, using the equation,1 cfu = 5 fg of chromosomal DNA, were used for qPCR measurements (n = 2), and the Ct values are presented as the means ± SD (n = 2).