CN108315440B - MiRNA marker for distinguishing buffalo milk from cow milk and application thereof - Google Patents

Abstract

The invention discloses a miRNA marker for distinguishing buffalo milk from cow milk and application thereof, and relates to the technical field of biology. The miRNA marker is miR-2285t, and the sequence of the miRNA marker is shown in SEQ ID NO: 1 is shown. The result of the invention shows that the miR-2285t gene has extremely obvious high expression in the milk exosome of the dairy cow. The miR-2285t can be applied to preparation of a detection reagent for distinguishing buffalo milk from cow milk, and provides a new identification method for distinguishing buffalo milk from cow milk at a molecular level in agricultural production.

Description

MiRNA marker for distinguishing buffalo milk from cow milk and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a miRNA marker for distinguishing buffalo milk from cow milk and application thereof.

Background

miRNA is also called microRNA, and is a single-stranded non-coding RNA composed of about 22 nucleotides, which mainly utilizes direct cleavage of messenger RNA or indirect inhibition of translation to regulate expression of target genes at post-transcriptional level. The miRNA has different expression modes in different stages of ontogeny, such as cell differentiation, proliferation, apoptosis and the like, and different tissues, and shows that the miRNA plays an important role in regulation and control in development and differentiation. Through research on tissue miRNA expression profiles, the miRNA has strong cell, tissue or disease specificity, and the specifically expressed miNRA is not only the basis of function research but also a marker of different cell, tissue, organ or disease models.

Milk is an important source of nutrition for all mammalian newborns, and contains many biologically active substances, such as: cytokines, growth factors and hormones, which regulate the development of the newborn. The fat, protein and lactose contents of buffalo milk are several times of those of ordinary milk, and the mineral and vitamin contents are also several tens times of those of cow milk and human milk. It has rich fragrance, low cholesterol, rich vitamins and trace elements, especially high casein content, and can be used for deep processing of high-quality dairy products. As a high-grade nutritional food, buffalo milk products are gradually becoming a new favorite for people to consume. The exosome is a small vesicle on a membrane with the diameter of 30-100 nm secreted by various cells, tissues or organisms under normal or pathological states, is in a spherical structure and contains hydrophilic water-soluble and limited lipid bilayer components. Research shows that besides epithelial cells, B cells, T cells and other cells can secrete exosomes, exosomes also exist in various physiological fluids such as plasma, urine, saliva, milk and the like, and the exosomes present pleiotropic biological functions including immunoregulatory activity, cell-to-cell communication regulation and signal transmission functions. miRNA is used as a non-coding small molecular RNA and can be wrapped in exosomes of milk. Studies have shown that mirnas are found in milk exosomes of different species, and that there are significant differences in composition and abundance of mirnas.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention mainly aims to provide the miRNA marker for distinguishing buffalo milk from cow milk.

The invention also aims to provide application of the miRNA marker.

It is still another object of the present invention to provide a test kit for distinguishing buffalo from cow's milk.

The invention provides a detection method for distinguishing buffalo milk from cow milk by using a buffalo and cow milk exosome miR-2285t and application thereof, and provides a new identification method for distinguishing buffalo milk from cow milk at a molecular level in agricultural production.

The purpose of the invention is realized by the following technical scheme:

the invention provides a miRNA marker for distinguishing buffalo milk and cow milk, wherein the miRNA marker is miR-2285t, and the sequence of the miRNA marker is as follows: 5'-agaaucuggaugaacuuuuugg-3', as shown in SEQ ID NO: 1 is shown.

The invention also provides a primer of the miRNA marker, and the sequence is as follows: (5'-3')

The upstream primer sequence of miR-2285t is as follows: ggccagaatctggatga, SEQ ID NO: 3;

the sequence of the miR-2285t downstream primer is as follows: gtgcagggtccgaggt, SEQ ID NO: 4.

the invention also provides application of the miRNA marker in distinguishing buffalo milk from cow milk, and particularly application in preparing a detection kit for distinguishing buffalo milk from cow milk.

Preferably, the miRNA marker primer is applied to distinguishing buffalo from milk, and particularly applied to preparing a kit for distinguishing buffalo from milk of dairy cow.

The invention also provides a detection kit for distinguishing buffalo milk from cow milk, which comprises a primer and/or a probe aiming at the miR-2285 t.

Further, when a stem-loop method is used for detecting the miR-2285t marker, the sequence of the reverse transcription primer is SEQ ID NO: 2. the sequence of the upstream primer is SEQ ID NO: 3 and the sequence of the downstream primer is SEQ ID NO: 4.

further, when the stem-loop method is used for detecting the miR-2285t marker, a fluorescent dye method can be adopted.

Specifically, the kit comprises a reverse transcription reagent for the miR-2285t, a reverse transcription primer, a specific primer, an internal reference primer and a fluorescent quantitative PCR reaction solution;

preferably, a miR-2285t specific probe is also included.

Wherein, the reverse transcription primer sequence aiming at the miR-2285t is SEQ ID NO: 2;

the specific primer comprises an upstream primer and a downstream primer, wherein the sequence of the upstream primer is SEQ ID NO: 3, the sequence of the downstream primer is SEQ ID NO: 4;

the internal reference primers comprise an upstream primer and a downstream primer of a GAPDH internal reference gene, and the sequence of the upstream primer is SEQ ID NO: 5, the sequence of the downstream primer is SEQ ID NO: 6.

the kit is applied to quantitative detection of miR-2285t in milk, and can be used for distinguishing buffalo from milk.

The invention also provides a detection method for distinguishing buffalo from milk, which comprises the following steps:

the step of detecting the expression level of miR-2285t comprises the following steps:

1) extracting total RNA in exosomes of buffalo and milk samples;

2) carrying out miR-2285t reverse transcription to cDNA by a reverse transcription primer under the action of reverse transcriptase;

3) carrying out amplification detection on the cDNA obtained in the step (2) and the reference gene on a fluorescent real-time quantitative PCR instrument;

4) analysis by dissolution Curve, 2^-ΔΔCtThe method is used for relative quantification.

Compared with the prior art, the invention has the following advantages and effects:

the invention provides a miRNA marker for distinguishing buffalo milk and cow milk, which is named miR-2285t and has the sequence as follows: 5'-agaaucuggaugaacuuuuugg-3', as shown in SEQ ID NO: 1 is shown. The result of the invention shows that the miR-2285t gene has extremely obvious high expression in the milk exosome of the dairy cow. The miR-2285t can be applied to preparation of a detection reagent for distinguishing buffalo milk from cow milk, and provides a new identification method for distinguishing buffalo milk from cow milk at a molecular level in agricultural production.

Drawings

FIG. 1 shows that the relative expression amount of miR-2285t in milk is detected by fluorescent quantitative PCR.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Those of ordinary skill in the art will understand that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.

Example 1 separation of milk exosomes from buffalo and cow

1) Thawing a milk sample stored in a refrigerator at the temperature of-80 ℃, centrifuging at the temperature of 4 ℃ for 30min at 2000g, collecting a supernatant, and removing milk fat protein and mammary gland cell fragments;

2) centrifuging the collected supernatant again at 12000g at 4 deg.C for 30min to collect supernatant, and removing milk fat protein, casein and other cell debris;

3) the finally collected supernatant was then subjected to ultracentrifugation at 50000rpm at 4 ℃ for 2 hours to obtain milk exosomes.

Example 2 exosome RNA extraction

Extracting total RNA in a milk exosome sample by adopting a Trizol one-step method, which comprises the following steps:

1) placing a milk exosome sample obtained by ultra-high speed centrifugation into a 2mL centrifuge tube added with 1mL Trizol reagent for rapid homogenization for 20s, and properly increasing the homogenization time according to the homogenization degree;

2) the homogenate was transferred to a 1.5mL centrifuge tube and centrifuged at 12000rpm for 15min at 4 ℃ to remove the tissue mass that was not homogenized completely.

3) The supernatant was transferred to another new 1.5mL centrifuge tube, 1/5 volume of about 0.2mL chloroform was added, the mixture was shaken vigorously for 30s to mix the samples well, allowed to stand at room temperature for 5min, and centrifuged at 12000rpm for 15min at 4 ℃.

4) The upper aqueous phase was carefully transferred to a new 1.5mL centrifuge tube, 0.5mL isopropanol was added, mixed well and precipitated at-80 ℃ overnight.

5) Centrifuge at 12000rpm at 4 deg.C for 15min, remove supernatant, add 1mL 75% ethanol. And blowing and washing twice. Centrifugation was carried out at 12000rpm and 4 ℃. And drying the RNA precipitate in a super clean bench for 5-10 min to ensure that the alcohol is completely volatilized, adding a proper amount of DEPC (diethyl phthalate) treated water into the dried RNA sample, and gently blowing and beating the mixture by sterilization until the RNA is completely dissolved.

Example 3

The miR-2285t screening method comprises the following steps:

1) selecting mid-lactation milk of the Moraxel buffalo and the Holstein cow as research objects;

2) respectively separating exosomes (exosomes) in buffalo and dairy cow milk;

3) identifying total miRNA genes of the milk exosomes of buffalo and dairy cow by a high-throughput sequencing technology;

4) performing differential expression analysis on total miRNA genes of the exosomes of the buffalo and the milk by bioinformatics;

5) the miR-2285t gene has extremely low expression in buffalo milk and extremely high expression in milk cow milk.

Therefore, miR-2285t can be used as a miRNA marker for distinguishing buffalo milk from cow milk.

Wherein, the Moraxella buffalo and the Holstein cow are disclosed in the document "determination of enzyme activity in serums of Areca Jiangshou buffalo, Moraxella buffalo and Holstein cow" [ J ]. Heilongjiang animal husbandry, 2016(5):212 and 214 ".

Example 4 fluorescent quantitative PCR detection of miR-2285t expression in buffalo and milk

Reagent:

a reverse transcription kit: PrimeScript^TMRT reagent Kit with gDNA Eraser(Perfect Real Time)，#RR047Q，Takara；

Quantitative PCR reagent:

Fast qPCR Mix，#RR430A，Takara。

related experimental article RNase removal treatment:

1) washing all glassware with DEPC before application, soaking at 120 deg.C under high pressure for 20min, and oven drying at 180 deg.C for more than 2 hr.

2) Plastic vessels (such as: EP tube/gun head) is soaked in 0.1% DEPC water overnight before use, and then liquid is drained, and the liquid is baked in an oven for standby after being heated at 120 ℃ and under high pressure for 20 min.

Synthesis of miR-2285t gene in milk

1) Milk total RNA is qualifiedImmediately thereafter, synthesis of miR-2285t gene was carried out. Reverse transcription kit (PrimeScript) from Dalibao Bio Inc. was used for reverse transcription^TMRT reagent Kit with gDNA Eraser), first prepare a genome-free reaction system in a 0.2mL PCR tube according to the following table, the total reaction system is 10. mu.L, and then place for 2 minutes at 42 ℃.

5×gDNA Eraser Buffer	2μL
		gDNA Erase	1μL
Total RNA	1000ng
		RNase-free dH2O	Adding to 10 μ L
Reaction system	10μL

2) Adding 5 XPrimeScript Buffer 4. mu.L, PrimeScript RT Enzyme Mix I1. mu.L, miR-2285t reverse transcription primer 0.5. mu.L, internal reference gene downstream primer 0.5. mu.L and RNase-free dH into the reaction solution in sequence₂O4. mu.L, for a total of 20. mu.L. The reaction was terminated at 37 ℃ for 30 minutes and at 85 ℃ for 15 seconds. Then 180. mu.L of RNase free dH was added₂O was diluted to 200. mu.L and stored at-20 ℃ for subsequent testing.

Quantitative analysis

1) Dalianbao bio-Co for this test

Fast qPCR Mix reagents were subjected to real-time quantitative PCR analysis. 3 replicates were set for each sample and 3 negative controls were set for each pair of primers. GAPDH is used as an internal reference gene, and the reaction system and conditions are as follows:

2×SYBR Premix Ex Taq II	12.5μL
		upstream primer (10. mu.M)	1μL
Downstream primer (10. mu.M)	1μL
		cDNA	1.5μL
RNase-free dH2O	Up to 25μL
		Reaction system	25μL

The reverse transcription primer sequence aiming at miR-2285t is SEQ ID NO: 2;

the specific primer aiming at miR-2285t comprises an upstream primer and a downstream primer, wherein the sequence of the upstream primer is SEQ ID NO: 3, the sequence of the downstream primer is SEQ ID NO: 4;

an upstream primer and a downstream primer of the GAPDH reference gene, wherein the sequence of the upstream primer is SEQ ID NO: 5, the sequence of the downstream primer is SEQ ID NO: 6.

2) real-time quantitative PCR reaction conditions: first, pre-denaturation at 95 ℃ for 30 seconds; 40 cycles of denaturation at 95 ℃ for 5 seconds and annealing at 60 ℃ for 30 seconds; after the circulation, the dissolution curve was recorded at 65-95 ℃.

3) Abundance of Gene expression 2^-ΔΔCtThe method shows that the relative expression result of miR-2285t in buffalo and cow milk is shown in figure 1. According to the figure 1, the expression quantity of miR-2285t in buffalo milk is extremely lower than that of cow milk, and the miR-2285t can be used as a marker for distinguishing the buffalo milk from the cow milk.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

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Claims (7)

1. The application of the miRNA marker for distinguishing the buffalo milk from the cow milk in distinguishing the buffalo milk from the cow milk is characterized in that:

the miRNA marker is miR-2285t, and the sequence of the miRNA marker is shown in SEQ ID NO: 1 is shown in the specification;

the buffalo is a Mora buffalo; the dairy cow is a Holstein dairy cow.

2. Use of a detection primer for the miRNA marker as defined in claim 1 for distinguishing between buffalo and cow's milk, characterized in that: the detection primer is as follows:

the upstream primer sequence of miR-2285t is as follows: 5'-ggccagaatctggatga-3', respectively;

the sequence of the miR-2285t downstream primer is as follows: 5'-gtgcagggtccgaggt-3', respectively;

the buffalo is a Mora buffalo; the dairy cow is a Holstein dairy cow.

3. Use of a test kit for distinguishing between buffalo and cow's milk in distinguishing between buffalo and cow's milk, characterized in that the kit comprises the detection primer of claim 2;

the buffalo is a Mora buffalo; the dairy cow is a Holstein dairy cow.

4. Use according to claim 3, characterized in that:

the detection kit comprises a reverse transcription reagent, a reverse transcription primer, a specific primer, an internal reference primer and a fluorescent quantitative PCR reaction solution aiming at the miR-2285t in claim 1.

5. Use according to claim 4, characterized in that:

the reverse transcription primer sequence aiming at the miR-2285t is SEQ ID NO: 2.

6. use according to claim 4, characterized in that:

the internal reference primers comprise an upstream primer and a downstream primer of a GAPDH internal reference gene, and the sequence of the upstream primer is SEQ ID NO: 5, the sequence of the downstream primer is SEQ ID NO: 6.

7. a test method for distinguishing buffalo from dairy milk comprising the steps of:

1) extracting total RNA in exosomes of buffalo and milk samples;

2) carrying out miR-2285t reverse transcription to cDNA by a reverse transcription primer under the action of reverse transcriptase;

3) carrying out amplification detection on the cDNA obtained in the step 2) and the reference gene on a fluorescent real-time quantitative PCR instrument;

4) analysis by dissolution Curve, 2^-ΔΔCtCarrying out relative quantification;

the buffalo is a Mora buffalo; the dairy cow is a Holstein dairy cow.

Images