Simultaneous detection of ovine and caprine DNA in meat and dairy products using triplex TaqMan real‐time PCR

Abstract In this study, we report a new approach for the detection of ovine and caprine DNA in meat and dairy products using real‐time PCR protocol. Our new approach is based on the use of endogenous control and species‐specific TaqMan fluorescence probes. With this methodology, we specifically detected ovine and caprine DNA in meat and dairy products, with limits of detection of 0.001 ng and 0.01 ng for fresh and processed ovine meats, respectively, and 0.00025 ng, 0.005 ng, and 0.01 ng for caprine meat, milk, and cheese, respectively. Artificial meat and milk mixtures from sheep and goat were used to validate the protocol. Our results support that TaqMan real‐time PCR with endogenous control is an efficient and accurate method to detect DNA from sheep and goat in meat and dairy products.


| INTRODUC TI ON
Meat and dairy products provide essential nutrients that support growth, functions, immunity, and general human well-being.
Xilingol grassland (203,000 km 2 ) in China is a natural grazing region, known for the free raising of animals for human consumption and its low pollution levels, resulting in the production of high-quality meat and dairy products from sheep and goat, among others. The average annual output of sheep and goat in Xilingol grassland is more than ten million of animals. Nevertheless, some producers and traders deliberately adulterate meat and dairy products with less expensive substitutes, which unbalances market competition and disregards consumer's interests. Further, product authentication is required to manage health risks and to comply with religious norms. Therefore, the development of efficient approaches for product authentication is essential to ensure the purity of food products. Here, we developed a specific and highly sensitive method to evaluate adulteration of meat and milk from sheep and goat based on DNA detection using real-time PCR with TaqMan technology.
Distinct analytical PCR-based methods have been developed for DNA detection in animal-derived products such as meat and dairy (Abbas et al., 2018;Bohme et al., 2019;Lo & Shaw, 2018). Conventional PCR (Fajardo et al., 2006;Kumar et al., 2014;Pfeiffer et al., 2004), multiplex PCR (Golinelli et al., 2014Safdar & Junejo, 2015;Xue et al., 2017), conventional real-time PCR López-Calleja et al., 2007;Tanabe et al., 2007), and multiplex real-time PCR (Agrimonti et al., 2015;Di Domenico et al., 2017;Guo et al., 2019) are standard techniques used in the detection of ovine and caprine DNA. Here, we developed a multiplex real-time PCR with endogenous control for simultaneous detection of ovine and caprine DNA in meat and dairy products. Multiplex real-time PCR is more efficient and less time-consuming than conventional real-time PCR and PCR due to lack of electrophoresis and simultaneous amplification of different target genes. Furthermore, the simultaneous amplification of an endogenous control avoids false-negative results.

| Sample preparation and DNA isolation
Fresh and processed meat samples from sheep, goat, beef, horse, pork, chicken, duck, goose, dog, rabbit, cat, and fish were purchased from 109 supermarkets and DKL shopping mall in Xilingol region in Inner Mongolia. Milk from goat, cow, and mare, as well as koumiss, was obtained from Plain Mountain Pasture from Xilingol region. Caprine cheese (Queserias Entrepinares) was purchased from the Shanghai Rongyue company, and bovine cheese was obtained from Ximulike dairy company from Xilingol region.
DNA from meat and dairy products was isolated with TaKaRa MiniBEST Universal Genomic DNA Extraction Kit, using a modified CTAB method (Guo et al., 2018 shares one identical forward primer (LP1) and two reverse primers (Ovine-RP1 and Caprine-RP1) with one different base, and control probe can anneal the same targeted amplified sequence with ovine probe and caprine probe in the triplex real-time PCR.
PCRs were performed in an ABI 7300 plus thermocycler (Applied Biosystems), with the following program: 94°C for 30 s followed by 40 cycles of 94°C for 5 s and 60°C for 31 s.

| Reaction specificity and sensitivity evaluation
Samples of raw meat from sheep, goat, beef, horse, pork, chicken, duck, goose, dog, rabbit, cat, and fish were used to validate our newly designed primers and probes. Ct values were calculated for eight different types of fresh ovine and caprine meats and four types of processed ovine meats, and three types of milk and three types of cheese from goat were utilized to verify the specificity.

| Specificity of triplex real-time PCR in the amplification of DNA from meat and dairy
In the TaqMan real-time PCR assay, the ovine-specific probe was labeled with FAM (fluorophore) and TAMRA (quencher). The caprine-specific probe was labeled with HEX and TAMRA. The endogenous control probe was labeled with ROX and BHQ-2. The use of different fluorophores (FAM, HEX, and ROX) allowed the simultaneous detection of three distinct fluorescence signals, thus allowing the identification of DNA from different sources (in this case, from sheep, goat, and the endogenous control). Figure 1 shows the amplification plots of triplex real-time PCR using DNA from fresh ovine and caprine meat as template. An endogenous control (Control-ROX) was also amplified to eliminate false-negative results. As shown in Figure Abbreviation: N/A, not applicable. a Data (average ± SD) represent three replicates.
The results show that the amplification of sheep-and goat-derived DNA was steadily showed. Furthermore, no amplification was observed when nontarget animal DNA was used as a template for the PCRs, supporting the specificity of the method. The above data illustrated that the triplex real-time PCR with the endogenous control was specific for the simultaneous detection of ovine and caprine DNA isolated from the respective fresh meat.
Compared with fresh meat, processed meat products are more likely to be adulterated due to physical processing.
Processed meat samples from sheep, cattle, horse, and pig were used to evaluate the specificity of the method, and the corresponding plots show that amplification was specific to ovine and caprine samples, whereas no amplification was observed in samples from other species, using our sheep-and goat-specific Corresponding Ct values are shown in Thus, our data show that triplex real-time PCR with an endogenous control is specific for the simultaneous detection ovine and caprine DNA in processed meat products.
Sheep and goat are important livestock in Xilingol grassland, being mainly raised for meat, milk, and fleece production. We then evaluated the specificity of our method in DNA isolated from caprine milk and cheese.   SD) represent three replicates.
bovine cheese ( Figure 2i) and koumiss (Figure 2j), was used as the negative control. The Ct values (average ± SD) of caprine milk, bovine milk, mare milk, caprine cheese, bovine cheese, and koumiss are illustrated in Table 3. The above data demonstrate that our triplex real-time PCR is a specific technique for the authentication of caprine milk and cheese products.
Generally, the identification of DNA from meat and dairy products is based on conventional PCR and real-time PCR with fluorescent dye.
However, when compared to TaqMan-based methods, conventional PCR shows limited specificity and is more time-consuming, as it only allows the identification of a single species in each reaction. Our multiplex real-time PCR was designed to detect several DNA species in a single PCR using species-specific TaqMan probes. For that, it is fundamental to design specific primers and probes that are compatible in the same reaction. In the context of the authentication of ovine and caprine meat and dairy products, this type of approach has not been used, so far. Our results show that our triplex real-time PCR is suitable for the specific identification of ovine and caprine DNA, both in fresh and in processed meat products, as well as in caprine milk and dairy products.
The simultaneous detection of DNA from different species contributes to lowering reagent cost, consumables, and reduces experimental time by half due to the addition of a species-specific probe. Furthermore, the performance of the PCR can be monitored by the inclusion of the endogenous control in the same reaction of the target template, thus avoiding false-negative results.

| Sensitivity of triplex real-time PCR in the amplification of DNA from meat and dairy products
As shown in Figure 3a,b, the LOD of ovine DNA was of 0.001 ng (DNA isolated from fresh ovine meat) and of 0.01 ng (DNA isolated from processed ovine meat; confidence limit: 95%). Moreover, as shown in Figure 3c-e, the LOD of caprine DNA identification was of 0.00025 ng (DNA isolated from fresh caprine meat) and of 0.005 ng and 0.01 ng (DNA isolated from caprine milk and cheese, respectively, confidence limit: 95%). As shown in Table 4

| Triplex real-time PCR in DNA detection from artificial meat and dairy mixtures
To evaluate the efficiency of the triplex real-time PCR in the detection of DNA from meat mixtures, we isolated DNA from artificial binary meat mixtures, containing ovine and porcine meat, and used it as template for PCR. The percentages of ovine meat in the mixtures were 99.9%, 99%, 90%, 70%, 30%, 10%, 1%, and 0.1% (w/w).

TA B L E 4
The Ct values in the real-time PCR assay for the sensitivity of identification of meat and dairy products from sheep and goat The corresponding percentages of porcine meat in the mixtures were 0.1%, 1%, 10%, 30%, 70%, 90%, 99%, and 99.9%. As shown in Figure 5a and Table 5 In a similar set of experiments as before, we used binary dairy mixtures containing caprine and bovine milk to evaluate the efficiency of the triplex real-time PCR. The percentages of caprine milk in the mixtures were 99%, 95%, 90%, 70%, 30%, 10%, 5%, and 1% (w/w). The corresponding percentages of bovine milk in the mixtures were 1%, 5%, 10%, 30%, 70%, 90%, 95%, and 99%. As shown in Figure 5c and Table 5, with the decreasing of caprine milk in the mixtures, the Ct values were increasing, and the Ct values for 5% and 1% caprine percentage were 0. The amplification plots of Goat-HEX were steadily appeared in ≥10% caprine percentages, not shown in the percentage of 5% and 1% (Figure 5c) (confidence limit: 95%).
Furthermore, the endogenous control (Control-ROX) was steadily amplified in all eight dairy mixtures ( Figure 5c). In conclusion, our data suggest that our newly developed triplex real-time PCR method with an endogenous control can simultaneously detect caprine DNA in dairy mixtures.

As our results show, probe amplification is observed in mixtures
where the corresponding meat percentage is at least of 10%, which in the context of meat adulteration in industry is common and profitable. Therefore, we propose that our PCR method can be used to certificate ovine and caprine meat and dairy products in mixtures.
Still, the limit of detection of our method did not reach 1%-0.1% discrimination (average level), which might be due to our specific primer and probe design. In our triplex real-time PCR method, com- of ovine meat. The lower limit of detection in the sheep-chicken meat mixture indicates that methods based on species-specific primer pairs are more sensitive in DNA identification in meat and dairy mixtures, compared to methods using species-conserved primers. Currently, our team is developing two types of real-time PCR systems for product authenticity certification. On the one hand, we developed species-conserved primers and multichannel probes for high-throughput evaluation. On the other hand, we also developed species-specific primers and a probe for qualitative and quantitative analysis of low abundant contamination (adulteration). and dairy products and to include an endogenous control to avoid false-negative results. The method is based on the design of speciesconserved primers, that is, a primer pair that targets a conserved genomic region across different species, an endogenous control probe, and species-specific probes. Oligonucleotide design was optimized to ensure all oligonucleotides were compatible to be amplified in the same real-time PCR. Our results show that our method allows the specific identification of ovine and caprine DNA, the limits of detection of 0.001 ng and 0.01 ng (ovine DNA in fresh and processed meat samples), and 0.00025 ng, 0.005 ng, and 0.01 ng (caprine DNA in fresh meat, milk, and cheese samples, respectively). Our triplex real-time PCR method with endogenous control is, thus, a specific and highly sensitive technique for the certification of meat and dairy products derived from sheep and goat.

ACK N OWLED G M ENTS
This work was supported by Natural Science Foundation of Inner Mongolia

Autonomous Region of China (2020MS03065) and Inner Mongolia
College Science Research Project (NJZY20312 and NJZY19337) and Xilingol Vocational College Key Research Project (ZD-2020-03).

CO N FLI C T O F I NTE R E S T
All authors declared 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
Data available on request from the authors.