CN111763714A - Kit for rapidly identifying donkey and horse-derived components and donkey and horse-derived components in product and application of kit - Google Patents
Kit for rapidly identifying donkey and horse-derived components and donkey and horse-derived components in product and application of kit Download PDFInfo
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Abstract
The invention provides a kit for rapidly identifying donkey-derived components and donkey-derived components in products thereof, and belongs to the technical field of molecular detection of animal-derived components. The kit comprises primers shown in SEQ-ID-Nos. 1-3, a probe, an RAA fluorescent universal reaction reagent, a reaction buffer solution and positive and negative quality control substances. In addition, the kit also comprises horse specific primers and probes, and the nucleotide sequences of the primers and probes are shown in SEQ-ID-Nos. 4-6. The method adopts an RAA fluorescence method to perform isothermal amplification at 30-42 ℃, completes detection within 5-20min, and can judge whether the sample contains donkey source components or not and whether horse source components are doped or not. The kit is simple to operate, and can provide a rapid, sensitive and accurate detection method for molecular identification of donkey-derived components in meat and skin and detection of adulterated components in the donkey-derived components; the kit is matched with a small portable instrument, is suitable for large-scale laboratory detection and rapid detection of a basic level and a site, and has good application prospect.
Description
Technical Field
The invention relates to the field of animal molecular biology, in particular to a kit for rapidly identifying donkey and horse-derived components and donkey and horse-derived components in products and application thereof.
Background
The donkey meat enjoys the reputation of 'Tianshanglong meat and overground donkey meat' since ancient times, and the taste and mouthfeel of the donkey meat are excellent. In fact, the nutritive value of the donkey meat is not lower than or even higher than that of the beef, the mutton and the pork. It has the characteristics of high protein, high essential amino acid, high essential fatty acid, low fat, low cholesterol and low calorie, and is rare healthy meat product. The ass meat has rich nutrition and delicious taste, and has good effect of preventing obesity and cardiovascular diseases.
With the rapid development of economy and the improvement of living standard of people, the demands of material and mental consumption are diversified, and the requirements on the quality of livestock products are higher and higher. The donkey meat has delicious meat and unique flavor, and related meat products are deeply loved by consumers, and the demand is increasing day by day. Meanwhile, the quality safety problem of donkey meat and products also becomes the key point of attention of the society and the supervision department. Adulteration of donkey meat and products has become a serious social problem, known as "economic-interest-driven adulteration". In pursuit of interest, some illegal vendors use low-quality horse meat to try out from high-quality donkey meat, and the safety of donkey meat product sources has become one of the most concerned problems for consumers. Therefore, it is necessary to establish a set of rapid, sensitive and accurate detection method for donkey-derived products.
The method of identifying animal products by means of sense and experience has not been able to achieve the purpose of controlling and supervising the adulteration of donkey meat and its processed products. In recent years, DNA having high stability and identity in various tissues has been used as a target for detection of animal-derived components. The method comprises the following steps of adopting the common Polymerase Chain Reaction (PCR) technology and the fluorescent quantitative PCR technology (RT-PCR) to become the mainstream method for identifying the animal-derived components (Caochiphon, florida, Zhang rainbow and the like, donkey or donkey-derived component real-time fluorescent PCR detection method, primers and probes for detection, Chinese patent: the PCR and the RT-PCR have a common defect that the time is long, and the result can be obtained generally within more than 2 hours, so that a detection method for more quickly, sensitively and accurately identifying meat adulteration is an urgent need.
At present, no report is found on a rapid detection method for simultaneously identifying donkey and horse meat derived components. In addition, the phenomenon of adulteration of animal-derived ingredients in donkey hide is also very common. Therefore, establishing a detection method for rapidly identifying donkey and horse source components has great significance for safety supervision of donkey meat food and products.
Disclosure of Invention
The invention aims to provide a primer, a probe or a kit for rapidly identifying donkey-derived components by an RAA fluorescence method, aiming at the defect of long time consumption in the prior art, can realize sample detection within 30min, has the characteristics of rapidness, sensitivity and accuracy, and can meet the requirements of large-scale laboratory detection and rapid field detection.
The invention also aims to provide a rapid detection method for identifying the horse meat adulterated with the donkey-derived product.
In order to achieve the purpose, the donkey genome specific DNA sequence is screened from the aspects of intraspecies consistency and stability, interspecific specificity, copy number and the like, and a donkey specific DNA fragment which does not exist in horses or other animal species or has low homology is screened as a detection target through detection in donkey and horse genome DNA. After a large amount of analysis and experiments, the donkey specific DNA sequence for detection is finally obtained.
The invention firstly provides a primer or a probe for rapidly detecting donkey specific DNA fragments by an RAA fluorescence method, wherein the sequence of the primer or the probe is as follows:
EA-A：5’-CAAGATGCTCCCATAATTGGAACACCTGATG-3’，SEQ-ID-NO.1；
EA-B：5’-GTAGAGCCTTGTGAAGCAGTGTCTGAGAAC-3’，SEQ-ID-NO.2。
EA-C：5’- CCACAACTGACGGAGACCTGTGCACTGGC[FAM-dT]G[THF]C[BHG1-dT]GGACCCACAGAAGC[C3-spacer]-3’；SEQ-ID-NO.3；
the probe is modified by adopting a fluorescence reporter group FAM-dT and a fluorescence quenching group BHG1-dT, and the fluorescence reporter group is modified at a position which is 30bp away from the 5' end base number of the probe sequence; the fluorescence quenching group is modified on the position 15bp away from the 3 'end base number of the probe sequence, 3 bases GCC are arranged between the fluorescence reporting group and the quenching group at intervals, the middle base C is replaced by tetrahydrofuran residue THF, and the 3' end of the probe EA-C is connected with C3-spacer.
Further, the present invention provides a detection kit comprising the above-mentioned specific primer or probe.
Further, the kit also comprises the following primers or probes:
the primer for detecting horse specific DNA fragments by the RAA fluorescence method has the following sequences:
EC-D：5'-CGGCCTCCATTCTCAGTGACAAGGCGGTGAA-3'，SEQ-ID-NO.4；
EC-E：5'-CGCAGGACGCAGTTCTCGAACGTCTTGGGTG-3'，SEQ-ID-NO.5。
EA-F：5’-tctcctcccaccttgtgggtatcccgggc[fam-dt]c[thf]a[bhg1-dt]gcggatgctgccag[c3-spacer]-3’；SEQ-ID-NO.6；
the probe EA-F is modified by adopting a fluorescence reporter group FAM-dT and a fluorescence quenching group BHG1-dT, and the fluorescence reporter group is modified at a position which is 30bp away from the 5' end base number of a probe sequence; the fluorescence quenching group is modified on the position 15bp away from the 3 'end base number of the probe sequence, 3 bases CAA are arranged between the fluorescence reporting group and the quenching group at intervals, the middle base A is replaced by tetrahydrofuran residue THF, and the 3' end of the probe EA-F is connected with C3-spacer.
Further, the kit also comprises one or more of the following reagents: RAA fluorescence method universal reaction reagent, negative and positive quality control product; the positive quality control product is donkey genome DNA template, and the negative quality control product is ddH2O or purified water.
Further, the positive quality control product also comprises an equine genomic DNA template.
Further, the invention provides application of the donkey specific primer or probe or detection kit used in the RAA fluorescence method in donkey-derived component identification or donkey-derived product adulteration detection.
The invention also provides a method for detecting donkey-derived components and horse-derived components in products thereof by using an RAA fluorescence method, which comprises the following steps:
1) preparing a sample genome DNA template;
2) isothermal amplification by RAA fluorescence method: isothermal amplification at 30-42 deg.C for 5-20 min;
A) RAA fluorescence method detection is carried out on a sample by using a primer for detecting donkey-derived components by using RAA fluorescence method and a probe for detecting donkey-derived components by using RAA fluorescence method, and ddH is used as positive control by using donkey genome DNA template2O or purified water as a negative control; obtaining a kit;
B) using the kit of A) to carry out RAA fluorescence detection on a sample, taking one or more of donkey genome DNA template and horse DNA template as positive control, and ddH2O or purified water as a negative control;
3) and (5) analyzing and judging the result.
The invention has the advantages of
1. Rapid detection method for simultaneously identifying donkey and horse meat derived components
The method can complete sample detection within 30min, has the characteristics of rapidness, sensitivity and accuracy, and can meet the requirements of large-scale laboratory detection and field rapid detection. The kit is simple to operate, and can provide a rapid, sensitive and accurate detection method for molecular identification of donkey-derived components in meat and skin and detection of adulterated components in the donkey-derived components; the kit is matched with a small portable instrument, is suitable for large-scale laboratory detection and rapid detection of a basic level and a site, and has good application prospect.
2. The role in food safety supervision
The phenomenon of adulteration of animal-derived ingredients in donkey hide is also very common. Therefore, establishing a detection method for rapidly identifying donkey and horse source components has great significance for safety supervision of donkey meat food and products.
Detailed Description
The following examples are provided to further illustrate the present invention and should not be construed as limiting the invention, as modifications and enhancements may be made thereto without departing from the spirit and spirit of the invention.
EXAMPLE 1 specific detection of donkey-derived Components in samples
1. Preparation and preservation of samples
1.1 sampling
Collecting donkey meat sample 1g, and storing at-20 deg.C for use.
1.2DNA template preparation
The DNA template can be prepared by a conventional extraction method or a commercial extraction-free kit, and the prepared template is stored at 4 ℃ for later use or stored at-20 ℃ for later use.
2. Primer design
The DNA sequence of the primer or probe of the present example is shown in SEQ-ID-NO: 1 to 3.
Donkey specific primer probe designed by the invention
EA-A：5’-CAAGATGCTCCCATAATTGGAACACCTGATG-3’，SEQ-ID-NO.1；
EA-B：5’-GTAGAGCCTTGTGAAGCAGTGTCTGAGAAC-3’，SEQ-ID-NO.2。
EA-C：5’- CCACAACTGACGGAGACCTGTGCACTGGC[FAM-dT]G[THF]C[BHG1-dT]GGACCCACAGAAGC[C3-spacer]-3’；SEQ-ID-NO.3；
3. Selection of fluorescence reporter and fluorescence quencher
The fluorescence detected by the RAA-F1620 fluorescence gene detector produced by Wuxi-Tian-bioscience instruments, Inc. is FAM fluorescence, so that the fluorescence reporter group is selected as FAM and the fluorescence quencher group is selected as BHQ 1.
The fluorescent reporter group can also be selected to be HEX, TET, JOE, VIC, ROX, Cy3 or Cy5 depending on the ability of the instrument to detect fluorescence. The fluorescence quenching group is selected from TAMRA, Eclipse, BHQ2, BHQ3 or DABCYL. However, the fluorescent reporter group is preferably FAM or HEX, and the fluorescent quencher group is preferably BHQ1 or BHQ 2.
The method for modifying the probe preferably includes: the fluorescent reporter group is modified on the position of the probe sequence which is 30bp away from the 5' end base number; the fluorescence quenching group is modified on the position 15bp away from the 3' end base number of the probe sequence, 3 bases GCC are arranged between the fluorescence reporting group and the quenching group, wherein the base C at the middle position is replaced by a tetrahydrofuran residue. The modified probe EA-C is: CCACAACTGACGGAGACCTGTGCACTGGC [ FAM-dT ] G [ THF ] C [ BHG1-dT ] GGACCCACAGAAGC [ C3-spacer ].
4 RAA fluorescence detection
In this example, RAA fluorescent universal reagent was purchased from Jiangsu Qitian gene Biotechnology GmbH, cat # F00001 and was strictly performed according to the instructions. The specific operation steps are described as follows:
1) and (3) switching on a power supply of an instrument (RAA-F1620) for preheating, and setting reaction parameters: the temperature is 39 ℃ and the time is 20 min.
2) The RAA reaction buffer solution was re-dissolved, and the reaction system was configured as shown in Table 3, while negative and positive controls were set.
TABLE 3RAA fluorometric reaction System
| Name (R) | Volume (μ l) |
| RAA Universal reaction buffer | 25 |
| ddH2O | 15.7 |
| EA-F | 2.1 |
| EA-R | 2.1 |
| EA-p | 0.6 |
| Magnesium sulfate | 2.5 |
| DNA template | 2 |
| Total up to | 50 |
3) And (3) fully and uniformly mixing the reaction system obtained in the step 2), and then putting the mixture into an RAA-F1620 for detection.
5 analysis and determination of the detection result
5.1 analysis of control assay results
According to the positive judgment method of the RAA-F1620 instrument, judging the slope value K to be positive if the slope value K is more than or equal to 20; the slope value K is less than 20, and the result is judged to be negative; the positive control rate of inclination K is more than or equal to 20, the negative control rate of inclination K is less than 20, which indicates that the reaction system is normal, and the result judgment can be carried out.
5.2 analysis and determination of the results of sample detection
If the sample slope value K is more than or equal to 20, the sample is judged to be positive, namely the donkey source component is detected in the sample; if the slope value K is less than 20, the sample is judged to be negative, namely the donkey-derived component is not detected in the sample.
Example 2 sensitivity test
The donkey genomic DNA templates with different concentrations of 0.1 ng/. mu.L, 1 ng/. mu.L, 10 ng/. mu.L and 100 ng/. mu.L were used for detection according to the conditions of example 1. The template concentration of 1 ng/. mu.L is amplified, which shows that the amplified fragment of the specific primer has higher sensitivity.
Example 3 primer set for detecting horse-derived component doped in donkey-derived product
The invention relates to detection of donkey and horse-derived components, which is characterized in that RAA fluorescence detection is carried out by using corresponding primer probes of various species to judge whether the horse-derived components are or contain DNA of horses, and further judge whether the horse-derived components are doped.
1 preparation of sample DNA template
The preparation and preservation of DNA templates for each species was as described in example 1 above.
2 design of primers
The DNA sequence of the primer or probe of the present example is shown in SEQ-ID-NO: 4 to 6.
Horse specific primer probe designed by the invention
EC-D：5'-CGGCCTCCATTCTCAGTGACAAGGCGGTGAA-3'，SEQ-ID-NO.4；
EC-E：5'-CGCAGGACGCAGTTCTCGAACGTCTTGGGTG-3'，SEQ-ID-NO.5。
EA-F：5’-tctcctcccaccttgtgggtatcccgggc[fam-dt]c[thf]a[bhg1-dt]gcggatgctgccag[c3-spacer]-3’；SEQ-ID-NO.6；
3. Selection of fluorescence reporter and fluorescence quencher
Selection of fluorescent reporter and quencher the modified probes were as described in example 1 below: EC-F: TCTCCTCCCACCTTGTGGGTATCCCGGGC [ FAM-dT ] C [ THF ] A [ BHG1-dT ] GCGGATGCTGCCAG [ C3-spacer ] -3'.
4 RAA fluorescence detection
The detection method of this example is the same as that described in example 1.
5 analysis and determination of the detection result
The results show that: the specific primers can only amplify DNA templates of corresponding species, and the specificity is good.
Example 4A kit for detecting donkey and equine derived components
The kit comprises the following components:
primers and probes (donkey primer, horse primer and probe SEQIDNo.1-6 and one copy each);
RAA fluorescence method general buffer solution;
positive quality control substances (one part of each donkey genome DNA template and one part of each horse genome DNA template);
ddH20。
the method of use of the kit is as described in example 1 or example 3 above.
Example 5 application of kit for detecting donkey and equine derived components
1. The preparation of the sample DNA template, the RAA fluorescence detection and the analysis and determination of the result are the same as those described in example 1 or example 3.
2. The detection method is applied to detection of donkey meat products on the market, donkey meat products such as donkey meat burnt meat and the like of different merchants are purchased, basic information is recorded, a DNA template is prepared, RAA fluorescence method detection is carried out, and real components in the donkey meat products are detected.
3. The kit provided by the invention is used for detecting 13 groups of samples obtained on the market, the result shows that only 6 of the samples are qualified products, the PCR detection result of 7 groups of samples is different from that of a food label, and the detection result shows that 7 products have the phenomenon of horse meat adulteration.
The method disclosed by the invention has a wide prospect when being applied to detection of donkey-derived products. The detection kit can be used for detecting donkey meat or donkey meat products, and can also be widely applied to the related fields of skin products, traditional Chinese medicine component detection and the like.
Sequence listing
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<120> kit for rapidly identifying donkey and horse-derived components and donkey and horse-derived components in products and application thereof
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Claims (10)
1. A primer for detecting donkey-derived components by an RAA fluorescence method is characterized by comprising the following sequences:
EA-A：5’-CAAGATGCTCCCATAATTGGAACACCTGATG-3’，SEQ-ID-NO.1；
EA-B：5’-GTAGAGCCTTGTGAAGCAGTGTCTGAGAAC-3’，SEQ-ID-NO.2。
2. a probe for detecting donkey-derived components by an RAA fluorescence method is characterized by comprising the following sequences:
EA-C：5’- CCACAACTGACGGAGACCTGTGCACTGGC[FAM-dT]G[THF]C[BHG1-dT]GGACCCACAGAAGC[C3-spacer]-3’；SEQ-ID-NO.3；
the probe EA-C is modified by adopting a fluorescence reporter group FAM-dT and a fluorescence quenching group BHG1-dT, and the fluorescence reporter group is modified at a position which is 30bp away from the 5' end base number of a probe sequence; the fluorescence quenching group is modified on the position 15bp away from the 3 'end base number of the probe sequence, 3 bases GCC are arranged between the fluorescence reporting group and the quenching group at intervals, the middle base C is replaced by tetrahydrofuran residue THF, and the 3' end of the probe EA-C is connected with C3-spacer.
3. A kit comprising the primer according to claim 1 and the probe according to claim 2.
4. The detection kit according to claim 3, further comprising the following primers: and (3) amplifying the equine genomic DNA to generate primer pairs of equine specific amplified fragments:
EC-D：5'-CGGCCTCCATTCTCAGTGACAAGGCGGTGAA-3'，SEQ-ID-NO.4；
EC-E：5'-CGCAGGACGCAGTTCTCGAACGTCTTGGGTG-3'，SEQ-ID-NO.5。
5. the detection kit according to claim 3, further comprising the following probes: horse source component probe:
EA-F：5’-tctcctcccaccttgtgggtatcccgggc[fam-dt]c[thf]a[bhg1-dt]gcggatgctgccag[c3-spacer]-3’；SEQ-ID-NO.6；
the probe EA-F is modified by adopting a fluorescence reporter group FAM-dT and a fluorescence quenching group BHG1-dT, and the fluorescence reporter group is modified at a position which is 30bp away from the 5' end base number of a probe sequence; the fluorescence quenching group is modified on the position 15bp away from the 3 'end base number of the probe sequence, 3 bases CAA are arranged between the fluorescence reporting group and the quenching group at intervals, the middle base A is replaced by tetrahydrofuran residue THF, and the 3' end of the probe EA-F is connected with C3-spacer.
6. The detection kit according to any one of claims 3 to 5, further comprising one or more of the following reagents: RAA fluorescence method universal reaction reagent, negative and positive quality control product; the positive quality control product is donkey genome DNA template, and the negative quality control product is ddH2O or purified water.
7. The kit of claim 6, wherein the positive control further comprises an equine genomic DNA template.
8. Use of the kit of any one of claims 3 to 7 in donkey-derived component identification or donkey-derived product adulteration detection.
9. Use of the kit of claim 7 for simultaneous detection of donkey, equine derived components or donkey derived product adulterated horse meat.
10. A detection method for identifying donkey-derived components and horse-derived components in products thereof by an RAA fluorescence method is characterized by comprising the following steps:
1) preparing the primer of claim 4, the probe of claim 5, and the reagent of claim 7;
2) isothermal amplification by RAA fluorescence method: isothermal amplification at 30-42 deg.C for 5-20 min;
3) and (5) analyzing and judging the result.
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