CN110029172B - Double PCR detection kit for equine and donkey-derived components - Google Patents
Double PCR detection kit for equine and donkey-derived components Download PDFInfo
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Abstract
The invention provides a double PCR detection kit for horse, donkey and mule-derived components in donkey-hide gelatin and raw materials, and belongs to the technical field of molecular detection of animal-derived components. The kit comprises horse and donkey specific primers, a reaction reagent, a positive control and a blank control. The primers comprise Horse specific primers (Horse-F, R) and Donkey specific primers (primer pair 1: Donkey-1-F, R or primer pair 2: Donkey-2-F, R). Can be used for identifying different source components of horse and donkey such as hide and donkey-hide gelatin. The detection method disclosed by the invention has the advantages of good stability, strong specificity, simplicity in operation and direct result judgment, can provide an effective, accurate and reliable identification method for donkey skins and donkey-hide gelatin products thereof for donkey-hide gelatin enterprises and food and drug supervision departments, and also provides raw material purchasing and authenticity protection for donkey-hide gelatin products for donkey-hide gelatin enterprises.
Description
Technical Field
The invention relates to the field of animal molecular biology, in particular to a double PCR detection kit for horse, donkey and mule-derived components in donkey-hide gelatin and raw materials.
Background
The colla Corii Asini is solid gum prepared from dried or fresh skin of Equus asinus of Equidae by decocting and concentrating. It has effects in improving body constitution, improving sleep, strengthening brain, improving intelligence, delaying aging, and preventing and treating cancer. However, because the breed volume of present donkey can not satisfy the demand in market far away, and some horse skins, mule skin are difficult to distinguish through naked eye and donkey skin for be difficult to guarantee the authenticity of skin in raw materials purchase, lead to appearing the phenomenon that donkey skin adulterated in a large number on the market. The adulteration of horse hide and mule hide in donkey-hide gelatin not only affects the efficacy of the donkey-hide gelatin, but also can harm the health of consumers. Meanwhile, donkey-hide gelatin is adulterated and is a fraudulent behavior, so that enterprises and governments lose confidence in consumers and extremely bad social influence is caused. Therefore, the simple, reliable and effective method for detecting the hide raw materials and the donkey-hide gelatin finished products is established, and has important significance for production enterprises, supervision departments and consumers.
Aiming at the identification of horses, donkeys and mules, the traditional method mainly comprises the steps of observing the appearance and the color of the skins and distinguishing the smell, but the method is difficult to distinguish horse skins and mule skins which are high in similarity with the donkey skins. In recent years, with the development of molecular biotechnology, identification can be performed by using different genomic information possessed by various species, and compared with the conventional analysis method, the DNA molecular detection technology has objectivity and accuracy. The publication No. CN104046700A patent adopts 5-fold multicolor fluorescence quantitative PCR detection technology, and utilizes 16SrRNA genes of CKM nuclear gene (nDNA) and mitochondrial genome DNA (mtDNA) to identify donkey, horse mule and donkey mule. The method has high detection cost, needs large-scale expensive instruments, is not suitable for large-scale identification of donkey skins, and does not relate to detection of animal-derived components in the donkey-hide gelatin.
Disclosure of Invention
The invention aims to provide a kit for identifying horses, donkeys and mules by aiming at the defects of the prior art;
the second purpose of the invention is to provide a kit for identifying the source components of horse and donkey in donkey-hide gelatin;
the invention also aims to provide a method for identifying horse, donkey and mule hide and a method for identifying horse and donkey-derived components in donkey-hide.
In order to achieve the purpose, the invention screens horse and donkey specific sequences from the aspects of intraspecies consistency, stability, interspecific specificity and the like, selects specific gene segments in horses and donkeys as detection targets and designs specific primers by detecting and screening non-target species and different varieties in target species, and tests the interspecific specificity, the interspecific conservation, the sensitivity and the like by utilizing PCR amplification and electrophoresis technology. Through a large amount of analysis and experimental verification, the horse and donkey genome specific DNA molecules for detection are finally obtained, and the nucleotide sequences of the horse and donkey genome specific DNA molecules are shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3.
Based on the primer, the invention firstly provides a horse and donkey specific primer which specifically amplifies the nucleotide sequence shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 or the specific fragment of the sequence.
Preferably, the length of the primer is 20-24 bp. The design of the primer requires consideration of the susceptibility to mismatching, the length of the amplified fragment, the reaction temperature, and the like.
Preferably, the primer sequences are as in table 1:
TABLE 1 PCR amplification primers designed according to the present invention
The invention further provides a detection kit containing the specific primer.
Preferably, the kit further comprises one or more of the following reagents: taq enzyme, dNTPs, MgCl2PCR buffer solution, positive control DNA and double distilled water.
The positive control DNA is horse or donkey genome DNA, and the blank control is double distilled water.
Further, the invention provides application of the horse specific primer pair and the donkey specific primer pair 1 or the detection kit in horse, donkey and mule skin identification.
The invention also provides a horse specific primer pair, a donkey specific primer pair 1, a kit and a PCR detection method thereof in horse, donkey and mule skin identification, which comprises the following steps:
1) extracting hide genome DNA;
2) performing double PCR amplification by using the horse specific primer pair and the donkey specific primer pair 1, and respectively taking horse and donkey DNA templates as positive controls and double distilled water as a blank control;
3) and detecting and judging the result of the PCR amplification product.
Preferably, the reaction system of the horse, donkey and mule skin identification PCR amplification in the step 2) is shown in Table 2
TABLE 2 PCR amplification reaction system for identifying horse, donkey and mule skin
The PCR reaction procedure was as follows:
pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 15s, and 35 cycles; final extension at 72 ℃ for 30 s; cooling at 4 deg.C for 2 min.
The method for judging the identification result of horse, donkey and mule skin comprises the following steps:
in the positive control PCR reaction, the specific sequences (SEQ ID No.1 and SEQ ID No.2) of the horse and the donkey are amplified, the sizes of the amplified fragments are 166bp and 102bp respectively, meanwhile, no amplified product exists in a blank control, the PCR reaction system is indicated to work normally, and otherwise, the detection is carried out again.
When the PCR reaction product of the detected sample is an amplification band of 166bp, which is consistent with the horse positive control amplification product, the sample is judged to be horse skin; when the PCR reaction product of the detected sample is an amplification band of 102bp, which is consistent with the amplification product of the donkey positive control, the sample is judged to be donkey skin; when the PCR reaction product of the detection sample has two amplification bands, namely 166bp and 102bp, which are respectively consistent with the horse positive control amplification product and the donkey positive control amplification product, the sample is judged to be a mule skin; when the PCR reaction product of the detection sample has no amplification band, judging that the sample is not any one of horse, donkey and mule.
Further, the invention provides application of the horse-specific primer pair and donkey-specific primer pair 2 or the detection kit in identification of horse and donkey-derived components in donkey-hide gelatin.
The invention also provides a horse specific primer pair, a donkey specific primer pair 2, a kit and a PCR detection method for identifying the source components of horse and donkey in donkey-hide gelatin, which comprises the following steps:
1) extracting donkey-hide gelatin genome DNA;
2) performing double PCR amplification by using the horse specific primer pair and the donkey specific primer pair 2, and respectively taking horse or donkey genome DNA as a positive control and double distilled water as a blank control;
3) and detecting and judging the result of the PCR amplification product.
Preferably, the reaction system for identifying PCR amplification of horse and donkey-derived components in donkey-hide gelatin in step 2) is shown in Table 3
The PCR reaction procedure was as follows:
pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 15s, and 35 cycles; final extension at 72 ℃ for 30 s; cooling at 4 deg.C for 2 min.
The judgment method of the identification result of the source components of the horse and the donkey in the donkey-hide gelatin comprises the following steps:
in the positive control PCR reaction, the specific sequences (SEQ ID No.1 and SEQ ID No.3) of the horse and the donkey are amplified, the sizes of the amplified fragments are 166bp and 130bp respectively, meanwhile, no amplified product exists in a blank control, the PCR reaction system is indicated to work normally, and otherwise, the detection is carried out again.
When the PCR reaction product of the detection sample is an amplification band of 166bp, which is consistent with the horse positive control amplification product, the donkey-hide gelatin sample is judged to only contain horse-derived components; when the PCR reaction product of the detection sample is a 130bp amplification band which is consistent with the donkey positive control amplification product, judging that the donkey-hide gelatin sample only contains donkey-derived components; when the PCR reaction product of the detection sample has two amplification bands, namely 166bp and 130bp which are respectively consistent with the amplification products of the horse positive control and the donkey positive control, judging that the donkey-hide gelatin sample contains both horse-derived components and donkey-derived components; when the PCR reaction product of the detected sample has no amplification band, the sample is judged to contain no horse and donkey-derived components.
In the invention, the detection result is presented by agarose gel electrophoresis; the leather DNA is extracted by phenol-chloroform extraction method, and the donkey-hide gelatin sample DNA is extracted by DNA extraction kit.
The detection method disclosed by the invention adopts a conserved region of horse and donkey specific sequences to carry out primer design, so that the specificity of the primers is ensured, and the primers are highly conserved among varieties, thereby ensuring the accuracy and credibility of detection results; meanwhile, whether the donkey-hide gelatin contains horse-derived components or not can be identified through a PCR reaction, and horse skins or mule skins are doped in the donkey-hide gelatin for detection through the method disclosed by the invention. The invention can realize the supervision of donkey-hide gelatin enterprises from raw materials to product monitoring and management departments. The kit and the detection method have the advantages of good stability, strong specificity, simple operation and direct result judgment.
Drawings
FIG. 1 shows the results of specific detection of primers specific to horse and donkey in 16 species, and specific fragments were amplified using genomic DNAs of equine, donkey and non-equine mammals (mouse, rat, hamster, guinea pig, cow, buffalo, sheep, goat, pig, rabbit, fox, dog, mink, racoon dog) as templates. Wherein FIG. 1A is the species-specific detection result of the horse-specific primers; the numbers in lanes are indicated below: m: BM50bp DNA Marker; 1: blank control; 2: a horse; 3: a mouse; 4: a rat; 5: a hamster; 6: guinea pigs; 7: common cattle; 8: buffalo; 9: sheep; 10: a goat; 11: a pig; 12: donkey; 13: a rabbit; 14: foxes; 15: a dog; 16: mink; 17: a raccoon dog. FIGS. 1B and 1C are the results of species-specific detection of donkey- specific primer pairs 1 and 2, respectively; the numbers in lanes are indicated below: m: BM50bp DNA Marker; 1: blank control; 2: donkey; 3: a mouse; 4: a rat; 5: a hamster; 6: guinea pigs; 7: common cattle; 8: buffalo; 9: sheep; 10: a goat; 11: a pig; 12: a horse; 13: a rabbit; 14: foxes; 15: a dog; 16: mink; 17: a raccoon dog.
FIG. 2 is a graph of the sensitivity of primers specific to horse and donkey, wherein FIG. 2A: is the sensitivity detection result of the equine genome nucleotide specific sequence SEQ ID No.1 in different DNA template concentrations of horses; FIG. 2B and FIG. 2C are the sensitivity detection results of donkey genome nucleotide specific sequences SEQ ID No.2 and SEQ ID No.3 in donkey different DNA template concentrations, respectively. The numbers in lanes are indicated below: m: BM2000bpDNA Marker; 1-3: blank control; 4-6: template concentration of 0.01 ng/. mu.L; 7-9: template concentration of 0.1 ng/. mu.L; 10-12: template concentration of 1 ng/. mu.L; 13-15: template concentration of 10 ng/. mu.L; 16-18: template concentration of 100 ng/. mu.L.
FIG. 3 shows the results of the duplex PCR test for horse, donkey and mule skin using horse, donkey and mule specific primer pair 1 as templates, wherein the horse amplification product is 166bp specific fragment, donkey amplification product is 102bp specific fragment, and the mule individuals have horse and donkey specific fragments. Lanes are illustrated below: m: BM2000bp DNA Marker; 1: blank control; 2-4: horse hide DNA sample; 5-7: DNA samples of donkey hide; 8-10: mule skin DNA samples.
FIG. 4 is a double PCR test result of donkey-hide gelatin derived components identified by horse specific primer pair and donkey specific primer pair 2, using horse and donkey genome DNA as positive template, and using 4 donkey-hide gelatin DNA samples to test, wherein all samples contain horse-derived components, the amplification product is 166bp specific fragment, donkey-derived components, and the amplification product is 130bp specific fragment. Lanes are illustrated below: m: BM2000DNA Marker; 1: blank control; 2: horse DNA positive control; 3: donkey DNA positive control; 4: DNA sample of donkey-hide gelatin; 5-7: donkey-hide gelatin DNA samples (containing equine derived components).
Detailed Description
The following examples are intended to further illustrate the present invention but should not be construed as limiting the invention and modifications or enhancements which can be made thereto without departing from the spirit and spirit of the invention are intended to be within the scope of the invention.
Example 1 establishment of species-specific PCR reaction System for horse and donkey
1. Preparation and preservation of samples
1.1 sampling
Collecting hide sample of 0.6cm2。
1.2 DNA template preparation
DNA template preparation was carried out by the commonly used phenol-chloroform crude extraction method (Samburuke J, Friedel E F, Mannich Atitis T. molecular cloning, laboratory Manual [ M ]. 2 nd edition. gold time goose, Rimeng. Beijing: scientific Press, 1999.465-467).
2. Primer design
The primer sequences of this example are shown in Table 1.
The product size of the horse specific primer reaction system is 166bp, the product size of the donkey specific primer pair 1 reaction system is 102bp, the product size of the donkey specific primer pair 2 reaction system is 130bp, and the nucleotide sequences are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 of the sequence table.
PCR detection
3.1 sample PCR reaction
3.1.1 adding the reaction reagents into the PCR reaction tube in sequence and mixing evenly.
3.1.2 centrifuge the PCR tube on a centrifuge for 10s at 500-3000 g, then take out the PCR tube and put into a PCR instrument.
3.1.3 PCR reactions were performed. The procedure is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 15s, and 35 cycles; final extension at 72 ℃ for 30 s; cooling at 4 deg.C for 2 min.
3.1.4 taking out the PCR tube after the reaction is finished, and carrying out electrophoresis detection on the PCR reaction product.
3.2 control PCR reaction
3.2.1 blank control, negative control and positive control were set simultaneously with the PCR reaction of the samples. In each control PCR reaction system, the components except the template and the PCR reaction conditions are the same as those of the experimental group, and the concentration of the negative and positive control DNA templates also meets the concentration requirement of the sample DNA template.
3.2.2 horse or donkey, and non-equine mammals (mouse, rat, hamster, guinea pig, common cow, buffalo, sheep, goat, pig, rabbit, fox, dog, mink, racoon dog) as the negative control template of PCR reaction system.
3.2.3 horse or donkey genomic DNA was used as a positive control template for PCR reaction system.
3.2.4 double distilled water is used as a blank control template of the PCR reaction system.
4, detection and result judgment of PCR amplification product
Agarose was weighed at a mass concentration of 20g/L, added to 1 XTAE buffer solution, and dissolved by heating to prepare an agarose solution. Add 1. mu.L GEL RED solution to each 100mL agarose solution, mix well, cool slightly, pour it into electrophoresis plate, insert comb plate, solidify into GEL at room temperature, put in 1 XTAE buffer solution, pull out comb plate vertically and slightly upwards. Mixing 5 mu L of PCR product with 3 mu L of sample adding buffer solution, adding the mixture into gel sample application holes, adding a DNA molecular weight standard into one sample application hole, switching on a power supply, and carrying out electrophoresis for 15-30 min under the condition of 2-5V/cm.
And after the electrophoresis is finished, taking out the agarose gel, and placing the agarose gel on a gel imager or an ultraviolet transmission instrument for imaging. Judging the size of the amplified band according to the DNA molecular weight standard, and forming an electronic file by the electrophoresis result or taking a picture by a photographic system.
5. Analysis and presentation of results
5.1 As shown in FIG. 1A, the horse specific sequence (SEQ ID No.1) was amplified and the amplified fragment size was 166bp consistent with the expected fragment size, while there was no amplified product in the negative control and blank control, indicating that the selected horse primer pair had better specificity.
5.2 As shown in FIG. 1B, specific sequence of donkey (SEQ ID No.2) was amplified and the amplified fragment size was consistent with the expected fragment size of 102bp, while there was no amplified product in negative control and blank control, indicating that the selected donkey primer pair 1 had better specificity.
5.3 As shown in FIG. 1C, specific sequence of donkey (SEQ ID No.3) was amplified and the amplified fragment size was identical to the expected fragment size of 130bp, while there was no amplified product in negative control and blank control, indicating that the specificity of the chosen donkey primer pair 2 was better.
Example 2 sensitivity test
1. Equine genome specific primer sensitivity detection
A nucleotide-specific fragment of SEQ ID NO.1 was amplified under the conditions of example 1 using 0.01 ng/. mu.L, 0.1 ng/. mu.L, 1 ng/. mu.L, 10 ng/. mu.L, and 100 ng/. mu.L of equine DNA as templates, respectively. As shown in FIG. 2A, the amplification occurred at a template concentration of 0.1 ng/. mu.L, i.e., the amplification occurred when 0.2ng template DNA was added to the reaction system, indicating that the specific reaction system had a good sensitivity.
2. Donkey genome specific primer pair 1 sensitivity detection
The nucleotide specific fragment of SEQ ID NO.2 was amplified using donkey DNA of 0.01 ng/. mu.L, 0.1 ng/. mu.L, 1 ng/. mu.L, 10 ng/. mu.L, and 100 ng/. mu.L, respectively, as a template under the conditions of example 1. As shown in FIG. 2B, amplification occurred at a template concentration of 10 ng/. mu.L, i.e., 20ng of template DNA was added to the reaction system, indicating that the specific reaction system had better sensitivity.
3. Donkey genome specific primer pair 2 sensitivity detection
The nucleotide specific fragment of SEQ ID NO.3 was amplified using donkey DNA of 0.01 ng/. mu.L, 0.1 ng/. mu.L, 1 ng/. mu.L, 10 ng/. mu.L, and 100 ng/. mu.L, respectively, as a template under the conditions of example 1. As shown in FIG. 2C, amplification occurred at a template concentration of 0.01 ng/. mu.L, i.e., the reaction system was tested by adding 0.02ng template DNA, indicating that the specific reaction system had better sensitivity.
EXAMPLE 3 kit Assembly
TABLE 4 composition of horse, donkey, mule skin test kit
TABLE 5 composition of kit for detecting horse and donkey-derived ingredients in donkey-hide gelatin
The kit does not affect the use effect after being stored for 12 months.
The application method of the kit comprises the following steps:
1. the PCR reaction system of example 4 or example 5 was loaded as required.
2. Loading according to PCR amplification conditions of Table 2 or Table 3
3. After the reaction is finished, 5 mu L of PCR amplification product is taken, 2% agarose GEL electrophoresis (technical parameters: 2V/cm-5V/cm, electrophoresis time is 15 min-30 min), GEL RED staining is carried out, and the result is detected by a GEL imaging system.
Blank control and positive control are required to be set for each reaction, and the reaction system and the amplification conditions are the same as those of the sample to be detected.
Example 4 horse, donkey and mule Pico Zhang Duplex PCR
In order to facilitate rapid detection, the invention simultaneously considers the amplification efficiency of each primer under the condition of ensuring effective amplification and good specificity of each primer, and mixes the primer amount of the horse specific primer pair and the donkey specific primer pair 1 according to the proportion of 5:6 for standby.
1. The total reaction system is as in Table 2.
DNA sample: taking horse, donkey, mule skin DNA with the concentration of 25 ng/mu L as a template.
3. The procedure was as in example 1.
The resulting gel imaging results are shown in FIG. 3. Experimental results show that the primers have good specificity and can distinguish horses, donkeys and mule skins.
The method for judging the identification result of horse, donkey and mule skin comprises the following steps:
in the positive control PCR reaction, the specific sequences (SEQ ID No.1 and SEQ ID No.2) of the horse and the donkey are amplified, the sizes of the amplified fragments are 166bp and 102bp respectively, meanwhile, no amplified product exists in a blank control, the PCR reaction system is indicated to work normally, and otherwise, the detection is carried out again.
When the PCR reaction product of the detected sample is an amplification band of 166bp, which is consistent with the horse positive control amplification product, the sample is judged to be horse skin; when the PCR reaction product of the detected sample is an amplification band of 102bp, which is consistent with the amplification product of the donkey positive control, the sample is judged to be donkey skin; when the PCR reaction product of the detection sample has two amplification bands, namely 166bp and 102bp, which are respectively consistent with the horse positive control amplification product and the donkey positive control amplification product, the sample is judged to be a mule skin; when the PCR reaction product of the detection sample has no amplification band, judging that the sample is not any one of horse, donkey and mule.
Example 5 Duplex PCR of horse and donkey derived Components in donkey-hide gelatin
In order to facilitate the rapid detection of the donkey-hide gelatin product, the invention simultaneously considers the amplification efficiency of each primer under the condition of ensuring the effective amplification and good specificity of each primer, and mixes the primer amount of the horse specific primer pair and the donkey specific primer 2 according to the proportion of 1:1 for later use.
1. The total reaction system is as in Table 3.
DNA sample: horse and donkey positive DNA samples, as well as donkey hide gelatin DNA samples and donkey gelatin DNA samples containing equine derived components (samples customized by a company).
3. The procedure was as in example 1.
The resulting gel imaging results are shown in fig. 4. Experimental results show that the primers have good specificity, and can conveniently and effectively identify whether donkey-hide gelatin contains horse or donkey-derived components.
The judgment method of the identification result of the source components of the horse and the donkey in the donkey-hide gelatin comprises the following steps:
in the positive control PCR reaction, the specific sequences (SEQ ID No.1 and SEQ ID No.3) of the horse and the donkey are amplified, the sizes of the amplified fragments are 166bp and 130bp respectively, meanwhile, no amplified product exists in a blank control, the PCR reaction system is indicated to work normally, and otherwise, the detection is carried out again.
When the PCR reaction product of the detection sample is an amplification band of 166bp, which is consistent with the horse positive control amplification product, the donkey-hide gelatin sample is judged to only contain horse-derived components; when the PCR reaction product of the detection sample is a 130bp amplification band which is consistent with the donkey positive control amplification product, judging that the donkey-hide gelatin sample only contains donkey-derived components; when the PCR reaction product of the detection sample has two amplification bands, namely 166bp and 130bp which are respectively consistent with the amplification products of the horse positive control and the donkey positive control, judging that the donkey-hide gelatin sample contains both horse-derived components and donkey-derived components; when the PCR reaction product of the detected sample has no amplification band, the sample is judged to contain no horse and donkey-derived components.
Description of sequence listing:
SEQ ID NO.1 is a nucleotide sequence amplified by a horse specific primer pair, and the sequence length is 166 bp;
SEQ ID NO.2 is the nucleotide sequence amplified by the donkey specific primer pair 1, and the sequence length is 102 bp;
SEQ ID NO.3 is the nucleotide sequence amplified by the donkey specific primer pair 2, and the sequence length is 130 bp;
SEQ ID NO.4-9 are primers Horse-F, Horse-R, Donkey-1-F, Donkey-1-R, Donkey-2-F and Donkey-2-R, respectively.
Sequence listing
<110> university of agriculture in Huazhong
<120> horse and donkey-derived component duplex PCR detection kit
<160> 9
<170> SIPOSequenceListing 1.0
<210> <211> <212> <213> 1 166 DNA Equus caballus
<400> <211> <212> <213> 1 166 DNA Equus caballus
caggtgagcc gagctgcgct gagctggtgc tagggaggag gggctggaga ccccgagggt 60
tggccctgcg ccccgcgggc ccgggagagc gggaagccgg gctgggcgtg ctttgcgcgg 120
gttgggctct cgctgagccg tcgggaggga cgaactgcgt gcgcca 166
<210> <211> <212> <213> 2 102 DNA Equus asinus
<400> <211> <212> <213> 2 102 DNA Equus asinus
gatttctgga aagtcgcaga agggctggag ggcctgggga ggagggggac ggctgaggtt 60
gggggtcccg ccccctccga gcccccaatg cttttcgcaa gc 102
<210> 3
<211> 130
<212> DNA
<213> Equus asinus
<400> 3
catcctacta actatagccg tgctatcaat cctagtagga ggctgaggtg gcctcaacca 60
aacccaacta cgaaaaatta tggcatactc gtcaattgcc catataggat gaatagcagc 120
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 4
caggtgagcc gagctgcgct 20
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 5
tggcgcacgc agttcgtccc 20
<210> <211> <212> <213> 624 DNA Artificial sequence
<400> <211> <212> <213> 624 DNA Artificial sequence
gatttctgga aagtcgcaga aggg 24
<210> <211> <212> <213> 720 DNA Artificial sequence
<400> <211> <212> <213> 720 DNA Artificial sequence
gcttgcgaaa agcattgggg 20
<210> <211> <212> <213> 822 DNA Artificial sequence
<400> <211> <212> <213> 822 DNA Artificial sequence
catcctacta actatagccg tg 22
<210> <211> <212> <213> 920 DNA Artificial sequence
<400> <211> <212> <213> 920 DNA Artificial sequence
cactaagatg gctgctattc 20
Claims (6)
1. The specific primer for identifying the components derived from horses and donkeys comprises the following primer pairs:
horse specific primer pairs:
Horse-F：5´- CAGGTGAGCCGAGCTGCGCT-3´
Horse-R: 5 '-TGGCGCACGCAGTTCGTCCC-3'; and the number of the first and second groups,
donkey-specific primer pair 1:
Donkey-1-F：5´- GATTTCTGGAAAGTCGCAGAAGGG-3´
Donkey-1-R: 5 '-GCTTGCGAAAAGCATTGGGG-3', and/or
Donkey-specific primer pair 2:
Donkey-2-F：5´- CATCCTACTAACTATAGCCGTG-3´
Donkey-2-R：5´-CACTAAGATGGCTGCTATTC-3´ 。
2. a test kit comprising the specific primer of claim 1.
3. The test kit of claim 2, further comprising one of the following reagentsOne or more of: taq enzyme, dNTPs, MgCl2PCR buffer, positive control, blank control.
4. The detection kit according to claim 3, wherein the positive control is horse or donkey genomic DNA and the blank control is double distilled water.
5. The detection method of horse, donkey and mule leather comprises the following steps:
1) extracting hide genome DNA;
2) and (3) PCR amplification: performing double PCR amplification by using the horse-specific primer pair and the donkey-specific primer pair 1 as claimed in claim 1, and taking horse and donkey DNAs as positive controls and double distilled water as a blank control respectively;
3) detecting and judging the result of the PCR amplification product, and judging that the sample is horse hide when the PCR reaction product of the detected sample is an amplification band of 166 bp; when the PCR reaction product of the detected sample is an amplification band of 102bp, judging that the sample is donkey skin; when the PCR reaction product of the detected sample has two amplification bands of 166bp and 102bp respectively, judging that the sample is a mule skin; when the PCR reaction product of the detection sample has no amplification band, judging that the sample is not any one of horse, donkey and mule.
6. The method for identifying the source components of horse and donkey in donkey-hide gelatin comprises the following steps:
1) extracting DNA of a donkey-hide gelatin sample;
2) and (3) PCR amplification: performing double PCR amplification by using the horse-specific primer pair and the donkey-specific primer pair 2 as claimed in claim 1, and taking horse and donkey DNAs as positive controls and double distilled water as a blank control respectively;
3) detecting and judging the result of the PCR amplification product, and judging that the sample only contains equine derived components when the PCR reaction product of the detected sample is an amplification band of 166 bp; when the PCR reaction product of the detected sample is a 130bp amplification band, judging that the sample only contains donkey-derived components; when the PCR reaction product of the detected sample has two amplification bands of 166bp and 130bp respectively, judging that the sample contains both equine-derived components and donkey-derived components; when the PCR reaction product of the detected sample has no amplification band, the sample is judged to contain no horse and donkey-derived components.
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| CN110964837B (en) * | 2019-12-19 | 2022-06-21 | 内蒙古农业大学 | Primer group and detection kit for detecting horse, donkey, horse mule and donkey mule-derived components |
| CN113755607B (en) * | 2021-09-15 | 2023-09-26 | 华中农业大学 | Universal primer for specifically identifying donkey, horse and mule source components in donkey-hide gelatin and raw materials thereof, detection method and application |
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