阅读说明：本技术 基于miRNA的山羊中β2-受体激动剂检测引物及检测方法 (MiRNA-based β in goats2Primer and method for detecting receptor agonist ) 是由 杨曙明 赵璐瑶 王济世 薛佳俐 刘晓夏 刘瑞 刘倩 王彦云 赵汝婷 于 2020-01-21 设计创作，主要内容包括：本发明公开了一种基于miRNA的山羊β<Sub>2</Sub>-受体激动剂检测引物及检测方法。本发明提供了引物组合,由序列表的序列1至序列10所示的单链DNA分子组成。本发明还建立了山羊中β<Sub>2</Sub>-受体激动剂的有效监测方法。本发明在检测山羊中β<Sub>2</Sub>-受体激动剂中的应用中有广阔的前景。(The invention discloses a goat β based on miRNA 2 The invention provides a primer combination which consists of single-stranded DNA molecules shown in a sequence 1 to a sequence 10 of a sequence table, and the invention also establishes β in goats 2 β in detecting goat 2 The application of the receptor agonist has wide prospect.)

1. A primer combination comprising a primer A, a primer B, a primer C, a primer D, a primer E, a primer F, a primer G, a primer H, a primer I and a primer J;

the primer A is a single-stranded DNA molecule shown in a sequence 1 of a sequence table;

the primer B is a single-stranded DNA molecule shown in a sequence 2 of a sequence table;

the primer C is a single-stranded DNA molecule shown in a sequence 3 of a sequence table;

the primer D is a single-stranded DNA molecule shown in a sequence 4 of the sequence table;

the primer E is a single-stranded DNA molecule shown in a sequence 5 of a sequence table;

the primer F is a single-stranded DNA molecule shown in a sequence 6 of a sequence table;

the primer G is a single-stranded DNA molecule shown in a sequence 7 of a sequence table;

the primer H is a single-stranded DNA molecule shown in a sequence 8 of a sequence table;

the primer I is a single-stranded DNA molecule shown in a sequence 9 of a sequence table;

the primer J is a single-stranded DNA molecule shown in a sequence 10 of a sequence table.

2. The primer combination of claim 1, wherein: the primer combination also comprises a primer pair A;

the primer pair A consists of a primer F and a primer R;

the primer F is a single-stranded DNA molecule shown in a sequence 11 of a sequence table;

the primer R is a single-stranded DNA molecule shown as a sequence 12 in a sequence table.

3. The primer combination of claim 1 or 2, which is any one of the following (a1) - (a 4):

(a1) detection β₂-a receptor agonist;

(a2) prepared for detection of β₂-a product of receptor agonists;

(a3) identification or auxiliary identification of β in test sample₂-a receptor agonist;

(a4) is prepared for identifying or assisting in identifying whether β is contained in the sample to be tested₂-a product of receptor agonists.

4. Product comprising a combination of primers according to claim 1 or 2, for use as (b1) or (b 2):

(b1) detection β₂-a receptor agonist;

(b2) identification or auxiliary identification of β in test sample₂-a receptor agonist.

5. The product of claim 4, wherein: the product also comprises a miRNA cDNA first strand synthesis kit and a miRNA fluorescent quantitative detection kit.

6. Identification or auxiliary identification of β in test sample₂-a method of receptor agonism comprising the steps of:

(1) extracting miRNA of a sample to be detected, and completing synthesis of a miRNA cDNA first chain by adopting a miRNAcDNA first chain synthesis kit;

(2) performing fluorescence real-time quantitative PCR detection on the template by respectively using the 10 primers and the miRNA fluorescent quantitative detection kit in claim 1 by using the miRNA cDNA first strand obtained in the step (1) as a template to obtain △ ct values of 10 reaction systems, which are sequentially marked as x1, x2, x3, x4, x5, x6, x7, x8, x9 and x 10;

the x1 is the difference value of the Ct value obtained by carrying out fluorescence real-time quantitative PCR detection on the template by adopting the primer A and the miRNA fluorescence quantitative detection kit and the Ct value of a control reaction system;

the x2 is the difference value of the Ct value obtained by carrying out fluorescence real-time quantitative PCR detection on the template by adopting the primer B and the miRNA fluorescence quantitative detection kit and the Ct value of a control reaction system;

the x3 is the difference value of the Ct value obtained by carrying out fluorescence real-time quantitative PCR detection on the template by adopting the primer C and the miRNA fluorescence quantitative detection kit and the Ct value of a control reaction system;

the x4 is the difference value of the Ct value obtained by carrying out fluorescence real-time quantitative PCR detection on the template by adopting the primer D and the miRNA fluorescence quantitative detection kit and the Ct value of a control reaction system;

the x5 is the difference value of the Ct value obtained by carrying out fluorescence real-time quantitative PCR detection on the template by adopting the primer E and the miRNA fluorescence quantitative detection kit and the Ct value of a control reaction system;

the x6 is the difference value of the Ct value obtained by carrying out fluorescence real-time quantitative PCR detection on the template by adopting the primer F and the miRNA fluorescence quantitative detection kit and the Ct value of a control reaction system;

the x7 is the difference value of the Ct value obtained by carrying out fluorescence real-time quantitative PCR detection on the template by adopting a primer G and a miRNA fluorescence quantitative detection kit and the Ct value of a control reaction system;

the x8 is the difference value of the Ct value obtained by carrying out fluorescence real-time quantitative PCR detection on the template by adopting a primer H and a miRNA fluorescence quantitative detection kit and the Ct value of a control reaction system;

the x9 is the difference value of the Ct value obtained by carrying out fluorescence real-time quantitative PCR detection on the template by adopting the primer I and the miRNA fluorescence quantitative detection kit and the Ct value of a control reaction system;

the x10 is the difference value of the Ct value obtained by carrying out fluorescence real-time quantitative PCR detection on the template by adopting a primer J and a miRNA fluorescence quantitative detection kit and the Ct value of a control reaction system;

the Ct value of the control reaction system is the Ct value obtained by carrying out fluorescence real-time quantitative PCR detection on the A pair of templates by adopting the primer in claim 2;

(3) substituting x1-x10 obtained in the step (2) into the following formula 1 and formula 2,

equation 1:

Y1＝23.629x1-196.449x2-104.946x3+60.133x4+369.088x5+19.466x6+12.09x7+54.674x8+133.242x9+8.04x10-3428.62。

equation 2:

Y2＝23.181x1-215.131x2-108.599x3+65.817x4+390.8x5+20.088x6+20.98x7+59.853x8+142.476x9+11.898x10-3992.44；

if Y1 is more than or equal to Y2, the sample does not contain β₂Receptor agonists, if Y1 < Y2, the sample contains β₂-a receptor agonist.

7. The use according to claim 2, or the kit according to claim 4, or the method according to claim 6, wherein β is the compound₂The receptor agonist is ractopamine and/or clenbuterol hydrochloride.

8. The use or kit or method of claim 7, wherein: the sample to be detected is animal tissue.

9. The use or kit or method according to claim 8, wherein: the animal tissue is animal muscle tissue.

10. The use or kit or method according to claim 9, wherein: the animal is a goat.

Technical Field

The invention relates to miRNA-based β in goat₂A receptor stimulant detection primer and a detection method.

Background

In recent years, problems of diseased animal products, drug residues, artificially added prohibited articles and the like have caused great harm to human health, and whether the quality safety of animal products is a sensitive problem concerned by consumers or not has become a hotspot and focus of international and domestic social concerns.

β₂The receptor agonists are a class of synthetic drugs including cimaterol, pentaerythrite, tulobuterol, sibutrol, brefelone, mabuterol, terbutaline, chlorpropaline, ractopamine, clenbuterol, salbutamol, bambuterol, ritodrine, clenbuterol, zilpaterol, mcocarpus et al β₂Receptor agonists are mainly used for the prevention and treatment of bronchial asthma and bronchospasm in humans and animals, but when used in amounts exceeding therapeutic doses, they promote protein synthesis and fat breakdown in animals, and are therefore commonly used as animal feed additives for increasing lean meat percentage in farm animals, β₂The use of receptor agonists causes residues in animal products, posing a threat to human health.

Although the current national standard and related laws and regulations have been well established, β is prohibited from being added into animal feed and drinking water₂Receptor agonists, but are still frequently forbidden, and currently mainstream drug residue analysis methods such as instrumental analysis, immunoassay, sensor and chip analysis, etc. are based on detection of known drugs, which results in only finding new β₂Receptor agonists only then can corresponding assays be established, with extremely low response efficiency and difficulty to cope with the current severe situation, and therefore, suitable β has been sought₂The receptor agonist detection marker and the establishment of the detection method can discover the known drug types in animal products and the novel drugs in the family, and are important for improving the supervision efficiency and early detection and early treatment.

Disclosure of Invention

The invention aims to provide a goat β based on miRNA₂A receptor stimulant detection primer and a detection method.

In the first aspect, the invention firstly protects a primer combination, which comprises a primer A, a primer B, a primer C, a primer D, a primer E, a primer F, a primer G, a primer H, a primer I and a primer J;

the primer A is a single-stranded DNA molecule shown in a sequence 1 of a sequence table;

the primer B is a single-stranded DNA molecule shown in a sequence 2 of a sequence table;

the primer C is a single-stranded DNA molecule shown in a sequence 3 of a sequence table;

the primer D is a single-stranded DNA molecule shown in a sequence 4 of the sequence table;

the primer E is a single-stranded DNA molecule shown in a sequence 5 of a sequence table;

the primer F is a single-stranded DNA molecule shown in a sequence 6 of a sequence table;

the primer G is a single-stranded DNA molecule shown in a sequence 7 of a sequence table;

the primer H is a single-stranded DNA molecule shown in a sequence 8 of a sequence table;

the primer I is a single-stranded DNA molecule shown in a sequence 9 of a sequence table;

the primer J is a single-stranded DNA molecule shown in a sequence 10 of a sequence table.

The primer combination can also comprise a primer pair A;

the primer pair A consists of a primer F and a primer R;

the primer F is a single-stranded DNA molecule shown in a sequence 11 of a sequence table;

the primer R is a single-stranded DNA molecule shown as a sequence 12 in a sequence table.

The application of the primer combination is any one of the following (a1) - (a 4):

(a1) detection β₂-a receptor agonist;

(a2) prepared for detection of β₂-a product of receptor agonists;

(a3) identification or auxiliary identification of β in test sample₂-a receptor agonist;

(a4) is prepared for identifying or assisting in identifying whether β is contained in the sample to be tested₂-a product of receptor agonists.

In a second aspect, the invention also protects the application of the primer combination, which is any one of the following (a1) - (a 4):

(a1) detection β₂-a receptor agonist;

(a2) prepared for detection of β₂-a product of receptor agonists;

(a3) identification or auxiliary identification of β in test sample₂-a receptor agonist;

(a4) is prepared for identifying or assisting in identifying whether β is contained in the sample to be tested₂-a product of receptor agonists.

In a third aspect, the invention also protects a product containing the primer combination, and the application of the product is as follows (b1) or (b 2):

(b1) detection β₂-a receptor agonist;

(b2) identification or auxiliary identification of β in test sample₂-a receptor agonist.

The product also comprises a miRNA cDNA first strand synthesis kit and a miRNA fluorescent quantitative detection kit. The miRNAcDNA first strand synthesis kit and the miRNA fluorescent quantitative detection kit are matched products, and can be used together with the primers to realize the synthesis and fluorescent quantitative detection of the miRNAcDNA first strand. The mirnaccat dna first strand synthesis kit may specifically be a miRcute enhanced mirnaccat dna first strand synthesis kit (TIANGEN, beijing, china). The miRNA fluorescent quantitative detection kit can be a miRcute enhanced miRNA fluorescent quantitative detection kit (TIANGEN, Beijing, China).

In a fourth aspect, the invention also protects and identifies or assists in identifying whether β is contained in the sample to be tested₂-a method of receptor agonism comprising the steps of:

(1) extracting miRNA of a sample to be detected, and completing synthesis of a miRNA cDNA first chain by adopting a miRNAcDNA first chain synthesis kit;

(2) performing fluorescent real-time quantitative PCR detection on the template by respectively using the 10 primers (from the primer A to the primer J) and the miRNA fluorescent quantitative detection kit by using the first chain of the mirnacDNA obtained in the step (1) as a template to obtain △ ct values of 10 reaction systems, which are sequentially marked as x1, x2, x3, x4, x5, x6, x7, x8, x9 and x 10;

the x1 is the difference value of the Ct value obtained by carrying out fluorescence real-time quantitative PCR detection on the template by adopting the primer A and the miRNA fluorescence quantitative detection kit and the Ct value of a control reaction system;

the x2 is the difference value of the Ct value obtained by carrying out fluorescence real-time quantitative PCR detection on the template by adopting the primer B and the miRNA fluorescence quantitative detection kit and the Ct value of a control reaction system;

the x3 is the difference value of the Ct value obtained by carrying out fluorescence real-time quantitative PCR detection on the template by adopting the primer C and the miRNA fluorescence quantitative detection kit and the Ct value of a control reaction system;

the x4 is the difference value of the Ct value obtained by carrying out fluorescence real-time quantitative PCR detection on the template by adopting the primer D and the miRNA fluorescence quantitative detection kit and the Ct value of a control reaction system;

the x5 is the difference value of the Ct value obtained by carrying out fluorescence real-time quantitative PCR detection on the template by adopting the primer E and the miRNA fluorescence quantitative detection kit and the Ct value of a control reaction system;

the x6 is the difference value of the Ct value obtained by carrying out fluorescence real-time quantitative PCR detection on the template by adopting the primer F and the miRNA fluorescence quantitative detection kit and the Ct value of a control reaction system;

the x7 is the difference value of the Ct value obtained by carrying out fluorescence real-time quantitative PCR detection on the template by adopting a primer G and a miRNA fluorescence quantitative detection kit and the Ct value of a control reaction system;

the x8 is the difference value of the Ct value obtained by carrying out fluorescence real-time quantitative PCR detection on the template by adopting a primer H and a miRNA fluorescence quantitative detection kit and the Ct value of a control reaction system;

the x9 is the difference value of the Ct value obtained by carrying out fluorescence real-time quantitative PCR detection on the template by adopting the primer I and the miRNA fluorescence quantitative detection kit and the Ct value of a control reaction system;

the x10 is the difference value of the Ct value obtained by carrying out fluorescence real-time quantitative PCR detection on the template by adopting a primer J and a miRNA fluorescence quantitative detection kit and the Ct value of a control reaction system;

the Ct value of the control reaction system is the Ct value obtained by carrying out fluorescent real-time quantitative PCR detection on the template A by adopting the primer;

(3) substituting x1-x10 obtained in the step (2) into the following formula 1 and formula 2,

equation 1:

Y1＝23.629x1-196.449x2-104.946x3+60.133x4+369.088x5+19.466x6+12.09x7+54.67 4x8+133.242x9+8.04x10-3428.62；

equation 2:

Y2＝23.181x1-215.131x2-108.599x3+65.817x4+390.8x5+20.088x6+20.98x7+59.853x 8+142.476x9+11.898x10-3992.44；

if Y1 is more than or equal to Y2, the sample does not contain β₂Receptor agonists, if Y1 < Y2, the sample contains β₂-a receptor agonist.

β of any of the above₂The receptor agonist may in particular be ractopamine and/or clenbuterol hydrochloride.

Any of the above samples to be tested may be animal tissue.

The animal tissue can be animal muscle tissue.

The animal may be a goat. The goat may be a cashmere goat.

The invention establishes β in the goat₂β in detecting goat₂The application of the receptor agonist has wide prospect.

Drawings

FIG. 1 shows the analysis results of the cell experiment principle components.

FIG. 2 shows the analysis results of animal experiment principal components.

FIG. 3 is a comparison of the results of sequencing (relative expression fold) and the results of fluorescent quantitative PCR detection (relative expression fold) at a drug concentration of 80. mu.g/kg.

FIG. 4 is a comparison of the results of sequencing (relative expression fold) and the results of fluorescent quantitative PCR detection (relative expression fold) at a drug concentration of 20. mu.g/kg.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Healthy cashmere goats: the Bayan Dan is commercially available.