Improved triplex real‐time PCR with endogenous control for synchronous identification of DNA from chicken, duck, and goose meat

Abstract The authentication and labeling of meat products, concerning origins and species, are key to fair trade and to protect consumer interests in the market. We developed an improved triplex real‐time PCR approach to simultaneously identify chicken, duck, and goose DNA in meat, including an endogenous control to avoid false negatives. Our method specifically detected DNA from chicken, duck, and goose, and showed no cross‐reaction with DNA extracted from other meat types. The detection limits of chicken, duck, and goose DNA were 0.001–0.00025 ng, 0.0025–0.0001 ng, and 0.001–0.00001 ng, respectively, and we were able to simultaneously identify DNA from two distinct origins using as little as 0.1% of total meat weight. Our newly generated triplex real‐time PCR method with endogenous control exhibited high specificity, sensitivity, and efficiency for simultaneous identification of DNA from chicken, duck, and goose in meat.


| INTRODUC TI ON
In the last 50 years, poultry production has increased at global scale; the rate of increase in number is over twice that in human population (Bazer et al., 2020). Poultry is a source of high-quality animal proteins and is often used as an ingredient in meat products. In addition, meat alteration with lower-cost products, such as chicken, duck, or goose meat, has been used to illegitimately achieve higher financial profits. These fraudulent practices of adulteration might affect fair competition and do not consider the consumer interests in the market (Abbas et al., 2018;Bohme et al., 2019). Regulations have been applied to impose restrictions on the adulteration of meat products.
To implement these legislations, there is an increasing demand for robust methods to analyze the authenticity of meat species claimed by manufacturers or distributors. Therefore, a specific, sensitive, and efficient method for the authentication of poultry meat (chicken, duck, and goose) is essential to the supervision of market practices.
The goal of this study was to develop a triplex real-time PCR method for the simultaneous identification of chicken, duck, and goose DNA in meat. More importantly, an endogenous control was designed to be compatible with chicken, duck, and goose probes, and was amplified with poultry-specific probes in order to eliminate false negative results, which is known to be a serious limitation to the legal impartiality of the report of authentication test.

| Preparation of meat samples and DNA extraction
Raw meat and products from chicken, duck, goose, quail, pigeon, cattle, buffalo, yak, sheep, goat, pig, horse, donkey, camel, and rabbit were obtained from agricultural market in Xilinhot, China. The meat samples were chopped into small pieces and stored at −80°C to prevent degradation of DNA.
The genomic DNA from meat samples was extracted by Takara MiniBEST Universal Genomic DNA Extraction Kit (TaKaRa, Dalian, China) according to manufacturer's protocol. The purity and concentration of extracted DNA were determined based on absorbance at A 260 /A 280 by Nanodrop2000 (Thermo Fisher Scientific).

| Development of primers and probes for the triplex real-time PCR
Triplex real-time PCR involves three amplification reactions, which require three independent primer pairs and three probes.
Functionally, three different probes anneal with the corresponding species-specific DNA and participate in the PCR reaction with primers. From our previous experience, we purpose that a speciesconserved forward primer, a species-specific reverse primer, and three species-specific probes are more compatible and stable than three independent primer pairs in the triplex real-time PCR. In this study, after alignment of mitochondrial DNA from different species (chicken, duck, goose, quail, pigeon, cattle, buffalo, yak, sheep, goat, pig, horse, donkey, camel, and rabbit), the species-conserved forward primer, the species-specific reverse primer, and the species-specific probes were designed to specifically target the mitochondrial gene sequences of chicken, duck, and goose, respectively ( Figure 1).
Additionally, a species-conserved probe for endogenous control was designed to monitor the amplification reaction, and eliminate false negative detection (Figure 1). The primers and probes for our triplex real-time PCR were designed to anneal with a limited DNA length between 100 and 150 nucleotides. Additionally, different fluorescent reporters, such as 6-carboxyfluorescein (FAM), hexacholoro-6-corboxyfluorescein (HEX), and carboxy-X-rhodamine (ROX), were introduced to label chicken, duck, goose, and the endogenous control probe, respectively. All oligonucleotides were synthesized and purified using HPLC by Ruibiotech Company, and showed in Table 1.

| Specificity assay
DNA from raw meat (chicken, duck, goose, quail, pigeon, cattle, buffalo, yak, sheep, goat, pig, horse, donkey, camel, and rabbit) and processed meat products (chicken sausage, spiced duck wing, goose jerky, chicken pork sausage, chicken beef sausage, beef jerky, mutton jerky, dried horse meat, and dried donkey meat) were used to confirm the specificity of the triplex real-time PCR, and the results were verified by three replicates. The triplex real-time PCR systems consisted of 10 μl of TransStart probe qPCR SuperMix (Tansgen), each of 1 μl forward primer (10 μM), 1 μl reverse primer (including reverse primers of chicken, duck, and goose), 0.5 μl of chicken probe (10 μM), 0.5 μl of duck probe (10 μM), 0.5 μl of goose probe or control probe (10 μM), 1 μl of DNA template (100 ng/μl), and distilled deionized water (Transgen) for a total volume of 20 μL. The amplification was performed using initial denaturation step at 94°C for 30 s, followed by 40 cycles of denaturation at 94°C for 5 s, and annealing and extension at 60°C for 34 s. (ABI 7300plus, Applied Biosystems).
Twenty replicates for each dilution were used for the evaluation of LOD of the triplex real-time PCR, and the results were analyzed as inferred from Probit analysis (Finney, 1971).

| Specificity evaluation of the triplex real-time PCR reaction
The specificity of the triplex real-time PCR assay was determined using the designed probes to identify the corresponding DNA from chicken, duck, and goose. As shown in Figure  and goose jerky ( Figure 2f). As shown in Table 2, the cycle threshold (Ct) values (average ± SD) of the triplex real-time PCR method with chicken, duck, and goose probes were consistent with the amplification curves. These results indicate that the chicken, duck, and goose probes can simultaneously identify their target DNA, and that the triplex real-time PCR method is specific in the detection of distinct poultry meats.
Although efforts have been made to develop efficient methods for the detection of poultry contamination in meat (Amaral et al., 2015;Furutani et al., 2017;Hou et al., 2015;Kesmen et al., 2012;Martin et al., 2007;Pegels et al., 2012;Thanakiatkrai et al., 2019), only two studies reported multiplex PCR techniques containing an end point PCR method (Hou et al., 2015) and a TaqMan-based multiplex realtime method (Köppel et al., 2013). However, the protocol based on multiplex PCR was shown to be less efficient, more time-consuming, and more laborious than our approach, which does not require electrophoresis of the amplified DNA (Hou et al., 2015).

| Sensitivity evaluation of the triplex real-time PCR reaction
According to the parameters required for the development and  Table 3).
The above results suggested that the LOD was different between meat samples. We speculated that the integrity of DNA influences F I G U R E 2 Triplex real-time PCR amplification curves for specificity evaluation. The results were confirmed by 3 replicates. ΔRn = change in normalized reported values the LOD of the real-time PCR reaction. The LODs observed in this study were lower than in previously reported multiplex PCR method (Hou et al., 2015) and were similar to other real-time PCR approaches (Guo et al., 2018;Kesmen et al., 2012). These results show that our newly developed real-time PCR is sensitive in the detection of poultry DNA in meat products.
To determine the linearity of the triplex real-time PCR assay, DNA from meat products was serially diluted and used as template. show that our method is in accordance with the established guidelines for the quantitative determination of the raw and processed poultry.

| Authentication evaluation of the triplex realtime PCR reaction
In order to simulate the poultry adulteration practice and to validate the simultaneous triplex real-time PCR method for authentication, the method was utilized to detect chicken, duck, and goose DNA in the ternary meat mixtures of chicken, duck, and goose. As shown in Figure 5 and Table 4, the three target poultry species were detected in the ternary meat mixtures, at levels as low as 0.1% of total meat weight. Moreover, the triplex real-time PCR method was successfully employed for simultaneous detection of two target species in the three types of ternary meat mixtures, at levels as low as 0.1% of total meat weight ( Figure 5). These results revealed that the triplex real-time PCR method is a sensitive and specific approach for the rapid and simultaneous identification of minimal percentages of two target species in meat mixtures. It is frequently observed that the inclusion of poultry products in other meats, for economic profit, corresponds to over 10% of total meat weight. Thus, we purpose that our method might be useful in the market supervision of meat adulteration.
PCR-based methods have been previously used to identify as little as 0.1% pork content (Karabasanavar et al., 2014), 0.1% dog meat (Rahman et al., 2014), and 0.1% chicken content in meat mixtures (Furutani et al., 2017). Yet, to our knowledge, no PCR-based methods for the simultaneous identification of two species, maintaining the same detection limit, have been developed so far. Previously developed triplex real-time PCR methods for the simultaneous identification of mare and cow, as well as goat and cow, in milk, showed less sensitivity (1%-10%) (Guo et al., 2018(Guo et al., , 2019. The triplex real-time PCR for mare, goat, and cow milk used a species-conserved primer pair. Conversely, we hypothesize that the species-conserved forward primer ensured the performance of the triplex real-time PCR, while the species-specific reverse primer increased the authentication ability of the method in this study.

| Endogenous control validation
We designed an endogenous probe to be amplified with chicken-, duck-, and goose-specific probes, to avoid false negative results.
The specificity of the triplex real-time PCR containing Chicken-FAM, Duck-HEX, and Control-ROX was validated using chicken, duck, and goose meats. As expected from our previous findings, the amplification curves of chicken-FAM were specifically observed for chicken meat ( Figure 6a) and processed chicken (Figure 6b), and the amplification curves of duck-HEX were specifically observed for duck meat ( Figure 6c) and spiced duck wing (Figure 6d). More importantly, the amplification curves of Control-ROX were detected in all poultry samples ( Figure 6). Although the assembled multiplex real-time PCR has been developed to detect simultaneously chicken, duck, and goose (Köppel et al., 2013), our results suggest that the triplex real- in the corresponding binary meat mixtures. As shown in Figure 7 and

TA B L E 3 (Continued)
F I G U R E 4 Quantification assays in poultry: chicken, duck meat, and goose meat (a) and chicken sausage, spiced duck wing, and goose jerky (b) F I G U R E 5 Validation assays in ternary meat mixtures. The results were confirmed by 20 replicates In this study, we used ABI 7300plus to perform the triplex realtime PCR. This instrument can simultaneously detect three different fluorescent probes. Therefore, the three triplex real-time PCR system was assembled from the four probes developed in this study.
Moreover, these probes can be combined into a quadruplex realtime PCR method, allowing for the simultaneous detection of meat origins and the amplification of the endogenous control.

| CON CLUS IONS
To study the adulteration in poultry and increase the efficiency and sensitivity of real-time PCR, we developed the triplex real-time PCR for the simultaneous identification of chicken, duck, and goose DNA. The improved triplex real-time PCR containing the speciesconserved forward primer, the species-specific reverse primer, TA B L E 4 Authentication assay of the triplex real-time PCR in the ternary and binary meat mixtures and three species-specific probes is more efficient and economical than previously reported methods using conventional real-time PCR. Furthermore, an endogenous probe has been designed to be amplified with poultry-specific probes in order to avoid false negative results. The limits of detection of chicken, duck, and goose in the improved triplex real-time PCR were 0.001-0.00025 ng, F I G U R E 6 Triplex real-time PCR amplification curves for specificity evaluation, with endogenous control probe. The results were confirmed by 3 replicates. ΔRn = change in normalized reported values F I G U R E 7 Validation assays in binary meat mixtures. The results were confirmed by 20 replicates 0.0025-0.0001 ng, and 0.001-0.00001 ng, respectively. In addition, 0.1% poultry adulteration can be steadily validated in the simulation of adulteration. In conclusion, our triplex real-time PCR method with an endogenous control shows higher specificity, sensitivity, and efficiency in the synchronous identification of chicken, duck, and goose DNA in meat products.

CO N FLI C T O F I NTE R E S T
All authors declare no conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.