Multiplex PCR primers, kits and methods for identifying emu and ostrich meat components

Abstract

The present invention discloses multiplex PCR specific identification primers for emu and ostrich meat source components, including emu specific primers, whose nucleotide sequence is 5'-TGTGGGGGCTTTCTTCTTAGT-3', and ostrich specific primers, whose nucleotide sequence is 5'-ACAGCGTATGAGAGTGCTATAGTC-3'. The present invention also discloses a kit comprising the multiplex PCR specific identification primers, and a multiplex PCR identification method for emu and ostrich meat source components. The emu and ostrich specific primers provided by the present invention respectively perform PCR amplification on mitochondrial 16S rRNA and ND1, and the amplification effect is stable, and specific bands of 236bp and 389bp can appear respectively, which effectively identifies the emu and ostrich meat sources.

Description

Technical Field

[0001] The invention relates to multiplex PCR identification of meat-derived components, in particular to multiplex PCR specific identification primers for emu and ostrich meat-derived components. Background Art

[0002] The ostrich, scientifically known as Struthio camelus, belongs to the class Aves, the order Ratite, the order Struthius, the family Struthidae, and the genus Struthius. It is an important economically characteristic poultry species that has been domesticated artificially. It is the largest existing bird in the world, with a maximum weight of 200kg. In the 1980s, my country began to introduce African black ostriches, red-necked ostriches, and blue-necked ostriches, which were included in the "Chinese Livestock and Poultry Genetic Resources - Special Livestock and Poultry" published in 2011, and were also included in the "National Livestock and Poultry Genetic Resources Catalog" published in 2020. Ostriches adapt to dry environments, and green roughage is the main part of their daily feed. They do not compete with humans for land and food, and can reach a weight of 100kg after being raised for about ten months. Ostrich meat is edible, its skin can be made into leather, and its hair can be made into decorations. Its whole body is a treasure, and its meat has high protein content, low fat content, and good taste, which is comparable to beef, etc., and has high economic utilization value.

[0003] The origin of emu is Australia, and it is a species endemic to Australia. Its scientific name is Dromaiusnovaehollandiae. It belongs to the class Aves, the order Ratite, the order Struthiomorpha, the family Emu, and the genus Emu. It weighs about 40-50kg and is the second largest bird after the ostrich. Due to its uniqueness and tolerance, emu has long been raised in my country and is also called "Australian ostrich" in my country. With the successful artificial breeding of emu in the 1950s, artificial large-scale breeding began in Guangdong Province, my country in 1989, and has now formed a large breeding scale in the country. Emu was included in the "Chinese Livestock and Poultry Genetic Resources-Special Livestock and Poultry" published in 2011, and was also selected in the "National Livestock and Poultry Genetic Resources Catalog" published in 2020. Emu meat looks similar to beef cattle, is rich in protein, low in cholesterol, helps reduce the occurrence of cardiovascular diseases, and is in line with the dietary trends of modern people. Emu skin and emu oil both have important development value. Emu is a new breed of livestock animal that is mainly herbivorous and has high comprehensive benefits.

[0004] With the rapid development of emu and ostrich breeding industry in my country in recent years, counterfeit emu and ostrich meat has also occurred from time to time. Therefore, the development of rapid detection technology for emu and ostrich meat ingredients is of great significance to the development of emu and ostrich industries in my country.

[0005] The identification of meat-derived ingredients is usually sampled by real-time fluorescence quantitative PCR method. This method has high sensitivity, is suitable for the detection of trace meat-derived ingredients in processed foods, and can be relatively quantitative, so this method is widely used. However, fluorescence quantitative PCR requires expensive instruments, the operation method is cumbersome, and the cost is high. In addition, sequencing methods can also be used to detect a variety of meat-derived ingredients, but this method is labor-intensive, costly, and inaccurate. Summary of the invention

[0006] The purpose of the present invention is to provide multiplex PCR specific identification primers for emu and ostrich meat source components, which have high specificity for emu and ostrich meat sources and can be effectively identified.

[0007] One of the purposes of the present invention is to provide multiplex PCR specific identification primers for ostrich meat source components, which have high specificity for ostrich meat source and can be effectively identified.

[0008] The multiplex PCR specific identification primers for ostrich meat-derived components include ostrich-specific primers, the nucleotide sequences of which are:

[0009] 5'-ACAGCGTATGAGAGTGCTATAGTC-3'.

[0010] Furthermore, the identification primers provided by the present invention include:

[0011] Ostrich-specific primer: 5'-ACAGCGTATGAGAGTGCTATAGTC-3'

[0012] Universal upstream primer: 5'-ACGACCTCGATGTTGGATCA-3'

[0013] Universal downstream primer: 5'-CGGTCTGAACTCAGATCACGTA-3'.

[0014] The second object of the present invention is to provide a kit comprising the above-mentioned multiplex PCR specific identification primers for ostrich meat-derived components.

[0015] Specifically, the kit includes at least ostrich-specific primers:

[0016] 5'-ACAGCGTATGAGAGTGCTATAGTC-3'.

[0017] Furthermore, the kit of the present invention also includes:

[0018] Universal upstream primer: 5'-ACGACCTCGATGTTGGATCA-3',

[0019] Universal downstream primer: 5'-CGGTCTGAACTCAGATCACGTA-3'.

[0020] The third object of the present invention is to provide a multiplex PCR identification method for ostrich meat-derived components based on the above-mentioned specific identification primers.

[0021] Specifically, the multiplex PCR identification method for ostrich meat-derived components is to take a meat-derived sample to be identified, place it in a lysis solution for lysis, extract genomic DNA, perform PCR amplification, and perform electrophoresis; when in the electrophoresis result, the amplification product only has a common amplification band of about 100 bp, the sample is determined to be non-ostrich; when the amplification product simultaneously has a 100 bp common amplification band and a 389 bp ostrich-specific band, the sample is determined to be ostrich.

[0022] Emu meat is similar to ostrich meat, and therefore, the fourth object of the present invention is to further provide specific identification primers capable of performing multiplex PCR on emu and ostrich meat components, that is, on the basis of the identification primers described above, an emu-specific primer is also included, and the nucleotide sequence thereof is: 5'-TGTGGGGGCTTTCTTCTTAGT-3'.

[0023] Specifically, the identification primers provided by the present invention include:

[0024] emu-specific primer: 5′-TGTGGGGGCTTTCTTCTTAGT-3′;

[0025] Ostrich-specific primer: 5′-ACAGCGTATGAGAGTGCTATAGTC-3′.

[0026] Further, including:

[0027] Universal upstream primer: 5′-ACGACCTCGATGTTGGATCA-3′;

[0028] Universal downstream primer: 5'-CGGTCTGAACTCAGATCACGTA-3'.

[0029] A fifth object of the present invention is to provide a kit comprising the above-mentioned multiplex PCR specific identification primers for emu and ostrich meat-derived components.

[0030] Specifically, the kit includes at least an emu-specific primer: 5'-TGTGGGGGCTTTCTTCTTAGT-3';

[0031] and ostrich-specific primers:

[0032] 5'-ACAGCGTATGAGAGTGCTATAGTC-3'.

[0033] Furthermore, the kit of the present invention also includes:

[0034] Universal upstream primer: 5′-ACGACCTCGATGTTGGATCA-3′;

[0035] Universal downstream primer: 5'-CGGTCTGAACTCAGATCACGTA-3'.

[0036] The sixth object of the present invention is to provide a multiplex PCR identification method for emu and ostrich meat-derived components based on the above-mentioned specific identification primers.

[0037] Specifically, the multiplex PCR identification method for emu and ostrich meat components is to take a meat sample to be identified, place it in a lysis solution for lysis, extract genomic DNA, PCR amplify, and electrophoresize; when the electrophoresis result shows that the amplified product has only one common amplification band of about 100 bp, the sample is judged to be non-emu and non-ostrich; when the amplified product has both a 100 bp common amplification band and a 236 bp emu-specific band or a 389 bp ostrich-specific band, the sample is judged to be emu or ostrich; the primers used in the multiplex PCR amplification are as follows:

[0038] Universal upstream primer: 5′-ACGACCTCGATGTTGGATCA-3′;

[0039] Universal downstream primer: 5′-CGGTCTGAACTCAGATCACGTA-3′;

[0040] emu-specific primer: 5′-TGTGGGGGCTTTCTTCTTAGT-3′;

[0041] Ostrich-specific primer: 5′-ACAGCGTATGAGAGTGCTATAGTC-3′.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The invention provides emu and ostrich specific primers for conventional PCR amplification of 16S rRNA and ND1, does not require expensive equipment such as a fluorescent quantitative PCR instrument, and has a stable amplification effect, can produce 236bp and 389bp specific bands, can effectively and independently (universal primer + one specific primer) identify emu or ostrich meat sources, and can also use a set of primers (universal primer + two specific primers) to simultaneously identify emu and / or ostrich meat sources; and the provided universal primers can efficiently amplify vertebrate meat sources, and can effectively avoid false negatives. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Electropherograms of PCR amplification products for separate identification of emu and ostrich meat sources;

[0045] M stands for DNA marker DL 2000.

[0046] Figure 2 Electrophoresis diagram of PCR amplification products for joint identification of emu and ostrich meat sources;

[0047] Lanes 1-2 are red-necked ostriches + emus, 3-4 are blue-necked ostriches + emus, 5-6 are African black ostriches + emus, 7-12 are emus, 13-14 are red-necked ostriches, 15-16 are blue-necked ostriches, 17-18 are African black ostriches, 19 are buffalo + Mongolian cattle + Simmental cattle + Holstein cattle, 20 are horses + donkeys + camels + sika deer, 21 are pigs + sheep + dogs + rabbits, 22 are chickens + ducks + geese, 23 are pigeons + quails, and M is DNA marker DL 2000.

[0048] Figure 3 Alignment of universal primers on vertebrate mitochondrial 16S rRNA sequences.

[0049] Figure 4 Electropherograms of primer-sensitive PCR assays for emu and ostrich;

[0050] Lanes 1-3 are 1 ng / μL, 4-6 are 0.1 ng / μL, 7-9 are 0.01 ng / μL, 10-12 are 0.001 ng / μL, 13-15 are 0.0001 ng / μL, 16-18 are blank controls, and M is DNA marker DL 2000.

[0051] Figure 5 This is the electrophoresis diagram of the PCR amplification products of emu meat samples.

[0052] Figure 6 This is the electrophoresis diagram of the PCR amplification product of red-necked ostrich meat sample.

[0053] Figure 7 This is the electrophoresis diagram of the PCR amplification product of the blue-necked ostrich meat sample.

[0054] Figure 8 This is the electrophoresis diagram of the PCR amplification products of African black ostrich meat samples.

[0055] Fig. 9 This is the electrophoresis diagram of the PCR amplification products of beef samples.

[0056] Fig.10 This is the electrophoresis diagram of the PCR amplification products of horse meat samples.

[0057] Fig.11 This is the electrophoresis diagram of the PCR amplification products of donkey meat samples.

[0058] Fig.12This is the electrophoresis diagram of the PCR amplification products of camel meat samples.

[0059] Fig.13 This is the electrophoresis diagram of the PCR amplification products of sika deer meat samples.

[0060] Fig.14 This is the electrophoresis diagram of the PCR amplification products of pork samples.

[0061] Fig.15 This is the electrophoresis diagram of the PCR amplification products of mutton samples.

[0062] Fig.16 This is the electrophoresis diagram of the PCR amplification products of dog meat samples.

[0063] Fig.17 This is the electrophoresis diagram of the PCR amplification products of rabbit meat samples.

[0064] Fig.18 This is the electrophoresis image of the PCR amplification products of chicken samples.

[0065] Fig.19 This is the electrophoresis diagram of the PCR amplification products of duck meat samples.

[0066] Fig. 20 This is the electrophoresis diagram of the PCR amplification products of goose meat samples.

[0067] Fig.21 This is the electrophoresis diagram of the PCR amplification products of pigeon meat samples.

[0068] Fig. 22 This is the electrophoresis diagram of the PCR amplification products of quail meat samples. DETAILED DESCRIPTION

[0069] The specific embodiments of the present invention will be described in more detail below. These embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0070] 1 Establishment of detection technology

[0071] 1.1 Sample source

[0072] 5g leg muscle of red-necked ostrich, blue-necked ostrich and African black ostrich were collected from ostrich farms in Jieyang City, Guangdong Province, Baoding City, Hebei Province and Zhengzhou City, Henan Province, respectively. The number of each species was 48. The sex ratio was 2:1 through gonad observation. The samples were placed in 1.5mL centrifuge tubes containing 95% alcohol. The samples were transported in a frozen environment and stored at -20℃ for later use.

[0073] 5g of leg muscle from slaughtered emus were collected from an emu farm in Qingyuan City, Guangdong Province. 32 males and 32 females were observed through the gonads and placed in a 1.5mL centrifuge tube filled with 95% alcohol. The samples were transported in a frozen environment and stored at -20℃ for later use.

[0074] At the same time, frozen / fresh meat samples of cattle, horses, donkeys, camels, sika deer, chickens, ducks, geese, pigeons, quails, rabbits, pigs, sheep, dogs and other species were selected as controls. Among them, the samples of chickens, ducks, geese, quails and pigeons were obtained after being slaughtered to confirm that they were alive, and the other samples were obtained after the accurate sources were determined.

[0075] 1.2 Primer design

[0076] The mitochondrial DNA genome sequences of ostrich, emu, horse, donkey, camel, chicken, duck, goose, pigeon, quail, rabbit, pig, cattle, sheep, dog, etc. were downloaded from GenBank. After sequence alignment using biological software MEGA 7.0, emu and ostrich specific sites were found, and then universal primers were designed on the flanks of emu and ostrich specific sites. Finally, mitochondrial 16SrRNA~ND1 was determined as the primer design region.

[0077] Universal upstream primer: 5′-ACGACCTCGATGTTGGATCA-3′;

[0078] Universal downstream primer: 5′-CGGTCTGAACTCAGATCACGTAT-3′;

[0079] emu-specific primer: 5′-TGTGGGGGCTTTCTTCTTAGT-3′;

[0080] Ostrich-specific primer: 5′-ACAGCGTATGAGAGTGCTATAGTC-3′.

[0081] Primer combination 1 (universal upstream primer + universal downstream primer) is used to amplify the common fragment of the sample to be tested, and primer combination 2 (universal upstream primer + emu-specific primer) and primer combination 3 (universal upstream primer + ostrich-specific primer) are used to amplify the specific fragments of emu and ostrich, respectively.

[0082] 1.3 Genomic DNA extraction

[0083] Cut a small amount of muscle (10 mg) and use a kit to extract genomic DNA. DNA is stored at 2-8°C and stored at -20°C for a long time, and can be used directly as a template for PCR amplification.

[0084] The DNA extraction kit was from the “Universal DNA Extraction Kit” of Guangzhou Magentec Biotechnology Co., Ltd. (http: / / www.magentec.com.cn / product_show.php?id=41).

[0085] 1.4 PCR amplification

[0086] First, the reaction conditions for identifying emu and ostrich meat components were determined separately. The reaction system was as follows: 1 μL of the template DNA prepared above, 15 μL of amplification buffer, 0.3 μL of universal primers and emu or ostrich specific primers (20 μmol / L) and 13.1 μL of sterile water were added to a 200 μL PCR thin-walled tube. The PCR reaction system was 30 μL, and the sample was centrifuged briefly after addition. The PCR reaction procedure was as follows: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 30 s, 30 cycles; 72°C extension for 5 min.

[0087] Primer mix for emu identification:

[0088] Universal upstream primer: 5′-ACGACCTCGATGTTGGATCA-3′;

[0089] Universal downstream primer: 5′-CGGTCTGAACTCAGATCACGTA-3′;

[0090] Emu-specific primer: 5′-TGTGGGGGCTTTCTTCTTAGT-3′.

[0091] Identification of ostrich primer mix:

[0092] Universal upstream primer: 5′-ACGACCTCGATGTTGGATCA-3′;

[0093] Universal downstream primer: 5′-CGGTCTGAACTCAGATCACGTA-3′;

[0094] Ostrich-specific primer: 5′-ACAGCGTATGAGAGTGCTATAGTC-3′.

[0095] The amplification buffer was from the "Premix Taq TM ”(https: / / www.takarabiomed.com.cn / ProductShow.aspx?m=20141215102854153148&productID=20141224135122093601).

[0096] After determining the reaction conditions for identifying emu and ostrich meat components, determine the reaction system for amplification of emu and ostrich specific primers in the same group. The reaction system is: in a 200μL PCR thin-walled tube, add 1μL of the template DNA prepared in 1.3 above, 15μL of amplification buffer, universal primers, 0.3μL of emu and ostrich specific primers (20μmol / L) and 12.8μL of sterile water, and the PCR reaction system is 30μL. Centrifuge briefly after adding the sample. The PCR reaction program is: 94℃ pre-denaturation for 3min; 94℃ denaturation for 30s, 60℃ annealing for 30s, 72℃ extension for 30s, 30 cycles; 72℃ extension for 5min.

[0097] Primer mix:

[0098] Universal upstream primer: 5′-ACGACCTCGATGTTGGATCA-3′;

[0099] Universal downstream primer: 5′-CGGTCTGAACTCAGATCACGTA-3′;

[0100] emu-specific primer: 5′-TGTGGGGGCTTTCTTCTTAGT-3′;

[0101] Ostrich-specific primer: 5′-ACAGCGTATGAGAGTGCTATAGTC-3′.

[0102] The amplification buffer was from the "Premix Taq TM ”(https: / / www.takarabiomed.com.cn / ProductShow.aspx?m=20141215102854153148&productID=20141224135122093601).

[0103] 1.5 Agarose gel electrophoresis

[0104] Place 1.5% agarose gel (premixed with GelRed dye) in 1×TAE electrophoresis buffer. Spot 4 μL of the reaction product in the gel wells. Run electrophoresis at 100V and 150mA for 40 min at room temperature. Image and take photos in a gel imaging system.

[0105] 1.6 Interpretation of results

[0106] When only one common amplification band of about 100bp appears in the amplification product, it indicates that the PCR amplification is good and the sample is judged to be neither emu nor ostrich; when the amplification product simultaneously shows a common amplification band of about 100bp and an emu-specific band of 236bp or an ostrich-specific band of 389bp, it indicates that the PCR amplification is good and the sample is judged to be emu or ostrich. When the amplification product simultaneously shows a common amplification band of about 100bp and an emu-specific band of 236bp and an ostrich-specific band of 389bp, it indicates that the PCR amplification is good and the sample is judged to be emu and ostrich. If no band appears, the source of the test sample cannot be determined. The electrophoresis diagram of the PCR product of single identification (three primers: universal primer + emu or ostrich-specific primer) is shown in Figure 1 , combined identification (four primers: universal primer + emu and ostrich specific primers) PCR product electrophoresis diagram is shown in Figure 2 .

[0107] 2 Feasibility Verification

[0108] The method established by the present invention was used to identify the meat source of 48 ostriches of three species and 64 emus aged 400 days old that were identified through live identification. A small amount of breast muscle sample was taken from each individual, such as Figure 5-8 As shown, the meat source identification result provided by the present invention is completely consistent with the result of the live body identification. For specific methods, please refer to the examples in the following section 5.

[0109] 3 Specificity Verification

[0110] Ten muscle samples of horses, donkeys, camels, sika deer, chickens, ducks, geese, pigeons, quails, rabbits, pigs, cattle, sheep, and dogs were randomly selected and their genomic DNA was extracted using a "Universal DNA Extraction Kit" (Guangzhou Meiji Biotechnology Co., Ltd.). Then, the primers designed by the present invention were directly used for PCR amplification. The results are as follows: Figure 1 , Figure 2 As shown, two bands (100bp, 236bp) can be amplified from the emu sample and the ostrich sample, two bands (100bp, 389bp) can be amplified from the ostrich sample, while only one band (99bp or 100bp) can be amplified from the other tested samples.

[0111] To further verify the specificity of the emu and ostrich meat source detection technology established by the present invention, any two primers of the present invention were input into the primer design interface Primer-BLAST of NCBI (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / index.cgi) for electronic PCR test. The database was selected as "nr" (nucleotide fragment), the target organism was selected as "vertebrata (taxid:7742)" (Vertebrate subphylum), and other options were default parameters. Combination 1 (universal upstream primer + universal downstream primer) returned 998 results, covering all species used in the present invention. The target gene was 16S rRNA, and the target band was 99-100bp. The universal primers showed good amplification efficiency and compatibility, and the nucleic acid sequences of representative species of all vertebrate phyla were exactly the same ( Figure 3 ). Combination 2 (universal primer upstream + emu-specific primer) returned 4 results, all of which were amplified targets of emu, the target gene was 16S rRNA, and the amplification length was 236bp. Combination 3 (universal primer upstream + ostrich-specific primer) returned 4 results, all of which were amplified targets of ostrich, the target gene was 16S rRNA+tRNA-Leu+ND1, and the amplification length was 388-389bp. No specific amplification target was found in other combinations (matching results were zero).

[0112] 4 Sensitivity Verification

[0113] The emu and ostrich DNA were diluted to 1 ng / μL, 0.1 ng / μL, 0.01 ng / μL, 0.001 ng / μL, and 0.0001 ng / μL with ultrapure water, and PCR was performed according to the method described in Part 1 above. The results are shown in Figure 4 As shown in the figure, there is still a clear signal at a meat DNA concentration of 0.001ng / μL, indicating that the sensitivity of the primer can reach 0.001ng / μL pure emu and ostrich meat DNA samples. If emu and ostrich are added at the same time, the sensitivity can also reach 0.001ng / μL. It is worth noting that even if the DNA concentration is diluted to 0.0001ng / μL, the amplification band of the universal primer is still obvious, indicating that the amplification efficiency of the universal primer is high.

[0114] 5. Embodiment

[0115] Fresh / frozen muscle samples of horses, donkeys, camels, sika deer, chickens, ducks, geese, pigeons, quails, rabbits, pigs, cattle, sheep, and dogs that have been verified in living or in appearance were selected for testing. The number of samples is shown in the following table. After the genomic DNA was extracted using the "Universal DNA Extraction Kit" (Guangzhou Meiji Biotechnology Co., Ltd.), the primer set designed by the present invention (four primers: universal primer + emu and ostrich specific primers) was directly used for PCR amplification. The results are shown in the following table. Figure 5-22 As shown, only one band (99 bp or 100 bp) was amplified for all samples. The total number of samples was 827, and emu and ostrich were successfully identified, while other samples were identified as non-emu or non-ostrich, with a success rate of 100%.

[0116] Species Sample type Number of varieties Sample quantity Emu Fresh Muscle 1 64 ostrich Fresh Muscle 3 144 ox Fresh Muscle 4 99 horse Fresh Muscle 1 24 donkey Fresh Muscle 1 24 camel Fresh Muscle 1 25 Sika Deer Fresh Muscle 1 24 pig Fresh Muscle 1 46 sheep Freezing Muscle 1 42 dog Freezing Muscle 1 41 rabbit Freezing Muscle 1 43 chicken Fresh Muscle 2 86 duck Freezing Muscle 1 48 Goose Fresh Muscle 1 35 pigeon Fresh Muscle 1 34 quail Freezing Muscle 1 48

Claims

1. Specific primers for multiplex PCR identification of emu and ostrich meat ingredients, characterized in that: It comprises a universal primer, an ostrich-specific primer and an emu-specific primer, wherein the nucleotide sequence of the ostrich-specific primer is: 5'-ACAGCGTATGAGAGTGCTATAGTC-3'; the nucleotide sequence of the emu-specific primer is 5'-TGTGGGGGCTTTCTTCTTAGT-3'; The universal primers: Universal upstream primer: 5′-ACGACCTCGATGTTGGATCA-3′; Universal downstream primer: 5'-CGGTCTGAACTCAGATCACGTA-3'.

2. A kit for multiplex PCR identification of emu and ostrich meat ingredients, characterized in that: The following primers were included: Emu-specific primer: 5'-TGTGGGGGCTTTCTTCTTAGT-3', Ostrich-specific primer: 5′-ACAGCGTATGAGAGTGCTATAGTC-3′; Universal upstream primer: 5′-ACGACCTCGATGTTGGATCA-3′; Universal downstream primer: 5'-CGGTCTGAACTCAGATCACGTA-3'.

3. A multiplex PCR method for identifying emu and ostrich meat ingredients, characterized in that: Take the meat sample to be identified, place it in a lysis solution, lyse it, extract the genomic DNA, amplify it by PCR, and perform electrophoresis; When only one 100 bp common amplification band appears in the electrophoresis result, the sample is judged to be neither emu nor ostrich; when the amplification product simultaneously shows a 100 bp common amplification band and a 236 bp emu-specific band or a 389 bp ostrich-specific band, the sample is judged to be emu or ostrich; the primers used in the multiplex PCR amplification are as follows: Universal upstream primer: 5′-ACGACCTCGATGTTGGATCA-3′; Universal downstream primer: 5′-CGGTCTGAACTCAGATCACGTA-3′; emu-specific primer: 5′-TGTGGGGGCTTTCTTCTTAGT-3′; Ostrich-specific primer: 5′-ACAGCGTATGAGAGTGCTATAGTC-3′.

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