Muscle and organ tissue (including heart, liver, lung, spleen, and intestine) samples from cattle, sheep, and goat were provided by a local slaughter house. Raw meat samples from buffalo, yak, horse, donkey, pig, chicken, duck, goose, and rabbit were purchased from local markets. DNA samples of rat, mouse, hamster, guinea pig, and dog were obtained from Veterinary Hospital (Huazhong Agricultural University, Wuhan, China). DNA samples of deer, fox, mink, raccoon dog, zebu, small tailed Han sheep, Merinos sheep, Hu sheep, Mongolian sheep, and Mongolian goat were obtained from Laboratory Animal Center (Huazhong Agricultural University, Wuhan, China). Feed ingredients including fish meal, maize, and soybean meal were provided by Hubei Feed Quality Station of Analysis and Supervisor (Huazhong Agricultural University, Wuhan, China). All animal samples were transported to the laboratory under refrigeration, and processed immediately or frozen stored at –80 ℃ until used.
Preparation of binary meat mixture samples, artificial feed samples, and commercial feed samples
To evaluate the co-detection ability of our method, different percentages of binary meat mixture samples were made in laboratory. Cattle and sheep muscle samples were dried in oven (65 ℃ for 120 min), smashed in grinder, and mixed at a weight/weight (w/w) ratio of 100:0, 95:5, 90:10, 75:25, 50:50, 25:75, 10:90, 5:95, and 0:100, respectively. All binary meat mixture samples were made to reach a final weight of 100 g using a blender (Sunbeam Oster, Florida, USA) and stored at -20 ℃ until use.
To evaluate the sensitivity of our method, artificial feed samples were prepared by mixing different percentages of bovine or ovine/caprine meat and bone meal (MBM) with soybean meal. According to the practical production procedure of MBM (European Commission, 2009), bovine MBM was made by mixing equal amount of cattle organ tissues (liver, lung, spleen, and intestine) and treating them at 133 ℃, 300 kPa for 20 min in autoclave (GR60DA; Zealway Instrument Inc., Delaware, USA). Ovine/caprine MBM was a mixture of heart, liver, lung, and intestine tissues from sheep, whose processing method was same with that of bovine MBM. Subsequently, binary mixtures mixing 0.1%, 1%, 5%, 10%, 25%, and 50% (w/w) heat-treatment MBM (bovine or ovine/caprine) and soybean meal were prepared into artificial feed samples.
To further evaluate the applicability of our method in commercial feeds, a feed mill (Shandong Tianpu Sunshine Feed Co., Ltd., Shandong, China) was commissioned for preparing five commercial feed samples. The actual production process of commercial feed samples included crushing, puffing, steam quenching, and granulation (90 ℃, 800 kPa for 20 min). According to our request, 1% of bovine MBM, ovine/caprine MBM, or fish meal was randomly incorporated to five commercial feed samples, and the detail adulteration information was unavailable to us.
The extraction of sample genomic DNA was carried out according to the proteinase K-sodium dodecyl sulfate (SDS)-phenol/chloroform protocol (Reid, 1991), with minor modifications. The detailed descriptions were presented in E-Supplementary. The concentration and purity of all the genomic DNA extracts were measured using Nanodrop 2000 (Thermo Scientific, USA). In addition, the integrity of DNA extracted from raw and heated tissues was checked by 1% agarose gel electrophoresis in 0.5 × TBE buffer (4.45 mmol/L Tris-HCl, 100 mmol/L EDTA-Na2, 44.50 mmol/L boric acid, pH 8.0). All chemical reagents were purchased from Shanghai Hushi Testing Equipment Co., Ltd (Shanghai, China).
Screening of universal target DNA sequence
DNA sequences of cattle were aligned with homologous sequences of other species (without bovine, ovine and caprine) available in GenBank by using the Basic Local Alignment Search Tool (BLAST). The sequences with the alignment parameters of Identity < 90% and Query-length > 200 bp were further aligned with the whole genome sequences of other bovines (zebu, yak, bison, and buffalo), ovine (sheep), and caprine (goat), respectively. The sequences with Identity > 95% were selected as specific sequences for bovine, ovine, and caprine species (Figure 1). For the convenience and efficiency of detection, a DNA fragment with 18 bp sequence deletion in both ovine and caprine genomes was selected as species-specific sequence. Genome sequence alignments of 53 species including representative species of 4 kingdoms, 10 classes, 21 orders, 36 families and 47 genera (cattle, zebu, yak, bison, buffalo, horse, przewalskii horse, donkey, sheep, goat, deer, camel, pig, rabbit, fox, dog, wolf, dingo, cat, bear, ferret, monkey, human, mouse, rat, jerboa, hamster, guinea pig, squirrel, beaver, pika, dolphin, whale, elephant, bat, hedgehog, chicken, turkey, duck, goose, ostrich, anole, frog, zebrafish, salmon, fruit fly, soybean, maize, rice, wheat, yeast, Escherichia coli, and Salmonella) were performed by BLAST and ClustalW.
Species-specific primer pair were designed in the conserved region using Primer Premier 5.0 (Premier, San Francisco, CA, USA) for specific detection of target sequences of bovine, ovine, and caprine species. The size of amplified fragment was set to be shorter than 200 bp with at least 10% difference between bovine and ovine/caprine DNA. The sequences of novel universal primer pair were shown in Table. 1. Moreover, a previously reported internal reference primer pair were used for the amplification of a conserved fragment of 18S rRNA from animal and plant species in this study (Yun et al., 2018). All the primer pairs were diluted to 10 µmol/L for preservation.
To optimize PCR system, the concentrations of universal primer pair were set as 0.1 µmol/L, 0.2 µmol/L, 0.3 µmol/L, and 0.4 µmol/L to amplify the target fragment of cattle and sheep, and annealing temperatures were set as 58 ℃, 60 ℃, 62 ℃, and 64 ℃, respectively. As shown in E-Supplementary Figure S1, the amount of amplified products reached the maximum for both cattle and sheep without dimers produced, when the concentration of universal primer pair was 0.2 µmol/L and the annealing temperature was 62 ℃. Thus, the PCR amplification was carried out at a total volume of 20 µL containing 50 ng of template DNA, 0.2 µmol/L of each primer, 1 × PCR buffer, 2 mmol/L MgCl2, 0.2 mmol/L of deoxynucleoside triphosphates (dNTPs), 0.5 U rTaq polymerase (TaKaRa Bio Inc., Otsu, Japan), and sterile distilled water. PCR conditions of universal primer pair were as follows: an initial heat denaturation at 95 ℃ for 5 min, followed by 35 cycles of DNA denaturation at 95 ℃ for 30 s, primer pair annealing at 62 ℃ for 30 s, and DNA extension 72 ℃ for 15 s. The last step was the extension at 72 ℃ for 3 min. In addition, the PCR reaction system and PCR procedures of internal reference primer pair referred to previous report by Yun et al. (2018). The PCR was performed in a thermal cycler (Bio-Rad Laboratories, Inc., USA). The amplified products were analyzed by 2.5% gel electrophoresis in 0.5 × TBE and were sequenced by TSINGKE Biotech co., Ltd (Beijing, China).