Multiplex PCR Identification Primers, Kits and Methods for Pearl Chicken Source Components

Abstract

The present invention discloses multiplex PCR specific identification primers for pearl chicken meat source components, including a pearl chicken specific primer with a nucleotide sequence of 5'-GGTGATGGGGGTCTTCTTAGC-3'. The present invention also discloses a kit containing the multiplex PCR specific identification primers, as well as a multiplex PCR identification method for pearl chicken meat source components. The pearl chicken specific primer provided by the present invention performs PCR amplification on mitochondrial 16S rRNA, with a stable amplification effect, capable of producing a 240bp specific band, and effectively identifying the pearl chicken meat source.

Description

Technical Field

[0001] The present invention relates to multiplex PCR technology, and particularly to multiplex PCR specific identification primers for pearl chicken meat source components. Background Art

[0002] Pearl chicken, also known as guinea fowl or common guinea fowl, belongs to the family Numididae of the order Galliformes, and its scientific name is Numida meleagris. Pearl chicken is native to places such as Kenya and Guinea in Africa and is a wild fowl living in tropical grasslands. After decades of artificial domestication, it has been cultivated into a domesticated poultry. Pearl chicken has delicate meat, delicious taste, strong game flavor, high protein content, low fat and cholesterol content, and has high nutritional value. Pearl chicken has low requirements for feeding conditions, strong adaptability, strong cold and heat resistance, strong disease resistance and foraging ability, and a wide and miscellaneous diet. Therefore, the pearl chicken breeding industry has the characteristics of less investment, low cost, fast turnover and high efficiency. China began to introduce and breed from abroad in the 1950s, and large-scale artificial domestication and breeding began in the 1980s. It has been listed as a special poultry by the National Committee for Animal Genetic Resources and has a certain influence in the poultry market. With the continuous improvement of living standards, people's demand for meat has changed from quantity to quality. With the gradual cancellation of the live poultry market and the continuous increase in breeding costs, the market share of chilled / frozen meat and cut meat has been continuously increasing, and the phenomenon of passing off inferior goods as good ones has emerged in an endless stream. Therefore, it is particularly important to study animal-derived food safety detection technology and accurately identify food animal-derived components.

[0003] For the identification of meat-derived components, the commonly used method is real-time fluorescence quantitative PCR. This method has high sensitivity, is suitable for detecting trace meat source components in processed foods, and can relatively quantify, so this method is widely used. However, it is difficult to design specific probes and primers for some species in fluorescence quantitative PCR, and a set of probes and primers only correspond to a specific single species component. There are also methods using universal primers or specific primers for amplification and sequencing to detect multiple meat-derived components, but this method has a large workload, high cost, and there is also a situation of missed detection. Summary of the Invention

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

[0005] The technical solution to achieve the present invention is: multiplex PCR specific identification primers for pearl chicken meat source components, including pearl chicken specific primers, and their nucleotide sequences are:

[0006] 5’-GGTGATGGGGGTCTTCTTAGC-3’.

[0007] Furthermore, the identification primers provided by the present invention include:

[0008] Specific primers for guinea fowl: 5'-GGTGATGGGGGTCTTCTTAGC-3'

[0009] Universal upstream primer: 5'-ACGACCTCGATGTTGGATCA-3'

[0010] Universal downstream primer: 5'-CGGTCTGAACTCAGATCACGTA-3'.

[0011] Another object of the present invention is to provide a kit containing the above-mentioned multiplex PCR specific identification primers for guinea fowl meat source components.

[0012] Specifically, the kit at least includes specific primers for guinea fowl:

[0013] 5'-GGTGATGGGGGTCTTCTTAGC-3'.

[0014] Furthermore, the kit of the present invention further includes:

[0015] Universal upstream primer: 5'-ACGACCTCGATGTTGGATCA-3'

[0016] Universal downstream primer: 5'-CGGTCTGAACTCAGATCACGTA-3'.

[0017] The third object of the present invention is to provide a multiplex PCR identification method for guinea fowl meat source components based on the above-mentioned specific identification primers.

[0018] Specifically, for the multiplex PCR identification method of guinea fowl meat source components, take the meat source sample to be identified, place it in a lysis solution for lysis, extract genomic DNA, perform PCR amplification, and electrophoresis; when in the electrophoresis result, only one common amplification band of about 100 bp appears in the amplification product, it is determined that the sample is not guinea fowl; when both a common amplification band of about 100 bp and a specific guinea fowl band of 240 bp appear in the amplification product, it is determined that the sample is guinea fowl; the primers used for the multiplex PCR amplification are as follows:

[0019] Universal upstream primer: 5'-ACGACCTCGATGTTGGATCA-3'

[0020] Universal downstream primer: 5'-CGGTCTGAACTCAGATCACGTA-3'

[0021] Specific primers for guinea fowl: 5'-GGTGATGGGGGTCTTCTTAGC-3'.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides a specific primer pair for guinea fowl to perform conventional PCR amplification on 16S rRNA, without the need for expensive equipment such as a fluorescence quantitative PCR instrument, and the amplification effect is stable. A 240bp specific band can appear, effectively identifying the meat source of guinea fowl; while the provided universal primer can effectively avoid false negatives. Description of the Drawings

[0024] Figure 1 It is an electrophoresis diagram of PCR amplification products of common commercially available meat samples;

[0025] Lanes 1-2 are guinea fowl, 3-4 are chickens, 5-6 are ducks, 7-8 are geese, 9 is pigeon, 10 is quail, 11 is pheasant, 12 is partridge, 13 is ostrich, 14 is emu, 15 is pig, 16 is cow, 17 is sheep, 18 is dog, 19 is rabbit, 20 is horse, 21 is donkey, 22 is camel, 23 is sika deer, and M is DNA marker DL 2000.

[0026] Figure 2 Electrophoresis diagram of PCR detection of the sensitivity of guinea fowl primers;

[0027] Lanes 1-3 are 1 ng / μL (guinea fowl meat DNA), 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.

[0028] Figure 3 It is an electrophoresis diagram of PCR amplification products of guinea fowl meat samples.

[0029] Figure 4 It is an electrophoresis diagram of PCR amplification products of chicken, duck, goose, and pigeon meat samples.

[0030] Figure 5 It is an electrophoresis diagram of PCR amplification products of quail, pheasant, partridge, and ostrich meat samples.

[0031] Figure 6 It is an electrophoresis diagram of PCR amplification products of emu, pig, cow, and sheep meat samples.

[0032] Figure 7 It is an electrophoresis diagram of PCR amplification products of dog, rabbit, horse, and donkey meat samples.

[0033] Figure 8 It is an electrophoresis diagram of PCR amplification products of camel and sika deer meat samples. Detailed Embodiments

[0034] 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.

[0035] 1 Establishment of detection technology

[0036] 1.1 Sample source

[0037] A small amount of pectoral muscle (0.5 g) was cut from slaughtered individuals in a guinea fowl farm, with 48 males and 48 females, and placed in 1.5 mL centrifuge tubes containing 95% alcohol. The samples were transported in a frozen environment and stored at -20 °C for later use.

[0038] Meanwhile, frozen / fresh meat samples of species such as chicken, duck, goose, pigeon, pheasant, quail, rabbit, pig, cow, sheep, dog, horse, donkey, camel, ostrich, emu, sika deer, etc. were selected in the farmers' market as controls. Among them, the samples of chicken, duck, goose, quail, pigeon, pheasant, and partridge were obtained after slaughtering after confirming the live animals, and the other samples were obtained after slaughtering with the determined sources.

[0039] 1.2 Primer design

[0040] The mitochondrial DNA genome sequences of guinea fowl, chicken, duck, goose, pigeon, pheasant, quail, rabbit, pig, cow, sheep, dog, etc. were downloaded from GenBank. After sequence alignment using the biological software MEGA7.0, specific sites of guinea fowl were searched, and then universal primers were designed on the flanks of the specific sites of guinea fowl. Finally, mitochondrial 16S rRNA was determined as the primer design region.

[0041] Universal upstream primer: 5’-ACGACCTCGATGTTGGATCA-3’;

[0042] Universal downstream primer: 5’-CGGTCTGAACTCAGATCACGTA-3’;

[0043] Guinea fowl specific primer: 5’-GGTGATGGGGGTCTTCTTAGC-3’;

[0044] Among them,

[0045] Primer combination 1: Universal upstream primer + Universal downstream primer, for amplifying the common fragment of the sample to be detected;

[0046] Primer combination 2: Universal upstream primer + Guinea fowl specific primer, for amplifying the specific fragment of guinea fowl.

[0047] 1.3 Genomic DNA extraction

[0048] Cut a small amount of muscle (10 mg) and extract genomic DNA using a kit. The DNA is stored at 2 - 8°C and stored at -20°C for long-term storage, and can be used as a template directly for PCR amplification.

[0049] The DNA extraction kit is the "Universal DNA Extraction Kit" from Guangzhou Magene Biotechnology Co., Ltd. (http: / / www.magentec.com.cn / product_show.php?id=41).

[0050] 1.4 PCR Amplification

[0051] The reaction system is as follows: In a 200 μL PCR thin-walled tube, add 1 μL of the template DNA prepared above, 15 μL of amplification buffer, 0.9 μL of mixed primers (20 μmol / L), and 13.1 μL of sterilized water. The PCR reaction system is 30 μL. After adding the samples, centrifuge briefly. The PCR reaction program is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, for 30 cycles; extension at 72°C for 5 min.

[0052] Mixed Primers:

[0053] Universal upstream primer: 5’-ACGACCTCGATGTTGGATCA-3’;

[0054] Universal downstream primer: 5’-CGGTCTGAACTCAGATCACGTA-3’;

[0055] Guinea fowl specific primer: 5’-GGTGATGGGGGTCTTCTTAGC-3’.

[0056] The amplification buffer is "Premix Taq" from Takara Bio Inc. (Dalian) TM ”(https: / / www.takarabiomed.com.cn / ProductShow.aspx?m=20141215102854153148&productID=20141224135122093601).

[0057] 1.5 Agarose Gel Electrophoresis

[0058] Place a 1.5% agarose gel (pre-mixed with GelRed dye) in 1×TAE electrophoresis buffer. Take 3 μL of the reaction product and load it into the gel wells. At room temperature, perform electrophoresis at 100 V and 150 mA for 40 min. Image and take photos in a gel imaging system.

[0059] 1.6 Result Interpretation

[0060] 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 determined to be non-pearl chicken; when both the common amplification band of about 100bp and the 240bp pearl chicken specific band appear in the amplification product, it indicates that the PCR amplification is good, and the sample is determined to be pearl chicken. If no band appears, the source of the test sample cannot be determined.

[0061] 2 Feasibility verification

[0062] The method established by the present invention was used to identify the meat sources of 48 male and 48 female pearl chickens at 300 days old whose sources were identified by in vivo identification. A small amount of pectoral muscle samples were taken from each individual, and the results of meat source identification provided by the present invention were completely consistent with the results of in vivo identification. For the specific method, see the examples in Part 5 below.

[0063] 3 Specificity verification

[0064] Randomly select 2 muscle samples each of chickens, ducks, geese, pigeons, quails, rabbits, pigs, cows, sheep, dogs, etc. that have been verified by in vivo or external appearance, extract genomic DNA with the "Universal DNA Extraction Kit" (Guangzhou Meiji Biotechnology Co., Ltd.), and directly perform PCR amplification with the primer set designed by the present invention. The results are as Figure 1 shown. Two bands (100bp, 240bp) can be amplified from the pearl chicken samples, while only one band (99bp or 100bp) can be amplified from other test samples.

[0065] To further verify the specificity of the pearl chicken 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 in silico PCR experiments. The database was selected as "nr" (nucleotide fragment), and other options were set as default parameters. When the target organism was selected as "vertebrata (taxid: 7742)" (subphylum Vertebrata), combination 1 (universal upstream primer + universal downstream primer) returned nearly a thousand results, covering all the species used in the present invention. The target band was at 99 - 100bp, and the target gene was mitochondrial 16S rRNA. Combination 2 (universal upstream primer + pearl chicken specific primer) returned 4 results, and the amplification targets were all pearl chicken, and the target gene and the amplification length were consistent. Other combinations (universal downstream primer + pearl chicken specific primer) returned zero results, indicating that no specific amplification target was matched.

[0066] 4 Sensitivity verification

[0067] Dilute guinea fowl DNA with ultrapure water to 1 ng / μL, 0.1 ng / μL, 0.01 ng / μL, 0.001 ng / μL, 0.0001 ng / μL, and perform PCR according to the method described in 1 above. The results are as Figure 2 shown. There is still a weak signal at a meat sample DNA concentration of 0.001 ng / μL, indicating that the sensitivity of this primer can reach 0.001 ng / μL of pure guinea fowl meat DNA sample.

[0068] 5 Examples

[0069] Select fresh / frozen muscle samples of guinea fowl, chicken, duck, goose, pigeon, quail, rabbit, pig, cow, sheep, dog, etc. that have been verified by in vivo or external appearance for detection. The number of samples is shown in the following table. After extracting genomic DNA with the "Universal DNA Extraction Kit" (Guangzhou Meiji Biotechnology Co., Ltd.), directly perform PCR amplification with the primer set designed by the present invention. The results are as Figures 3 - 8 shown. All guinea fowl samples can amplify two bands (100 bp, 240 bp), while other test samples can only amplify one band (99 bp or 100 bp). The total number of samples is 951, among which all 96 guinea fowl are accurately identified, and other samples are classified as non-guinea fowl samples, with an accuracy rate of 100%.

[0070] species sample type number of varieties number of samples guinea fowl fresh muscle 1 96 chicken fresh muscle 2 86 duck frozen muscle 1 48 goose fresh muscle 1 35 pigeon fresh muscle 1 34 quail frozen muscle 1 48 pheasant fresh muscle 1 80 partridge fresh muscle 1 71 ostrich frozen muscle 3 74 emu frozen muscle 1 64 pig fresh muscle 1 46 sheep frozen muscle 1 42 dog frozen muscle 1 41 rabbit frozen muscle 1 43 pig fresh muscle 1 46 horse fresh muscle 1 24 donkey fresh muscle 1 24 camel fresh muscle 1 25 sika deer fresh muscle 1 24

Claims

1. A multiplex PCR specific identification primer set for pearl chicken meat source components, characterized in that, Comprising guinea fowl specific primers and universal primers, the nucleotide sequence of the guinea fowl specific primers is 5’-GGTGATGGGGGTCTTCTTAGC-3’; the universal primers are as follows: Universal upstream primer: 5’-ACGACCTCGATGTTGGATCA-3’; Universal downstream primer: 5’-CGGTCTGAACTCAGATCACGTA-3’.

2. A kit comprising the multiplex PCR specific identification primer set for guinea fowl meat source components described in claim 1.

3. Multiplex PCR identification method for pearl chicken meat source components, characterized in that, Take the meat source sample to be identified, place it in the lysis solution for lysis, extract genomic DNA, perform PCR amplification, and electrophoresis; When only one common amplification band of about 100 bp appears in the electrophoresis result, the sample is determined to be non-guinea fowl; when both a common amplification band of about 100 bp and a guinea fowl specific band of 240 bp appear in the amplification product, the sample is determined to be guinea fowl; the primer set used for the multiplex PCR amplification is as follows: Universal upstream primer: 5’-ACGACCTCGATGTTGGATCA-3’ Universal downstream primer: 5’-CGGTCTGAACTCAGATCACGTA-3’ Guinea fowl specific primer: 5’-GGTGATGGGGGTCTTCTTAGC-3’.

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