A Heptaplex PCR Assay for Molecular Traceability of Species Origin With High Efficiency and Practicality in Both Raw and Heat Processing Meat Materials

Abstract

Frequent meat frauds have become a global issue because adulteration risks the food safety, breaches market rules, and even threatens public health. Multiplex PCR is considered to be a simple, fast, and inexpensive technique that can be applied for the identification of meat products in food industries. However, relatively less is known about a multiplex PCR method authenticating seven animal species simultaneously in one reaction due to technological challenge. Through screening new species-specific primers and optimizing PCR system, a heptaplex PCR method was established, which could simultaneously detect seven meat ingredients of camel (128 bp), pigeon (157 bp), chicken (220 bp), duck (272 bp), horse (314 bp), beef (434 bp), and pork (502 bp) in a single-tube reaction. DNA sequencing solidly validated that each set of primers specifically amplified target species from total DNA mixtures of seven meat species. The developed multiplex assay was stable and sensitive enough to detect 0.01-0.025 ng DNA from various meat treatments including raw, boiled, and autoclaved meat samples or target meat content of 0.1% total meat weight, suggesting the suitability of the heptaplex PCR technique for tracing target meats with high accuracy and precision. Most importantly, a market survey validated the availability of this multiplex PCR technique in real-world meat products with a good application foreground.

Keywords: adulteration; commercial foodstuffs; heptaplex PCR; meat species; mitochondrial sequence.

Figure 1

Verification of primer specificity with conventional simplex PCR. (A) Simplex PCR detection using species-specific primers for camel, pigeon, chicken, duck, horse, beef, and pork origin and respective genomic DNA as a template. (B) PCR amplification with premixed universal primers of eukaryotic 12S rRNA, 16S rRNA, and 18S rRNA genes for each meat species, respectively. (C) PCR amplification using individual template DNA from camel, pigeon, chicken, duck, horse, beef, and pork species. MIX, a mixture of seven primer pairs of camel, pigeon, chicken, duck, horse, beef, and pork species; 1–7, a mixture of six primer pairs of six nontarget species. (D) PCR amplification with species-specific primers for camel, pigeon, chicken, duck, horse, beef, and pork species. CM, a complete mixture of seven species including camel, pigeon, chicken, duck, horse, beef, and pork; 1–7, a complete DNA mixture of six meat species except target species. Lane M is ladder DNA.

Figure 2

Validation of the sensitivity of multiplex PCR assay. (A) Gel image of PCR fragments amplified by multiplex PCR using premixed DNA templates of seven species (10, 5, 2.5, 1, 0.5, 0.25, 0.1, 0.05, 0.025, and 0.01 ng) with species-specific primers of seven meat species in a single PCR. (B) The corresponding electropherogram of gel image represented pork, beef, horse, duck, chicken, pigeon, and camel in each lane. Lanes 1–10 are presented with labels (10, 5, 2.5, 1, 0.5, 0.25, 0.1, 0.05, 0.025, and 0.01) in (A). The value of number at the horizontal line means the relative position of peaks distant from the top of agarose gel. The value of number at the vertical line means the fluorescent intensity of DNA-bound dyes using 4S GelRed nucleic acid stain. Lane M is ladder DNA.

Figure 3

Validation of the sensitivity of multiplex PCR assay. (A) Gel image of PCR fragments amplified by multiplex PCR using model mixtures of pork, horse, duck, chicken, pigeon, and camel added to beef at 15, 10, 5, 2.5, 1, 0.5, 0.25, and 0.1% of total weight with species-specific primers of seven meat species in a single PCR. (B) The corresponding electropherogram of gel image represented pork, beef, horse, duck, chicken, pigeon, and camel in each lane. Lanes 1–8 are presented with labels (15, 10, 5, 2.5, 1, 0.5, 0.25, and 0.1%) in (A). The value of number at the horizontal line means the relative position of peaks distant from the top of agarose gel. The value of number at the vertical line means the fluorescent intensity of DNA-bound dyes using 4S GelRed nucleic acid stain. Lane M is ladder DNA.