Primers, kits, and methods for multiplex PCR identification of partridge meat components.

Abstract

This invention discloses multiplex PCR-specific primers for the identification of partridge meat-derived components, including partridge-specific primers with the nucleotide sequence 5'-GGCCCACCTATGTAAATCATCT-3'. This invention also discloses a kit containing the aforementioned multiplex PCR-specific primers, and a multiplex PCR method for the identification of partridge meat-derived components. The partridge-specific primers provided by this invention provide stable PCR amplification of 16S rRNA, producing a specific 167bp band, effectively identifying partridge meat as a source.

Description

Technical Field

[0001] This invention relates to multiplex PCR, and more specifically to multiplex PCR primers for the specific identification of partridge meat components. Background Technology

[0002] The partridge, also known as the American partridge, is scientifically named *Alectoris chukar*. Partridge meat is high in protein and low in fat, possessing extremely high nutritional value, and is often described as "one partridge is worth nine chickens." my country began importing and raising partridges from abroad as early as the 1980s, and it has been listed as a special poultry species by the National Livestock and Poultry Genetic Resources Committee. The annual output nationwide is nearly 100 million birds, giving it considerable influence in the poultry market. With the continuous improvement of living standards, people's demand for meat has shifted from quantity to quality. With the gradual elimination of live poultry markets and the continuous increase in breeding costs, the market share of chilled / frozen meat and cut meat is constantly increasing, leading to rampant instances of inferior products being sold as high-quality ones. Therefore, researching animal-derived food safety testing technologies and accurately identifying the animal-derived components of food is particularly important.

[0003] For the identification of meat-derived components, real-time quantitative PCR (qPCR) is commonly used. This method has high sensitivity, is suitable for detecting trace amounts of meat-derived components in processed foods, and can provide relative quantification, hence its widespread use. Chinese invention patent application CN102337335A, published on February 1, 2012, discloses primers and probes for real-time fluorescence PCR detection of meat-derived components in domestic partridges (rock partridges), but this method cannot distinguish false negatives and cannot identify meat-derived components in some species. Chinese invention patent application CN102337336A, also published on February 1, 2012, discloses a pair of primers for PCR detection of meat-derived components in domestic partridges (rock partridges), but this method cannot distinguish false negative samples and cannot identify meat-derived components in some species. However, it is difficult to design species-specific probes and primers for quantitative PCR, and a set of probes and primers only corresponds to a specific single species component. Methods using universal primers and cloning sequencing are also available for detecting multiple meat-derived components. However, this method requires screening for positive clones, which is labor-intensive, costly, and prone to false negatives. Summary of the Invention

[0004] The purpose of this invention is to provide multiplex PCR specific identification primers for partridge meat source components, which have high specificity for partridge meat source and can be effectively identified.

[0005] The technical solution for realizing the present invention is: multiplex PCR specific identification primers for partridge meat source components, including partridge-specific primers, whose nucleotide sequence is 5'-ggcccacctatgtaaatcatct-3'.

[0006] Furthermore, the identification primers provided by this invention include:

[0007] Partridge-specific primer: 5'-GGCCCACCTATGTAAATCATCT-3'

[0008] Universal upstream primer: 5'-CGCGGTATCCTAACCGTGC-3'

[0009] Universal downstream primer: 5'-CTGGGGTAGCTTGGTCCATT-3'.

[0010] Another object of the present invention is to provide a kit containing the above-described primers for the specific identification of partridge meat source components by multiplex PCR.

[0011] Specifically, the kit includes at least partridge-specific primers:

[0012] 5'-GGCCCACCTATGTAAATCATCT-3'.

[0013] Furthermore, the kit of the present invention also includes:

[0014] Universal upstream primer: 5'-CGCGGTATCCTAACCGTGC-3'

[0015] Universal downstream primer: 5'-CTGGGGTAGCTTGGTCCATT-3'.

[0016] A third objective of this invention is to provide a multiplex PCR method for identifying partridge meat components based on the aforementioned specific identification primers.

[0017] Specifically, the multiplex PCR identification method for partridge meat components involves taking the meat sample to be identified, lysing it in a lysis buffer, extracting genomic DNA, performing PCR amplification, and electrophoresis. If the electrophoresis results show only one 377bp common amplification band, the sample is determined to be non-partridge. If the amplification products simultaneously show a 377bp common amplification band and a 167bp partridge-specific band, the sample is determined to be partridge. The primers used for the multiplex PCR amplification are as follows:

[0018] Universal upstream primer: 5'-CGCGGTATCCTAACCGTGC-3'

[0019] Universal downstream primer: 5'-CTGGGGTAGCTTGGTCCATT-3'

[0020] Partridge-specific primer: 5'-GGCCCACCTATGTAAATCATCT-3'.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention provides partridge-specific primers for conventional PCR amplification of 16S rRNA, which does not require expensive equipment such as a real-time PCR instrument, and the amplification effect is stable, producing a 167bp specific band, effectively identifying partridge meat as the source; while the provided universal primers can effectively avoid false negatives. Attached Figure Description

[0023] Figure 1 This is the result of the identification of meat source components using partridge breast muscle as a sample in this invention;

[0024] Lanes 1-6 are male, lanes 7-12 are female, and M stands for DNA marker DL 2000.

[0025] Figure 2 Electrophoresis images of PCR amplification products from 10 common commercially available meat samples;

[0026] Lanes 1-2 are chickens, 3-4 are ducks, 5-6 are geese, 7-8 are pigeons, 9-10 are quails, 11-12 are rabbits, 13-14 are dogs, 15-16 are sheep, 17-18 are cows, 19-20 are pigs, 21-22 are partridges, and M stands for DNA marker DL 2000.

[0027] Figure 3 Electrophoresis diagram of PCR amplification of common commercially available meat samples using primer CN102337335A;

[0028] Lanes 1-3 are partridges, lanes 4-6 are pigs, lanes 7-9 are sheep, lanes 10-12 are cows, lanes 13-15 are dogs, lanes 16-18 are rabbits, lanes 19-21 are Muscovy ducks, lanes 22-24 are ducks, lanes 25-27 are geese, lanes 28-30 are quails, lanes 31-33 are pigeons, lanes 34-36 are chickens, lanes 37-39 are blank controls, and M is DNAmarker DL 2000.

[0029] Figure 4 Electrophoresis diagram of PCR amplification of common commercially available meat samples using primers CN102337336A;

[0030] Lanes 1-3 are partridges, lanes 4-6 are pigs, lanes 7-9 are sheep, lanes 10-12 are cows, lanes 13-15 are dogs, lanes 16-18 are rabbits, lanes 19-21 are Muscovy ducks, lanes 22-24 are ducks, lanes 25-27 are geese, lanes 28-30 are quails, lanes 31-33 are pigeons, lanes 34-36 are chickens, lanes 37-39 are blank controls, and M is DNAmarker DL 2000.

[0031] Figure 5 Electrophoresis diagram of PCR detection using partridge primers;

[0032] Lanes 1-3 were 10 ng / μL (partridge meat DNA), lanes 4-6 were 1 ng / μL, lanes 7-9 were 0.1 ng / μL, lanes 10-12 were 0.01 ng / μL, lanes 13-15 were 0.001 ng / μL, lanes 16-18 were blank controls, and M was DNA marker DL 2000.

[0033] Figure 6 This is an electrophoresis image of the PCR amplification products from a partridge meat sample.

[0034] Figure 7 This is an electrophoresis image of the PCR amplification products from a chicken sample.

[0035] Figure 8 This is an electrophoresis image of the PCR amplification products from a duck meat sample.

[0036] Figure 9 This is an electrophoresis image of the PCR amplification products from a goose meat sample.

[0037] Figure 10 This is an electrophoresis image of the PCR amplification products from a pigeon meat sample.

[0038] Figure 11 This is an electrophoresis image of the PCR amplification products from a quail meat sample.

[0039] Figure 12 This is an electrophoresis image of the PCR amplification products from a rabbit meat sample.

[0040] Figure 13 This is an electrophoresis image of the PCR amplification products from a dog meat sample.

[0041] Figure 14 This is an electrophoresis image of the PCR amplification products from a mutton sample.

[0042] Figure 15 This is an electrophoresis image of the PCR amplification products from a beef sample.

[0043] Figure 16 This is an electrophoresis image of the PCR amplification products from a pork sample. Detailed Implementation

[0044] Specific embodiments of this application will now be described in more detail. These embodiments are provided to enable a more thorough understanding of this application and to fully convey the scope of this application to those skilled in the art.

[0045] 1. Establishment of detection technology

[0046] 1.1 Sample Source

[0047] A small amount of pectoral muscle (1g) was taken from slaughtered partridges at the partridge farm, 60 males and 60 females, and placed into 1.5mL centrifuge tubes containing 95% alcohol. The samples were transported under frozen conditions and stored at -20℃ for later use.

[0048] Meanwhile, frozen / fresh meat samples of various species, including chickens, ducks, geese, pigeons, quails, rabbits, pigs, cattle, sheep, and dogs, were selected from farmers' markets as controls. Among them, the chicken, duck, goose, quail, and pigeon samples were obtained after slaughtering the animals after confirming they were alive, while the rabbit, pig, cattle, sheep, and dog samples were obtained after slaughtering animals whose origins had been identified.

[0049] 1.2 Primer Design

[0050] Mitochondrial DNA genome sequences of partridges, chickens, ducks, geese, pigeons, quails, rabbits, pigs, cattle, sheep, and dogs were downloaded from GenBank. After sequence alignment using the MEGA 7.0 software, partridge-specific sites were identified, and universal primers were designed flanking these sites. Finally, the 16S rRNA region was determined as the primer design region.

[0051] Universal upstream primer: 5'-CGCGGTATCCTAACCGTGC-3';

[0052] Universal downstream primer: 5'-CTGGGGTAGCTTGGTCCATT-3';

[0053] Partridge-specific primer: 5'-GGCCCACCTATGTAAATCATCT-3';

[0054] Primer combination 1 (universal upstream primer + universal downstream primer) amplifies the common fragment of the sample to be tested, while primer combination 2 (partridge-specific primer + universal downstream primer) amplifies the partridge-specific fragment.

[0055] 1.3 Genomic DNA Extraction

[0056] Cut a small amount of muscle and add it to a 0.2 mL 96-well PCR plate pre-filled with 100 μL of lysis buffer. Mince the muscle, add 4 μL of proteinase K, gently vortex for a few seconds, and briefly centrifuge. Incubate at 65°C for 10 min in a PCR instrument, then at 95°C for 5 min, and finally cool to 12°C. After a brief centrifugation, transfer the supernatant to a new 0.2 mL 96-well PCR plate, which can be used directly as a template for PCR amplification.

[0057] The lysis buffer was obtained from Chengdu Foregene Biotechnology's "Animal Tissue Direct PCR Kit" (http: / / www.foregene.com / scientific_detail.aspx?t=19&pid=5&cid=68).

[0058] 1.4 PCR Amplification

[0059] The reaction system was as follows: In a 200 μL PCR thin-walled tube, add 1 μL of the prepared template DNA, 15 μL of amplification buffer, 0.9 μL of mixed primers (20 μmol / L), and 13.1 μL of sterile water. The total PCR reaction volume was 30 μL. After adding the sample, the tube was briefly centrifuged. The PCR reaction program was: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 59℃ annealing for 30 s, 72℃ extension for 50 s, 33 cycles; 72℃ extension for 5 min.

[0060] Mixed primers:

[0061] Universal upstream primer: 5'-CGCGGTATCCTAACCGTGC-3';

[0062] Universal downstream primer: 5'-CTGGGGTAGCTTGGTCCATT-3';

[0063] Partridge-specific primer: 5'-GGCCCACCTATGTAAATCATCT-3'.

[0064] The amplification buffer was obtained from Chengdu Foregene Biotechnology's "Animal Tissue Direct PCR Kit" (http: / / www.foregene.com / scientific_detail.aspx?t=19&pid=5&cid=68).

[0065] 1.5 Agarose Gel Electrophoresis

[0066] Place a 1.5% agarose gel (premixed with GelRed dye) in 1×TAE electrophoresis buffer. Spot 3 μL of the reaction product into each gel well. Perform electrophoresis at 150 V, 150 mA for 30 min at room temperature. Image and photograph using a gel imaging system.

[0067] 1.6 Result Interpretation

[0068] When only one 377bp common amplification band appears in the amplification product, it indicates good PCR amplification, and the sample is determined to be non-partridge. When both a 377bp common amplification band and a 167bp partridge-specific band appear in the amplification product, it indicates good PCR amplification, and the sample is determined to be partridge. If no bands appear, the origin of the sample cannot be determined.

[0069] 2. Feasibility Verification

[0070] The method established in this invention was used to identify the meat source of six 200-day-old male and six female American partridges that had been identified through live animal testing. A small amount of pectoral muscle samples were taken from each individual. The meat source identification results provided by this invention were completely consistent with the results of live animal testing. For specific methods, please refer to the examples in Part 5 below.

[0071] 3. Specificity verification

[0072] Ten muscle samples from chickens, ducks, geese, pigeons, quails, rabbits, pigs, cattle, sheep, and dogs were randomly selected after in vivo or morphological verification. Genomic DNA was extracted using the lysis buffer of the "Animal Tissue Direct PCR Kit" (Chengdu Fuji Biotechnology), and then PCR amplification was performed directly using the primer set designed in this invention. The results are as follows: Figure 2 As shown, the partridge sample can amplify two bands (377bp, 167bp), while other samples can only amplify one band (377bp).

[0073] To further verify the specificity of the partridge meat source detection technology established in this invention, any two primers of this invention were input into the NCBI primer design interface Primer-BLAST (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / index.cgi) for electronic PCR. The database was selected as "nr" (nucleotide fragment), the target organism was "Vertebrata (taxid:7742)" (vertebrata), and other options were left at default. Combination 1 (universal upstream primer + universal downstream primer) returned 981 results. Except for pigs, cattle, dogs, and sheep, whose specificity was relatively poor (but the PCR amplification effect was good in this patent), the primer sequences of other species, especially birds, were basically completely matched. The target band was about 377 bp, and the target gene was 16S rRNA. Combination 2 (partridge-specific primer + universal downstream primer) returned 5 results. The amplification target was the genus Partridge, and the target gene and amplification length were completely consistent. Combination 3 (universal upstream primer + partridge-specific primer) did not find a specific amplification target.

[0074] Primers 5'-AGCACACTTCGGAGTTATGTTTGT-3' and 5'-AGTATCGGCGAGGTATGCCG-3' (CN102337335A) were used. The PCR reaction program was: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 5 min. Results are as follows: Figure 3As shown, partridge samples can amplify a band (89 bp), and some pig and chicken samples can also amplify a weak 89 bp band. Primer-BLAST results show that, in addition to partridges, it can also amplify wild bird samples such as grey-backed doves, blood chickens, parrots, woodpeckers, glossy starlings, and swiftlets. Furthermore, this method cannot detect false negatives.

[0075] Primers 5'-GCCCCGGCTAAAGACGAGGTAA-3' and 5'-GAATTTTTGATATTTAGGGTGAG-3' (CN102337336A) were used. The PCR reaction program was: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 5 min. Results are as follows: Figure 4 As shown, partridge samples can amplify a band (184bp), and some Muscovy duck samples can also amplify a weak 184bp band. Furthermore, this method cannot identify false negative test samples.

[0076] 4. Sensitivity Verification

[0077] Partridge DNA was diluted with ultrapure water to concentrations of 10 ng / μL, 1 ng / μL, 0.1 ng / μL, 0.01 ng / μL, and 0.001 ng / μL, and then PCR was performed as described in section 1 above. The results are as follows... Figure 5 As shown, a weak signal was still present at a meat sample DNA concentration of 0.01 ng / μL, indicating that the primer's sensitivity can reach 0.01 ng / μL pure partridge meat DNA sample.

[0078] 5 Examples

[0079] Fresh / frozen muscle samples from partridges, chickens, ducks, geese, pigeons, quails, rabbits, pigs, cattle, sheep, and dogs, which had undergone live or morphological verification, were selected for single-blind testing. After sample numbering, the samples were given to testers whose source was unknown for meat component analysis. The number of samples is shown in the table below. Genomic DNA was extracted using the lysis buffer of the "Animal Tissue Direct PCR Kit" (Chengdu Fuji Biotechnology), and PCR amplification was performed directly using the primer set designed in this invention. The results are as follows: Figure 6-16 As shown, all partridge samples amplified two bands (377bp, 167bp), while other samples only amplified one band (377bp). Out of a total of 731 samples, 730 were successfully identified, a success rate approaching 100%.

[0080]

[0081]

Claims

1. Primers for specific identification of partridge meat-derived components by multiplex PCR, characterized in that, This includes partridge-specific primers with the nucleotide sequence 5'-GGCCCACCTATGTAAATCATCT-3'; it also includes the following universal primers: Universal upstream primer: 5'-CGCGGTATCCTAACCGTGC-3'; Universal downstream primer: 5'-CTGGGGTAGCTTGGTCCATT-3'.

2. A kit containing the multiplex PCR specific identification primers for partridge meat source components as described in claim 1.

3. A multiplex PCR method for identifying partridge meat components, characterized by: Take meat samples to be identified, lyse them in lysis buffer, extract genomic DNA, amplify by PCR, and perform electrophoresis; When the electrophoresis results show only one 377bp common amplification band, the sample is determined to be non-partridge; when the amplification products show both a 377bp common amplification band and a 167bp partridge-specific band, the sample is determined to be partridge. The primers used for the multiplex PCR amplification are as follows: Universal upstream primer: 5'-CGCGGTATCCTAACCGTGC-3' Universal downstream primer: 5'-CTGGGGTAGCTTGGTCCATT-3' Partridge-specific primer: 5'-GGCCCACCTATGTAAATCATCT-3'.

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