Inhibitory Effect of Chocolate Components Toward Lard Detection in Chocolate Using Real Time PCR

Figures & data

TABLE 1 List of primers used in this work

Primer	Sequence (5’–3’)	Amplicon size (bp)	Tm (°C)	Reference
SUS_FWD	AGC TGC ACT ACA AGC ATT CC	89	65	US 2010/0216136 A1
SUS_RVS	ATG CGT TTG AGT GGG TTA GG
UF1	ATG GTG CAA CCG CTA TTA	93	51.5	This work
UR1	ATA GAA ACC GAC CTG GAT TG

TABLE 2 DNA yield and mean number of quantification cycles obtained in porcine-specific and universal real time PCR using DNA extracted from lard-adulterated samples

Samples	Lard (%)	DNA Yield	Mean number of quantification cycles (Cq)		Number of replicates
			Porcine-specific	Universal
Lard-adulterated chocolate	1	503.80 ± 9.24	ND	29.4 ± 0.51	3/3
	10	342.03 ± 20.88	ND	29.89 ± 0.31	3/3
	20	220.21 ± 25.28	ND	29.21 ± 0.42	3/3
	50	260.10 ± 34.87	ND	29.52 ± 0.13	3/3
Lard-adulterated cocoa butter	50	1.51 ± 1.50	31.77 ± 0.70	29.48 ± 0.08	3/3
Lard-adulterated sugar	50	2.00 ± 1.50	30.81 ± 0.33	29.70 ± 0.13	3/3
Lard-adulterated cocoa powder	50	521.20 ± 3.83	ND	ND	0/3
Lard-adulterated milk powder	50	47.90 ± 1.60	25.59 ± 0.02	29.92 ± 0.17	3/3

ND: Not detected.

FIGURE 1 Amplification plot and standard curve of 10-fold serially diluted porcine DNA, designated as (a) 1 ng, (b) 0.1 ng, (c) 0.01 ng, (d) 0.001 ng, and (e) 0.0001 ng; with Cq value of 16.89, 19.51, 22.91, 26.32 and 29.72, respectively.

FIGURE 2 Amplification plot of lard-adulterated cocoa powder, milk powder, cocoa butter and icing sugar using porcine-specific real-time PCR assay. Only lard-adulterated cocoa powder failed to give positive amplification.

FIGURE 3 Amplification plot of binary DNA admixture containing mixture of DNA from pure lard and DNA from cocoa powder. Positive amplification being obtained proves that the extracted DNA is applicable for PCR (free or insignificant amount of PCR inhibitor).