Rapid Genetic Identification of Meat

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As the demand for food research and monitoring continues to increase, simple and fast solutions are needed. This article describes a workflow that rapidly determines the identity of meat in the field using the Qamp_mini Thermal cycler for PCR-mediated DNA amplification and the Qsep₁ Bio-Fragment Analyzer (both from BiOptic, Inc., New Taipei City, Taiwan) for the analysis of PCR product by capillary gel electrophoresis (Figure 1).¹

Figure 1 – Three simple steps to results: sample preparation, PCR, and capillary gel electrophoresis.

Small pieces of raw meat and processed meat were pretreated by the simple and fast Q-dip method (BiOptic, Inc.). PCR amplifications were set up using specific primer sets and the Qamp_mini thermal cycler. PCR products of different species-specific lengths (goat-159 bp, chicken-228 bp, cow-273 bp, sheep-334 bp, and pig-396 bp) were analyzed by capillary gel electrophoresis (CGE) using disposable gel cartridges and fluorescence detection with the portable Qsep₁ Bio-Fragment Analyzer.² Q-Analyzer software (BiOptic, Inc.) was used to automatically call peaks and calculate PCR product sizes. PCR product sizes (numbers of base pairs) were used to verify the identity and origin of the meat. In this analysis, raw meat was identified, and even processed meats showed meaningful results. In addition, manufactured meat made of different species were detected simultaneously in one multiplex PCR reaction.

Portability of the instruments and straightforward sample preparation provides a complete and systematic workflow that can be easily employed in field applications, slaughterhouses, or food- or meat-processing facilities. It is envisioned that other applications focused on plant or fish identification, or screening for GMOs in a cost-effective manner, will be developed in the near future.

Instrumentation

PCR amplification

The Qamp_miniThermal cycler is controlled using a programmable memory chip and can process 1–8 samples, starting with the push of a button. The maximum heating rate for the mini-PCR is 4.6 °C/sec and maximum cooling rate is 3.4 °C/sec. Weight of the Qamp_mini instrument is ~1 kg.

Capillary gel electrophoresis

The automated, easy-to-use Qsep₁ CGE system provides fast separation; high detection sensitivity; and high resolution of DNA, RNA fragments, and proteins. Ready-to-use pen-shaped capillary gel cartridges eliminate the need for gel preparation, staining, and photography.³ The system provides accurate results within 3–5 minutes in three simple steps: 1) insert cartridge, 2) place sample, and 3) choose method and run. Q-Analyzer software with a simple user interface (UI) facilitates qualitative and quantitative data analysis of the CE electropherograms.

Materials

Materials used included 2× DirectGO PreMix-CE (BiOptic, Inc.)—a premixed, ready-to-use solution containing reaction buffer, dNTPs, glycerol, PCR enhancers—and a Taq DNA polymerase as a twofold concentration. Compared to conventional Taq polymerase, DirectGO Taq prevents nonspecific amplification due to mispriming and/or formation of primer dimers before thermal cycling, as well as a 2× faster elongation rate. Polymerase activity is restored during the initial denaturation step when amplification reactions are heated at 94–95 °C for 10 minutes. In addition, 2× DirectGO PreMix-CE is suitable for PCR amplification directly from whole blood, and samples from cell lines or tissues. The user can eliminate prior DNA purification steps, applying only a tiny amount of raw material directly into the PCR reaction.

Method

Sample preparation

To a clean 1.5-mL centrifuge tube, 500 μL lysis buffer was added. Next, 500 μL wash buffer was placed into two other individual clean 1.5-mL centrifuge tubes. A small (~1 mm diam) of meat (or sample) was excised and mixed into the lysis buffer. The stick end of the Q-dip was used to press the meat; after 5-min lysis, the membrane side of the Q-dip was used to insert into the lysate. The Q-dip was transferred into wash buffer 1 and stirred gently. The same step was repeated for wash buffer 2. After 2× washing, the membrane was released into the PCR mixture, followed by thermal cycling (Tables 1 and 2). PCR primers (BiOptic, Inc.) match sequences in the cytochrome b gene encoded by vertebrate mitochondrial DNA.

PCR product analysis

PCR products were analyzed using the Qsep₁ CGE system. All samples were introduced into the capillary by electrokinetic injection, i.e., DNA taken up directly from the PCR tubes into the capillary of the gel cartridge. Separations were carried out at ambient temperature applying 8 kV for 180 seconds or 6 kV for 300 seconds.

Results

The method for sample preparation is simple and quick, taking only a few minutes with no devices needed. Different meat samples had species-specific PCR products that could be distinguished and sized by the Qsep series CGE instruments (Figure 2A). Vertebrate mtDNA encoding the cytochrome b gene was conserved. The recognition sequence of turkey and chicken mtDNA are the same; hence the kit can detect both turkey and chicken, but for duck mtDNA, the detection sequence is different. Several processed meats were also identified, verifying that the samples tested were indeed meat.

Figure 2 – Meat species identification. Electropherograms obtained by analyzing PCR products derived from raw meat samples purchased from a local butcher or processed meats from a local restaurant. (A) (above) Lysates of raw meats were prepared with BiOptic lysis buffer. (B) (below) Lysates of processed meat were prepared with Q-dip and BiOptic lysis buffer. Even the processed meats were detected. For both (A) and (B), DirectGO PCR Master Mix was used for all PCR amplifications, and PCR products were analyzed using Qsep series CGE instruments.

The results indicate that even cooked meat can be identified (Figure 2B). Figure 3 shows that the kit can identify five different species of meat mixture at the same time with no need to test the sample individually. The Peak Calling function of the Q-Analyzer software can automatically identify each peak associated with the type of meat, which makes it simpler to screen a mixture of meat; the Comparison function can align multiple results in one chart for ease of visual identification; and the Automate Report function is useful for meat inspection labs that need to generate official reports.

Figure 3 – Meat species identification–mixture. Shown are the detection result of a meat mixture (including cow, pig, chicken, sheep, and goat) by the Peak Calling function of Qsep1 using the S1 high-resolution cartridge after multiplex PCR. Lower marker (LM = 20 bp) and upper markers (UM = 1000 bp) are shown in pink.

Conclusion

The ability to verify the identity and origin of the meats tested and simple sample treatment make the Qsep series CGE systems powerful and cost-effective tools for genotyping, restriction digests or PCR product (multiplex PCR) analyses, and quality control of assays. The workflow of these portable, easy-to-use, and cost-effective technologies will find use in many applications.

References

1. www.americanlaboratory.com/914-Application-Notes/339957-A-Field-Portable-Bioanalysis-System/
2. Colombo, F.; Marchisio, E. et al. A preliminary trial using multi-target polymerase chain reaction (multiplex PCR) and restriction fragment length polymorphism (PCR-RFLP) on the same feedstuffs to detect tissues of animal origin. Vet. Res. Commun. 2004, 28(6), 461–6.
3. www.americanlaboratory.com/914-Application-Notes/118163-Use-of-a-Pen-Shaped-Capillary-Gel-Electrophoresis-Cartridge-for-Cost-Effective-DNA-Fragment-Analysis/

The authors are with BiOptic, Inc., New Taipei City, Taiwan (ROC), and BiOptic, Inc., 1409½ Foothill Blvd., La Canada Flintridge, Calif. 91011, U.S.A.; tel.: 818- 679-4413; e-mail: [email protected]; www.bioptic.com.tw