Evaluation of alternative DNA extraction protocols for the species determination in turkey salami authentication tests

Figures & data

Figure 1. Amount and standard deviation of DNA extracted from six Tunisian brands of turkey salami. (A.1) Bardakci and Skibinski² Standard Protocol, (A.2) with homogenization by a ball mill (vibro mill MM 400).

¹DNA yield is calculated by multiplying the DNA concentration measured by nanodrop (ng/µl) by the elution volume (µl) divided by the sample mass (mg). ²Phenol Chloroform Isoamyl alcohol protocol corresponding to Bardakci and Skibinski protocol.

Figure 2. DNA yield¹ and purity (A260/280) obtained from six Tunisian brands of turkey salami using NaOH extraction protocol (Protocol 1), Phenol Chloroform Isoamyl protocol² (using 3.5 ml of the acqueous phase) (Protocol 2), Phenol Chloroform Isoamyl protocol² (using the rest of the acqueous phase) (Protocol 3), CTAB protocol (Protocol 4), Chloroform protocol (Protocol 5).

¹DNA yield is calculated by multiplying the DNA concentration measured by nanodrop (ng/µl) by the elution volume (µl) divided by the amount of starting material (mg). ²Phenol Chloroform Isoamyl alcohol protocol corresponding to Bardakci and Skibinski protocol with homogenization by a ball mill (vibro mill MM 400). Median values are indicated by the solid line within each box, and the box extends to upper and lower quartile values. The dashed line represents the median of the different DNA yields obtained for all tests for all Tunisian turkey salami types.

Figure 3. Agarose gel electrophoresis of total DNA isolated from brand 2 of Tunisian turkey salami using Bardakchi and Skibinski¹ with homogenization using only 3.5 ml of acqueous phase (Protocol 2) (lane 1), Bardakchi and Skibinski¹ with homogenization using the rest of the acqueous phase (Protocol 3) (lane 2), NaOH extraction protocol (Protocol 1) (lane 3), CTAB method (Protocol 4) (lane 4), Choloroform extraction method (Protocol 5) (lane 5) and DNeasy blood and tissue kit (Protocol 6) (lane 6). Lane 7: DNA KB-Ladder includes 9 fragments ranging from 0.5 to10 kilobases (kb) (BioBasic Inc). (a) using the same volume of 10 µl for all DNA extract; b: using the same concentration of 200 ng for all DNA extract

¹Phenol Chloroform Isoamyl alcohol protocol corresponding to Bardakci and Skibinski protocol.

Figure 4. (a) PCR product generated by D-loop primers amplification of DNA extracted from chicken and turkey peripheral blood. (b) PCR product generated by cytochrome b primers amplification of DNA extracted from chicken and turkey peripheral blood.

P1: negative control of primers, P2: 100% chicken (45 ng), P3: 100% turkey (45 ng), P4: 10% chicken and 90% turkey, P5: 5% chicken and 95% turkey, P6: 2% chicken and 98% turkey, P7: 1% chicken and 99% turkey, L: 100 bp DNA ladder (Catalog Number: 15628019; ThermoFisher Scientific)

Table 1. DNA yields (ng DNA/mg sample), A260/A280 and A260/A230 Ratios of DNA extracted from 6 brands of Tunisian turkey salami with five DNA extraction methods using an amount of 50 mg of starting material. The mean value and standard deviation are shown for each sample.

Extraction method	DNA yields (ng DNA/mg Sample)	A260/A280 Ratio	A260/A230 Ratio	Time consuming	Principles of DNA separation
NaOH extraction	375.85 ± 192.78	1.68 ± 0.27	0.36 ± 0.07	10 minutes	Cell lysis at elevated temperature (95°C) using NaOH. DNA precipitation with TrisHcl
Phenol Chloroform Isoamyl alcohol protocol (3,5)	15561 ± 8923.82	1.49 ± 0.15	1.006 ± 0.45	2 hours	Cell lysis at elevated temperature (65°C) using STE buffer and anionic detergent (SDS) and protein degrading enzyme (proteinase K). DNA purification with phenol chloroform isoamylalcohol. DNA precipitation with ethanol.
Phenol Chloroform Isoamyl alcohol protocol (rest)	15697.55 ± 1234.07	1.47 ± 0.13	1.38 ± 0.31	2 hours
CTAB protocol	140.23 ± 36.16	1.32 ± 0.11	0.14 ± 0.02	4 hours	Cell lysis at 65°C using CTAB extraction and protein degrading enzyme (proteinase K). DNA purification with chloroform. DNA precipitation with CTAB precipitation followed by precipitation of DNA with isopropanol,with salt (NaCl) and by ethanol.
Chloroform Method	206.96 ± 89.83	1.36 ± 0.10	0.26 ± 0.09	3 hours and 30 minutes	Cell Lysis at 65°C by detergent (SDS), precipitation of protein with salt (NaCl). DNA purification with chloroform.DNA precipitation with ethanol.
DNeasy Blood and Tissue kit	106.6 ± 33.45	1.69 ± 0.11	0.76 ± 0.65	1 hour and 30 minutes	Cell Lysis at 56°C and using protein degrading enzyme (Proteinase K). Precipitation of DNA by ethanol

Figure 5. (a) PCR product generated by D-loop and cytochromeb primers amplification of DNA extracted from brand 2 of turkey salami using 50 mg as an amount of starting material: P1→P5: D-loop primers. P6→P10: Cytochrome b primers. L:100 bp DNA ladder (Catalog Number:15628019; ThermoFisher Scientific). P1: negative control of D-loop primers. P6: negative control of cytochrome b primers, P2 and P7: NaOH extraction protocol (Protocol 1), P3 and P8: Phenol Chloroform Isoamyl alcohol extraction protocol¹ (using 3.5 ml of the acqueous phase) (Protocol 2), P4 and P9: Phenol Chloroform Isoamyl alcohol extraction protocol¹ (using the rest of the acqueous phase) (Protocol 3), P5 and P10: DNeasy blood and tissue kit (Protocol 6). (b) Turkey salami heteroplasmy exploration, cytochrome b sequence validation: turkey salami, chicken reference and turkey reference.

¹Phenol Chloroform Isoamyl alcohol protocol corresponding to Bardakci and Skibinski protocol with homogenization by a ball mill (vibro mill MM 400).

Table 2. DNA Yields (ng DNA/mg sample), A260/A280, A260/A230 Ratios and results of PCR using cytochrome b and D-loop primers of DNA extracted from brand 2 of Tunisian turkey salami with the Phenol Chloroform Isoamyl Alcohol protocol using different amount of starting material (2, 5, 7, 10 and 25 mg). The Mean Value and Standard Deviation are shown for each sample.

Amount of starting material (mg)	Sampled part*	DNA Yields (ng DNA/mg Sample)	A260/A280 Ratio	A260/A230 Ratio	Result of PCR (cytochromeb)	Score	Result of PCR (Dloop)	Score
2	1	4183.33 ± 693.42	1.33 ± 0.11	1.023 ± 0.15	±/+	3/6	-/-/-	1/6
	2	15633.33 ± 1271.15	1.18 ± 0.07	0.756 ± 0.16	+/-/-		+/-/-
5	1	3646.66 ± 1781.90	1.16 ± 0.06	0.69 ± 0.175	+/+/+	5/6	+/+/+	6/6
	2	2846.66 ± 610.68	1.23 ± 0.04	0.86 ± 0.14	+/-/+		+/+/+
7	1	2880.95 ± 200.33	1.20 ± 0.07	0.716 ± 0.176	+/+/-	4/6	+/+/+	5/6
	2	4303.76 ± 555.58	1.17 ± 0.08	0.78 ± 0.19	+/+/-		-/+/+
10	1	2716.66 ± 200.33	1.16 ± 0.125	0.73 ± 0.25	-/+/-	3/6	+/+/-
	2	5536.66 ± 2155.23	1.22 ± 0.03	0.81 ± 0.08	-/+/+		+/+/+	5/6
25	1	1248 ± 138.04	1.213 ± 0.02	0.85 ± 0.09	-/-/+	3/6	+/+/+	4/6
	2	2030.66 ± 495.48	1.22 ± 0.06	0.88 ± 0.20	+/-/+		+/-/-

*Sampled part: 1: Taken from the upper part of the aqueous phase (3.5 ml).

2: Taken from the rest of volume of the aqueous phase

Figure 6. (a) Histogram of DNA yields¹ obtained from brand 2 of Tunisian turkey salami using four extraction protocols (A) NaOH extraction protocol (Protocol 1) (B) Phenol Chloroform Isoamyl alcohol extraction protocol² (using 3.5 ml of the acqueous phase) (Protocol 2) (C) Phenol Chloroform Isoamyl alcohol extraction protocol² (using the rest of the acqueous phase) (Protocol 3) (D) DNeasy blood and tissue kit (Protocol 6) using differents masses of samples (2, 5, 7, 10 and 25 mg). (b) and (c) PCR product generated by D-loop and cytochrome b primers amplification of DNA extracted from brand 2 of turkey salami using different masses of starting material. (b): NaOH extraction protocol (Protocol 1), (c): DNeasy blood and tissue kit (Protocol 6): 2 mg (Lane 2 and 8), 5 mg (Lane 3 and 9), 7 mg (Lane 4 and 10), 10 mg (Lane 5 and 11) and 25 mg (Lane 6 and 12). P1→P6: D-loop primers, P7→P12: cytochrome b primers, L: 100 bp DNA ladder (Catalog Number:15628019; ThermoFisher Scientific). P1: negative control of D-loop primers, P7: negative control of cytochrome b primers.

¹DNA yield is calculated by multiplying the DNA concentration measured by nanodrop (ng/µl) by the elution volume (µl) divided by the sample mass (mg). ²Phenol Chloroform Isoamyl alcohol protocol corresponding to Bardakci and Skibinski protocol with homogenization by a ball mill (vibro mill MM 400).

Figure 7. PCR product generated by D-loop and cytochrome b primers amplification of DNA extracted from brand 2 of turkey salami from swabs using NaOH extraction protocol and Phenol Chloroform Isoamyl alcohol protocol. P1→P4: D-loop primers, P5→P8: Cytochrome b primers, P1: negative control of D-loop primers protocol corresponding to Bardakci and Skibinski protocol with homogenization by a ball mill (vibro mill MM 400)., P5: negative control of cytochrome b primers, P2 and P6: NaOH extraction protocol (Protocol 1), P3 and P7: Phenol Choloroform Isoamyl alcohol extraction protocol¹ (using 3.5 ml of the acqueous phase) (Protocol 2), P4 and P8: Phenol Choloroform Isoamyl alcohol extraction protocol¹ (using the rest of the acqueous phase) (Protocol 3), L: 100 bp DNA ladder (Catalog Number: 15628019; thermoFisher Scientific).

¹Phenol Chloroform Isoamyl alcohol protocol corresponding to Bardakci and Skibinski protocol with homogenization by a ball mill (vibro mill MM 400).