Identification and quantification of cassava starch adulteration in different food starches by droplet digital PCR

Abstract

We report a rapid and accurate quantitative detection method using droplet digital PCR (ddPCR) technology to identify cassava adulteration in starch products. The ddPCR analysis showed that the weight of cassava (M) and cassava-extracted DNA content had a significant linear relationship-the correlation coefficient was R2 = 0.995, and the maximum coefficient of variation of replicates was 7.48%. The DNA content and DNA copy number (C) measured by ddPCR also had a linear relationship with R2 = 0.992; the maximum coefficient of variation of replicates was 8.85%. The range of cassava ddPCR DNA content was 25 ng/μL, and the formula M = (C + 32.409)/350.579 was obtained by converting DNA content into the median signal. The accuracy and application potential of the method were verified using the constructed adulteration model.

Fig 1. Validation of the specificity of cassava primers.

The specificity of cassava primers were tested using the following samples: 1, beef; 2, lamb; 3, hazelnut; 4, soybean; 5, walnut; 6, sesame; 7, corn starch; 8, potato starch; 9, cassava starch; 10, sweet potato starch; and 11, ddH₂O.

Fig 2. Correlation between tapioca dry weight and extracted DNA.

Fig 3. Correlation between the content and copy number of cassava starch DNA.

DNA concentration was measured by NanoDrop 2000 and DNA copy number was determined by ddPCR.

Fig 4. Relationship between cassava DNA content and copy number.

Fig 5. Copy number of cassava and sweet potato ratio.

Channels 1–3: starch mixture containing 90% cassava starch; channels 4–6: starch mixture containing 80% cassava starch; channels 7–9: starch mixture containing 70% cassava starch; channels 10–12: starch mixture containing 60% cassava starch; channels 13–15: starch mixture containing 50% cassava starch; channels 16–18: starch mixture containing 40% cassava starch; channels 19–21: starch mixture containing 30% cassava starch; channels 22–24: starch mixture containing 20% cassava starch; channels 25–27: starch mixture containing 10% cassava starch; and channel 28: sterile double-distilled water.

Fig 6. Actual test of commercially available samples.

Channel 1–3: sample1; channel 4–6: sample2; channel 7–9: sample3; channel 10–12: sample4; channel 13–15: sample5; channel 16–18: sample6; channel 19–21: sample7; channel 22–24: sample8; channel 25–27: sample9; channel 28–30: sample10; channel31 negative; channel32 positive.

Fig 7. Actual test of commercially available samples.

Channel 1–3: sample11; channel 4–6: sample12; channel 7–9: sample13; channel 10–12: sample14; channel 13–15: sample15; channel 16–18: sample16; channel 19–21: sample17; channel 22–24: sample18; channel 25–27: sample19; channel 28–30: sample20; channel31 negative; channel32 positive.

Fig 8. Actual test of commercially available samples.

Channel 1–3: sample21; channel 4–6: sample22; channel 7–9: sample23; channel 10–12: sample24; channel 13–15: sample25; channel 16–18: sample26; channel 19–21: sample27; channel 22–24: sample28; channel 25–27: sample29; channel 28–30: sample30; channel31 negative; channel32 positive.

Fig 9. Actual test of commercially available samples.

Channel 1–3: sample31; channel 4–6: sample32; channel 7–9: sample23; channel 10–12: sample24; channel 13–15: sample25; channel 16–18: sample26; channel 19–21: sample27; channel 22–24: sample28; channel 25–27: sample29; channel 28–30: sample30; channel31 negative; channel32 positive.

Fig 10. Actual test of commercially available samples.

Channel 1–3: sample41; channel 4–6: sample42; channel 7–9: sample43; channel 10–12: sample44; channel 13–15: sample45; channel 16–18: sample46; channel 19–21: sample47; channel 22–24: sample48; channel 25–27: sample49; channel 28–30: sample50; channel31 negative; channel32 positive.