Detection method for simultaneously identifying raw meat and cooked meat of livestock and poultry based on multiple PCR (Polymerase Chain Reaction)

Abstract

The invention discloses a multiplex PCR (Polymerase Chain Reaction) system for identifying raw meat and cooked meat of livestock and poultry, which is used for simultaneously detecting target genes of sheep, goats, pigs, chickens and ducks as COX-2, NADH6, 16S rRNA, 16S rRNA and ATP6, and the lengths of PCR amplification fragments of the target genes are 306bp, 113bp, 173bp, 379bp and 240bp respectively. The method has the beneficial effects that meat sources of five common target species can be simultaneously identified, the adulteration conditions of sheep meat and goat meat can be rapidly detected, the types of common adulterated meat can be qualitatively identified, and the method is outstanding in sensitivity, specificity, repeatability and practicability and is more reliable and convenient.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular biology, and relates to a detection method for simultaneously identifying raw meat and cooked meat of livestock and poultry based on multiplex PCR, in particular to a multiplex PCR system for detecting sheep meat, goat meat and common adulterated pork, chicken and duck, detection primers and application thereof. BACKGROUND

[0002] Mutton occupies an important position in the diet culture due to its unique flavor, rich nutritional value and significant food therapy effect. In recent years, with the popularity of mutton consumption scenarios such as barbecue and hotpot, the market demand for mutton has been growing. However, adulteration of mutton in the market is becoming increasingly serious, and unscrupulous businessmen are making profits by adding cheap meat to mutton, which not only disrupts the market order, but also poses a serious threat to food safety.

[0003] Currently, consumers have significantly increased their attention to food authenticity, but traditional detection methods generally involve sensory evaluation, chemical detection and immunodetection, which have obvious limitations in dealing with processed foods or mixed samples, such as insufficient specificity, strong subjectivity, limited scope of application, and high professional requirements for operators. Therefore, developing a rapid, accurate, simple and widely applicable meat source identification technology has become an urgent need to ensure the safety of mutton products in the market.

[0004] The rapid development of molecular biology technology provides a new solution for identifying food adulteration. PCR technology has become the main method for meat species identification due to its strong specificity, good repeatability, high sensitivity and wide applicability. However, traditional PCR and fluorescent quantitative PCR methods often face challenges in detecting heat-processed, pH-adjusted or mechanically treated foods due to DNA degradation.

[0005] Selecting the appropriate DNA target is important for detection sensitivity. Mitochondrial DNA is an ideal choice due to its high copy number and species-specific sequence variation. Among various PCR technologies, multiplex PCR methods stand out due to their ability to simultaneously detect multiple meat species, providing higher cost-effectiveness compared to single-target detection systems. Currently, multiplex PCR technology has been widely used in food adulteration detection due to its simplicity, speed and economy.

[0006] Therefore, to effectively ensure the safety of the mutton and its products market, it is urgent to establish a multiplex PCR detection system that can simultaneously identify sheep meat, goat meat, pork, chicken meat and duck meat. SUMMARY

[0007] The present application aims at the technical problems to be solved, overcomes the deficiencies of the prior art and provides a detection method for simultaneously identifying raw and cooked meat of livestock and poultry based on multiplex PCR, which establishes a multiplex PCR system for identifying mutton and common adulterated meat, and distinguishes sheep meat, goat meat, pork, chicken meat and duck meat through PCR amplification and agarose gel electrophoresis.

[0008] One of the purposes of the present application is to provide a multiplex PCR system for identifying raw and cooked meat of livestock and poultry, which is used for simultaneously detecting sheep, goat, pig, chicken and duck, the target gene of the sheep is COX-2, the primer is designed for PCR amplification with GenBanK accession number NC_001941.1 as the reference genome, and the length of the amplified fragment is 306 bp; the target gene of the goat is NADH6, the primer is designed for PCR amplification with GenBanK accession number NC_005044.2 as the reference genome, and the length of the amplified fragment is 113 bp; the target gene of the pig is 16S rRNA, the primer is designed for PCR amplification with GenBanK accession number NC_000845.1 as the reference genome, and the length of the amplified fragment is 173 bp; the target gene of the chicken is 16S rRNA, the primer is designed for PCR amplification with GenBanK accession number chicken NC_053523.1 as the reference gene, and the length of the amplified fragment is 379 bp; and the target gene of the duck is ATP6, the primer is designed for PCR amplification with GenBanK accession number NC009684.1 as the reference gene, and the length of the amplified fragment is 240 bp.

[0009] The multiplex PCR system for identifying raw and cooked meat of livestock and poultry comprises the following primers: the upstream primer of the sheep is 5'-TGCTCTTCCATCCTTGCGAAT-3', and the downstream primer is 5'-CGACCTGGAATTGCGTCTGT-3'; the upstream primer of the goat is 5'-CTCATCCTCGTCACCGCAAA-3', and the downstream primer is 5'-GTGTTTGCGTCTGTTCGTCC-3'; the upstream primer of the pig is 5'-TCGCACACGCTTACATCAGT-3', and the downstream primer is 5'-TTGGTAAACAGGCGGGGTTT-3'; the upstream primer of the chicken is 5'-TGCGTCAAAGCTCCCTCATT-3', and the downstream primer is 5'-TTCGCACGGTTAGGATACCG-3'; The upstream primer of the duck is 5'-AAAACGGCCACAAATGAGCC-3', and the downstream primer is 5'-GGATTAGTGCGGGGATCAGG-3'.

[0010] The application further provides application of the primers of the multiplex PCR system in detection of mutton and common adulterated meat.

[0011] The application further provides a detection method for simultaneously identifying livestock and poultry raw meat and cooked meat based on multiplex PCR, comprising the following steps: (1) The raw meat and cooked meat samples of sheep, goats, pigs, chickens and ducks are respectively stirred and crushed, and then DNA of the raw meat and cooked meat samples of sheep, goats, pigs, chickens and ducks is extracted by a gene extraction kit; (2) The mitochondrial whole genome sequences of the five species of sheep, goats, pigs, chickens and ducks are downloaded, and ClustalW is used to screen out the species-specific genes thereof, the species-specific gene of sheep is COX-2, the species-specific gene of goats is NADH6, the species-specific gene of pigs is 16S rRNA, the species-specific gene of chickens is 16S rRNA, and the species-specific gene of ducks is ATP6; (2) The specific genes of the five species of sheep, goats, pigs, chickens and ducks are used as templates to design multiplex PCR primers, and the designed multiplex PCR primers are used for PCR amplification of the sample DNA of the raw meat and cooked meat of sheep, goats, pigs, chickens and ducks respectively; (3) After PCR amplification, agarose gel electrophoresis is performed, the agarose electrophoresis result is observed, and it is found that the mitochondrial DNA in the raw meat and cooked meat of goats, pigs, ducks, sheep and chickens has and only has a single, clear, bright and non-tailing band at 113bp, 173bp, 240bp, 306bp and 379bp respectively after multiplex PCR amplification; (4) The primers of the multiplex PCR system are used for PCR amplification of the DNA of the to-be-detected mutton, and the PCR amplification product is subjected to agarose gel electrophoresis.

[0012] The multiplex PCR primers include: The upstream primer of sheep is 5'-TGCTCTTCCATCCTTGCGAAT-3', and the downstream primer is 5'-CGACCTGGAATTGCGTCTGT-3'; The upstream primer of goats is 5'-CTCATCCTCGTCACCGCAAA-3', and the downstream primer is 5'-GTGTTTGCGTCTGTTCGTCC-3'; The upstream primer of pigs is 5'-TCGCACACGCTTACATCAGT-3', and the downstream primer is 5'-TTGGTAAACAGGCGGGGTTT-3'; Chicken upstream primer 5'-TGCGTCAAAGCTCCCTCATT-3', downstream primer 5'-TTCGCACGGTTAGGATACCG-3'; Duck upstream primer 5'-AAAACGGCCACAAATGAGCC-3', downstream primer 5'-GGATTAGTGCGGGGATCAGG-3'.

[0013] The application takes species-specific genes as templates, designs primers with species specificity through BLAST of NCBI and Primer Primer 5, and uses Autodimer to simulate multiple primer amplification, and then optimizes test conditions, verifies method specificity, repeatability and sensitivity, and combines with simulation adulteration test, comprehensively evaluates the actual application effect of the detection system, and provides reliable technical support for meat food safety supervision.

[0014] The application has the beneficial effects that five common target species of meat sources can be identified at the same time, the adulteration of sheep meat and goat meat can be quickly detected, and the types of common adulterated meat can be qualitatively identified, which is outstanding in sensitivity, specificity, repeatability and practicability, and is more reliable and convenient. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 It is a multiple PCR system annealing temperature optimization diagram of the application.

[0016] Figure 2 It is a one-reaction PCR specificity detection diagram in the multiple PCR system of the application.

[0017] Figure 3 It is a two-reaction PCR specificity detection diagram in the multiple PCR system of the application.

[0018] Figure 4 It is a three-reaction PCR specificity detection diagram in the multiple PCR system of the application.

[0019] Figure 5 It is a five-reaction + four-reaction PCR specificity detection diagram in the multiple PCR system of the application.

[0020] Figure 6 It is a repeatability detection diagram of the multiple PCR system of the application.

[0021] Figure 7 It is a sensitivity detection diagram of the multiple PCR system of the application.

[0022] Figure 8 It is a simulation sheep meat mixed duck meat test detection diagram of the application.

[0023] Figure 9The test result chart of simulating cooked meat according to the present application is shown in the following figure.

[0024] Figure 10 The test result chart of simulating cooked meat according to the present application is shown in the following figure. DETAILED DESCRIPTION

[0025] The technical solutions of the present application are further described in detail in combination with the following examples: The present embodiment is implemented on the premise of the technical solutions of the present application, and detailed implementation modes and specific operation processes are given, but the protection scope of the present application is not limited to the following examples. The test methods not specified in the following examples are selected according to the conventional methods and conditions in the art or the instructions of the commercial products. The reagents and materials involved in the following examples are commercially available, which are not listed one by one here.

[0026] Example 1 Species-specific gene fragment amplification (1) Primer design The mitochondrial whole genome sequences of sheep (NC_001941.1), goat (NC_005044.2), chicken (NC_053523.1), pig (NC_000845.1) and duck (NC_009684.1) were downloaded from the NCBI GenBank database. ClustalW was used to screen gene fragments with intraspecific conservation and interspecific specificity. The target genes of sheep, goat, pig, chicken and duck were COX-2, NADH6, 16S rRNA, 16S rRNA and ATP6, respectively. The criteria for including specific DNA sequences are: comprehensive and accurate information of different species can be obtained in NCBI; there is no difference in base length between different strains of the same species; there are enough differences in base correspondence and length between different species; the length of DNA sequence is more than 400 bp to facilitate primer design. By evaluating the primer structure itself, the interaction between primers (dimer), annealing temperature (the difference between upstream and downstream primers and between different species) and product length, five pairs of specific primers were initially selected. NCBI BLAST and Autodimer were used to ensure that the primers would not cause non-specific amplification or cross-reaction during the test. The primers and their amplified fragment lengths are shown in Table 1. All primers were synthesized by Beijing Qikeng Biotechnology Co., Ltd.

[0027] Table 1 Specific primer information of multiplex PCR system

[0028] (2) DNA extraction Six samples were extracted from goat, sheep, pig, chicken and duck meat, respectively. 100 mg of each meat sample was made brittle with liquid nitrogen, and then ground into powder with a high-pressure sterilization mortar and pestle. DNA was extracted using the TIANamp Genomic DNA Kit. The extracted DNA was analyzed using a ultramicro spectrophotometer, and then diluted to 20 ng / μl and stored at -20 °C for later use.

[0029] (3) PCR amplification and product accuracy verification The single PCR amplification system was composed of 12.5 μl 2x TSINGKE Master Mix, 0.5 μl upstream primer and 0.5 μl downstream primer (5 primers mixed), 10.5 μl ddH2O and 1 μl template DNA (nmol), with a total volume of 25 μl.

[0030] The multiplex PCR amplification system was composed of 12.5 μl 2x TSINGKE Master Mix, 1 μl upstream and downstream primers (5 primers mixed), 10.5 μl ddH2O, 1 μl template DNA (5 randomly mixed), with a total volume of 25 μl.

[0031] Using a BIO-RAD gradient PCR thermal cycler, the multiplex PCR reaction was performed by adjusting the annealing temperature and cycle number while keeping other conditions unchanged, to ensure that the amplified band was clear and bright, without tailing or diffusion, located at the target position, and presented as a single entity. The multiplex PCR amplification conditions were 94 °C, 3 min; 94 °C, 30 s; 56-60 °C (see Figure 1 ), 30 s; 72 °C, 1 min; 30 cycles; 72 °C, 5 min. The blank control group was amplified with ddH2O. Figure 1 Among them, 1 is 500 bp Maker, 2 is 60 °C, 3 is 59.7 °C, 4 is 59.2 °C, 5 is 58.5 °C, 6 is 57.5 °C, 7 is 56.8 °C, 8 is 56.3 °C, 9 is 56 °C, and 10 is a blank control.

[0032] A 2% agarose gel containing nucleic acid dye (Gel Green) was prepared and placed in a DYY-6C electrophoresis apparatus. Each PCR amplified sample and DNA ladder was loaded at 7 μl, the voltage was 120 V, the current was 150 mA, and the electrophoresis time was about 30 min. After electrophoresis, the electrophoresis results were observed under the gel imaging system to see if clear, bright, single and non-tailing bands appeared at the expected position.

[0033] Example 2 Specificity, repeatability, sensitivity and adaptability of multiplex PCR system (1) Specificity The DNA of eight different test animals, such as sheep, goats, pigs, ducks, chickens, cows, geese, and rabbits, was subjected to single-plex PCR, and the mixed DNA of five different test animals, such as sheep, goats, pigs, ducks, and chickens, was subjected to two-plex, three-plex, four-plex, and five-plex PCR amplification, and the amplification products were detected by 2% agarose gel electrophoresis. The results are shown in Figures 2-5 The amplification products of sheep, goats, pigs, chickens, ducks, and different random combinations of meat samples all met the expected results, there was no non-specific amplification, and there was no non-specific amplification in non-target meat and blank controls.

[0034] Figure 2 1 is a 1000bp Maker, 2 is a goat, 3 is a pig, 4 is a duck, 5 is a sheep, 6 is a chicken, 7 is a cow, 8 is a goose, 9 is a rabbit, and 10 is a blank control; Figure 3 1 is a 500bp Maker, 2 is a sheep + a goat, 3 is a duck + a goat, 4 is a pig + a goat, 5 is a chicken + a goat, 6 is a pig + a sheep, 7 is a chicken + a sheep, 8 is a duck + a sheep, and 9 is a blank control; Figure 4 1 is a 500bp Maker, 2 is a goat + a pig + a sheep, 3 is a goat + a duck + a sheep, 4 is a goat + a sheep + a chicken, 5 is a sheep + a pig + a duck, 6 is a sheep + a chicken + a pig, 7 is a sheep + a chicken + a duck, 8 is a goat + a chicken + a duck, 9 is a goat + a pig + a chicken, 10 is a goat + a pig + a duck, 11 is a blank control; Figure 5 1 is a 500bp Maker, 2 is a goat + a pig + a sheep, 3 is a goat + a duck + a sheep, 4 is a goat + a sheep + a chicken, 5 is a sheep + a pig + a duck, 6 is a sheep + a chicken + a pig, 7 is a sheep + a chicken + a duck, 8 is a goat + a chicken + a duck, 9 is a goat + a pig + a chicken, 10 is a goat + a pig + a duck, 11 is a blank control;

[0035] (2) Reproducibility The DNA samples were diluted to 20ng / μl and mixed using a SIVortex Genie2 vortex mixer. Under the condition that the primer concentration, PCR reaction program, system conditions, etc. remain unchanged, the DNA samples of 5 target species (6 samples for each species) were subjected to multiplex PCR amplification, and the amplification products were subjected to gel electrophoresis analysis. As shown in Figure 6 Under the same conditions, the target bands obtained for each species were uniform and consistent with the expected results, showing high reproducibility. These bands can accurately and effectively determine the source of the meat sample.

[0036] Figure 6 1 is a 500bp DNA molecular weight standard, 2-7 are mixtures of 6 groups of goat, sheep, pig, chicken, and duck DNA, and 8 is a blank control.

[0037] (3) Sensitivity The DNA of one animal was diluted with ddH2O in a 1:1 series. Then mixed with the remaining four animal DNA templates to obtain a target DNA template concentration gradient from 4 ng / μL to 0.125 ng / μL for multiplex PCR amplification. The detection process was carried out by 2% gel electrophoresis. As shown in Figure 7 , although the band intensity gradually weakened with the decrease of DNA concentration, at the concentration of 2 ng / μL, the amplification products of all five test animals could be observed. At the concentration of 1 ng / μL, the amplification products of the other four animals except chicken could be detected. At the lower concentration of 0.5 ng / μL, the products of sheep, duck and pig could still be detected. At the lowest concentration of 0.125 ng / μL, the DNA products of sheep and duck could still be detected.

[0038] Figure 7 A is chicken, B is duck, C is pig, D is goat, E is sheep, 1 is 100 bp / 50 bp DNA molecular weight marker, 2 is 4 ng / ml, 3 is 2 ng / ml, 4 is 1 ng / ml, 5 is 0.5 ng / ml, 6 is 0.25 ng / ml, 7 is 0.125 ng / ml, 8 is blank control.

[0039] Example 3 Method for detecting sheep meat and common adulterated meat by multiplex PCR system (1) Adulteration (simulation) test The duck, sheep meat and goat meat samples were cut into small pieces and ground into powder with a mortar; Duck and sheep meat mixed samples, sheep and goat meat mixed samples, and duck and goat meat mixed samples were prepared. The proportion of duck meat in the mixed samples was controlled at 50%, 25%, 10% and 5% respectively by an analytical balance; the proportion of sheep meat in the mixed samples was controlled at 50%, 25%, 10% and 5% respectively; the proportion of duck meat in the mixed samples was controlled at 50%, 25%, 10% and 5% respectively, and the total mass of each simulated mixed sample was about 220 mg.

[0040] After thorough mixing, DNA was extracted, amplified using the multiplex PCR system, and detected by 2% agarose gel electrophoresis (for specific methods, see Example 1); The results are shown in Figure 8 and Figure 9 At a mixing ratio of 5% (the minimum mass of non-sheep meat or non-goat meat was 11 mg), the target meat component could still be detected, and there was no non-specific amplification.

[0041] Figure 8M1 is 50bp DNA marker, M2 is duck meat accounts for 50% of mixed meat (duck meat and sheep meat), M3 is duck meat accounts for 25% of mixed meat, M4 is duck meat accounts for 10% of mixed meat, M5 is duck meat accounts for 5% of mixed meat, M6 is blank control. Figure 9 M1 is 500bp Maker, M2 is sheep accounts for 50% of mixed meat (sheep meat and goat meat), M3 is sheep accounts for 25% of mixed meat, M4 is sheep accounts for 10% of mixed meat, M5 is sheep accounts for 5% of mixed meat, M6 is duck accounts for 50% of mixed meat (duck meat and goat meat), M7 is duck accounts for 25% of mixed meat, M8 is duck accounts for 10% of mixed meat, M9 is duck accounts for 5% of mixed meat, M10 is blank control.

[0042] (2) Heat treatment (simulation) test Five kinds of target meat products are treated by microwave and then heat treated at 100℃; DNA extraction is carried out, and the extracted DNA is diluted to a concentration of 20ng / μL, PCR amplification is carried out, and the results are detected by gel electrophoresis (for specific methods, see implementation 1); The results are shown in Figure 10 Compared with fresh meat samples, there is almost no difference in DNA of cooked meat samples, the target band is still clear and bright, and there is no non-specific amplification.

[0043] Figure 10 M1 is 500bp Maker, M2 is chicken (fresh), M3 is chicken (cooked), M4 is sheep (fresh), M5 is sheep (cooked), M6 is duck (fresh), M7 is duck (cooked), M8 is goat (fresh), M9 is goat (cooked), M10 is pig (fresh), M11 is pig (cooked), M12 is blank control.

[0044] The above is only a specific implementation in the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can understand and think of the transformation or replacement within the technical range disclosed by the present application, which should be covered in the inclusive scope of the present application, therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A multiplex PCR system for discriminating raw meat of livestock and poultry from cooked meat, characterized by, The multiplex PCR system is used for simultaneously detecting sheep, goats, pigs, chickens and ducks, the target gene of the sheep is COX-2, the primer is designed according to the reference genome NC_001941.1 of GenBank, and the length of the amplified fragment is 306 bp; the target gene of the goat is NADH6, the primer is designed according to the reference genome NC_005044.2 of GenBank, and the length of the amplified fragment is 113 bp; the target gene of the pig is 16S rRNA, the primer is designed according to the reference genome NC_000845.1 of GenBank, and the length of the amplified fragment is 173 bp; the target gene of the chicken is 16S rRNA, the primer is designed according to the reference genome NC_053523.1 of GenBank, and the length of the amplified fragment is 379 bp; and the target gene of the duck is ATP6, the primer is designed according to the reference genome NC_009684.1 of GenBank, and the length of the amplified fragment is 240 bp.

2. The multiplex PCR system for identifying raw and cooked meat of livestock and poultry as described in claim 1, characterized in that, The primer of the multiplex PCR system comprises: the upstream primer of the sheep is 5'-TGCTCTTCCATCCTTGCGAAT-3', and the downstream primer is 5'-CGACCTGGAATTGCGTCTGT-3'; the upstream primer of the goat is 5'-CTCATCCTCGTCACCGCAAA-3', and the downstream primer is 5'-GTGTTTGCGTCTGTTCGTCC-3'; the upstream primer of the pig is 5'-TCGCACACGCTTACATCAGT-3', and the downstream primer is 5'-TTGGTAAACAGGCGGGGTTT-3'; the upstream primer of the chicken is 5'-TGCGTCAAAGCTCCCTCATT-3', and the downstream primer is 5'-TTCGCACGGTTAGGATACCG-3'; the upstream primer of the duck is 5'-AAAACGGCCACAAATGAGCC-3', and the downstream primer is 5'-GGATTAGTGCGGGGATCAGG-3'.

3. Application of the primer of the multiplex PCR system in claim 2 to detection of mutton and common adulterated meat.

4. A detection method for simultaneously identifying raw meat and cooked meat of livestock and poultry based on multiplex PCR, characterized in that, The method comprises the following steps: (1) stirring and crushing raw meat and cooked meat samples of sheep, goats, pigs, chickens and ducks, and then extracting DNA of the raw meat and cooked meat samples of the sheep, goats, pigs, chickens and ducks by using a DNA extraction kit; (2) downloading mitochondrial whole genome sequences of the five species of sheep, goats, pigs, chickens and ducks, and screening species-specific genes of the five species by using ClustalW, wherein the species-specific gene of the sheep is COX-2, the species-specific gene of the goat is NADH6, the species-specific gene of the pig is 16S rRNA, the species-specific gene of the chicken is 16S rRNA, and the species-specific gene of the duck is ATP6; (2) Using the specific genes of sheep, goat, pig, chicken and duck as templates, multiple PCR primers are designed, and the DNA samples of raw and cooked meat of sheep, goat, pig, chicken and duck are amplified by PCR using the designed multiple PCR primers; (3) After PCR amplification, agarose gel electrophoresis is carried out, and the agarose electrophoresis results are observed, and it is found that the mitochondrial DNA in the raw and cooked meat of goat, pig, duck, sheep and chicken amplified by multiple PCR is single, clear, bright and without tailing at 113bp, 173bp, 240bp, 306bp and 379bp respectively; (4) The DNA of the tested mutton is amplified by PCR using the primers of the multiple PCR system, and the PCR amplification products are subjected to agarose gel electrophoresis.

5. The method according to claim 4, wherein the method is used for simultaneously identifying raw meat and cooked meat of livestock and poultry. The multiple PCR primers comprise: The upstream primer of sheep is 5'-TGCTCTTCCATCCTTGCGAAT-3', and the downstream primer is 5'-CGACCTGGAATTGCGTCTGT-3'; The upstream primer of goat is 5'-CTCATCCTCGTCACCGCAAA-3', and the downstream primer is 5'-GTGTTTGCGTCTGTTCGTCC-3'; The upstream primer of pig is 5'-TCGCACACGCTTACATCAGT-3', and the downstream primer is 5'-TTGGTAAACAGGCGGGGTTT-3'; The upstream primer of chicken is 5'-TGCGTCAAAGCTCCCTCATT-3', and the downstream primer is 5'-TTCGCACGGTTAGGATACCG-3'; The upstream primer of duck is 5'-AAAACGGCCACAAATGAGCC-3', and the downstream primer is 5'-GGATTAGTGCGGGGATCAGG-3'.

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