Taqman-Based Duplex Real-Time Polymerase Chain Reaction Approach for the Detection and Quantification of Donkey and Pork Adulterations in Raw and Heat-Processed Meats

Figures & data

Figure 1 Simplex PCR assay for linearity test, regression line parameters, and sensitivity parameters of (a) donkey specific and (b) pork specific TaqMan PCR system using ten-fold dilution series ranging from 0.001 to 100 ng.

Figure 2 Duplex PCR assay for linearity test, regression line parameters, and sensitivity parameters of (a) donkey specific and (b) pork specific TaqMan PCR system using ten-fold dilution series ranging from 0.001 to 100 ng.

Table 1  Cycle of threshold (Ct) values for each species tested with the species-specific primer-probe sets

		Ct values of species-specific primer-probe sets
		Simplex PCR		Duplex PCR
Species	DNA concentration (ng)	Donkey	Pork	Donkey	Pork
Target species	0.001	34.72	33.19	35.12	33.70
	0.01	31.27	30.07	31.82	30.89
	0.1	27.76	26.74	28.36	27.10
	1	25.80	23.15	25.81	24.12
	10	21.06	19.85	22.01	20.74
Donkey	100	17.97	40.00	18.94	40.00
Pork	100	40.00	17.01	40.00	17.65
Horse	100	40.00	40.00	40.00	40.00
Bovine	100	40.00	40.00	40.00	40.00
Ovine	100	40.00	40.00	40.00	40.00
Turkey	100	40.00	40.00	40.00	40.00
Chicken	100	40.00	40.00	40.00	40.00

Table 2  Duplex PCR results for raw and heat-processed binary meat mixtures

				Ct (Cycle of threshold) values
	Ratio of target species in the meat mixtures					Autoclaved
Target species	Beef (%)	Donkey (%)	Pork (%)	Raw samples	Oven-cooked samples	samples (120°C)
Donkey (1st channel)	99.998	0.001	0.001	35.60	33.09	N/D
	99.98	0.01	0.01	32.62	32.88	33.72
	99.8	0.1	0.1	29.65	30.02	29.89
	98	1	1	25.97	26.27	27.15
	80	10	10	22.58	22.82	24.17
	0	50	50	18.45	18.93	21.24
Pork (2nd channel)	99.998	0.001	0.001	33.06	34.94	N/D
	99.98	0.01	0.01	32.61	31.44	34.36
	99.8	0.1	0.1	28.02	28.58	33.78
	98	1	1	23.33	23.72	28.57
	80	10	10	21.63	21.79	27.01
	0	50	50	17.57	17.82	23.84
N/D: Not detected.

Table 3  Linearity test parameters for duplex PCR results for raw and heat-processed binary meat mixtures

	Raw samples			Oven-cooked samples			Autoclaved samples
Target species	Intercept	Slope	Correlation	Intercept	Slope	Correlation	Intercept	Slope	Correlation
Donkey	25.77	−3.42	0.996	25.83	−2.99	0.959	27.63	−3.07	0.996
Pork	24.39	−3.29	0.970	24.67	−3.41	0.993	29.51	−2.78	0.954

Figure 3 Gel electrophoresis of simplex and duplex PCR products from reference dilutions. M: 600 bp ladder (Qiagen, Gelplot 100, Germany). Lanes D₁–D₆: Donkey (100, 10, 1, 0.1, 0.01, and 0.001%). Lanes (D + P₁)–(D + P₆): Donkey–Pork binary mixtures (100, 10, 1, 0.1, 0.01, and 0.001%). Lanes P₁–P₆: Pork (100, 10, 1, 0.1, 0.01, and 0.001%).

Figure 4 Gel electrophoresis of duplex PCR products from raw and heat-processed binary donkey/pork meat mixtures amplified with 40 cycles. M: 600 bp ladder (Qiagen, Gelplot 100, Germany). Lanes R₁–R₅: Raw donkey-pork binary mixtures in beef (50, 10, 1, 0.1, and 0.01%). Lanes O₁–O₅: Oven-cooked (200°C) donkey–pork binary mixtures (50, 10, 1, 0.1, and 0.01%). Lanes A₁–A₅: Autoclaved (120°C) donkey–pork binary mixtures (50, 10, 1, 0.1, 0.01%).