Kit for rapidly identifying donkey and horse-derived components and donkey and horse-derived components in product and application of kit
阅读说明：本技术 一种快速鉴定驴、马源成分及制品中驴、马源成分的试剂盒及其应用 (Kit for rapidly identifying donkey and horse-derived components and donkey and horse-derived components in product and application of kit ) 是由 齐鹏飞 张伟 朱曼玲 张燕 王怀中 黄迪海 于 2020-07-21 设计创作，主要内容包括：本发明提供一种快速鉴定驴源成分及其制品中驴源成分的试剂盒,属于动物种源性成分分子检测技术领域。试剂盒包括SEQ-ID-No.1～3所示引物、探针、RAA荧光通用反应试剂、反应缓冲液、以及阳性和阴性质控品。此外还包括马的特异性引物及探针,其核苷酸序列如SEQ-ID-No.4～6所示。本发明采用RAA荧光法在30-42℃下进行等温扩增,5-20min完成检测,可判定样品中是否含有驴源成分,以及是否掺有马源成分。本发明的试剂盒操作简单,可以为肉类、皮张中驴源成分的分子鉴定及其中掺假成分的检测提供快速、灵敏、准确的检测方法；配合小型便携式仪器,既适用于实验室大规模检测,也适合基层和现场的快速检测,具有良好的应用前景。(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.)
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.
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.