阅读说明：本技术 快速提取牛血液DNA进行β-酪蛋白基因型检测的方法 (Method for rapidly extracting bovine blood DNA (deoxyribonucleic acid) to carry out β -casein genotype detection ) 是由 刘继强 于 2019-12-05 设计创作，主要内容包括：本发明提供一种快速提取牛血液DNA进行β-酪蛋白基因型检测的方法,该方法包括：向采集的牛血液样本中加入样本10～30倍体积量的细胞裂解液A,混匀后加入与稳定液B,混匀后静置,取上清用水稀释后作为模板,用于PCR或实时荧光定量PCR反应,进行β-酪蛋白基因型的检测。本发明通过设计β-酪蛋白基因检测位点上、下游引物和Taqman探针,96孔板批量裂解血液,快速进行奶牛β-酪蛋白基因型的批量检测。该方法操作简单,无需加热、高速离心等DNA提取操作,成本较常规方法降低50％,获得DNA仅需10min左右,总检测耗时只需60min,大大缩短了检测时间,适合于奶牛场现场快速检测。(The invention provides a method for quickly extracting bovine blood DNA to detect a β -casein gene type, which comprises the steps of adding 10-30 times of cell lysate A of a sample into an acquired bovine blood sample, uniformly mixing, adding stabilizing solution B, uniformly mixing, standing, taking supernatant diluted by water as a template, using the supernatant as a PCR (polymerase chain reaction) or real-time fluorescence quantitative PCR reaction, and detecting the β -casein gene type.)

1. A method for rapidly extracting bovine blood DNA to carry out β -casein genotype detection is characterized in that 10-30 times of cell lysate A of a sample is added into an acquired bovine blood sample, the mixture is uniformly oscillated at room temperature, then, stabilizing solution B which is equal to the cell lysate A is added, the mixture is uniformly oscillated at room temperature and then stands, supernatant is taken and diluted by 10-20 times with water to be used as a template for PCR or real-time fluorescent quantitative PCR reaction, and the β -casein genotype detection is carried out;

wherein the cell lysate A is selected from at least one of 50-100 mM Tris-HCl, 50-75 mM NaCl, 0.5-5% TritonX-100 and 50-100 mM DTT, and the stabilizing solution B is selected from at least one of 10-30% glycerol, 10-25% DMSO and 0.5-5% Tween-20;

the primers used in the PCR reaction were designed for the 205 th base of the bovine β -casein gene.

2. The method of claim 1, wherein the collected bovine blood sample comprises an anticoagulant selected from at least one of EDTA, heparin, and potassium oxalate.

3. The method of claim 2, wherein the method comprises: adding 5 mu L of the bovine blood sample into a 96-well plate, adding 50 mu L of cell lysate A, and performing shaking lysis at room temperature for 10 min; then adding 50 mu L of stabilizing solution B, standing, taking the supernatant to dilute by 10-20 times and using the supernatant as a template to perform real-time fluorescence quantitative PCR reaction.

4. The method of claim 1, wherein the nucleotide sequences of the primers and probes used in the real-time fluorescent quantitative PCR reaction are as follows:

and (3) primer F: 5'-CCAGGATGAACTCCAGGATAAA-3'

And (3) primer R: 5'-CACAGGGGTTTGAGTAAGAGGA-3'

1, probe 1: 5'-CCCATCCATAACAGCCTCCC-3'

And (3) probe 2: 5'-CCCATCCCTAACAGCCTCC-3'

Wherein, the probe 1 and the probe 2 are respectively provided with different fluorescent groups and quenching groups.

5. The method of claim 4, wherein the PCR reaction system comprises: 2 × RealFAST Probe No-ROX Mix 2.5 μ L, ROXI0.1 μ L, template 1, 10 μ M primer F, primer R, probe 1, probe 2 each 0.2 μ L, and nuclease-free water 0.6 μ L.

6. The method of claim 4, wherein the PCR reaction is performed as follows: 5min at 95 ℃; 10sec at 95 ℃,20 sec at 60 ℃, 40 cycles; storing at 4 ℃.

7. The method of any one of claims 1 to 6, wherein the anticoagulant concentration in the blood sample is 1 to 2 mg/mL.

8. A kit for rapid extraction of bovine blood DNA for β -casein genotype testing, comprising cell lysate A of claim 1, stabilizing solution B, and primers and probes of claim 4.

Technical Field

The invention relates to the technical field of biology, in particular to a method for rapidly extracting bovine blood DNA (deoxyribonucleic acid) to carry out β -casein genotype detection.

Background

β -casein accounts for about 25-35% of the total amount of casein in milk, the coding gene is beta-casein (csn2), the main variants are A1 and A2, the first 7 amino acid residues of the A1 type β -casein can be cracked to generate β -casomorphin-7 (BCM-7) under the hydrolysis of protease, and the same position of the A2 type β -casein is proline (CCT) under the condition that the codon is CCT. the A1 type β -casein is hydrolyzed by protease, the former 7 amino acid residues can be cracked to generate β -casomorphin-7 (BCM-7), the A2 type β -casein can not be generated.

At present, A2 milk has great influence on milk industry of a plurality of countries such as Australia, New Zealand, England, America and the like, A2 type β -casein-producing dairy cows have higher economic value, for the dairy cow breeding and breeding industry, A2 individuals are identified and bred, and A2 milk core group is constructed with higher cost benefit.

The invention provides a method for quickly and conveniently obtaining DNA from milk cow blood for downstream Taqman genotyping, which can realize genotyping from blood in one step without DNA extraction, reduce cost, reduce time, labor and reagent consumption and realize large-scale on-site rapid detection.

Disclosure of Invention

The invention aims to provide a method for rapidly extracting bovine blood DNA to carry out β -casein genotype detection.

The technical principle of the invention is as follows: firstly, the rapid acquisition of DNA from the blood of a cow (cow) is realized, a lysate is provided to rapidly release DNA, and a stabilizing solution is added. Secondly, the inhibition of a blood lysate system on Taqman amplification is overcome, and the commercialized tolerant hot start Taq enzyme is selected.

In order to realize the purpose of the invention, the invention provides a method for rapidly extracting bovine blood DNA to carry out β -casein genotype detection, which comprises the steps of adding 10-30 times of cell lysate A of a sample into an acquired bovine blood sample, uniformly oscillating at room temperature, adding stabilizing solution B which is equal to the cell lysate A, uniformly oscillating at room temperature, standing, taking supernatant which is diluted by 10-20 times with water as a template, using the supernatant as a PCR or real-time fluorescence quantitative PCR reaction, and carrying out β -casein genotype detection.

The cell lysate A is selected from at least one of 50-100 mM Tris-HCl, 50-75 mM NaCl, 0.5-5% Triton X-100 and 50-100 mM DTT, and the stabilizing solution B is selected from at least one of 10-30% glycerol, 10-25% DMSO and 0.5-5% Tween-20.

Wherein, the primer for PCR reaction is designed aiming at 205 th base of bovine β -casein gene.

Preferably, the cell lysate a comprises: 50mM Tris-HCl and 50mM DTT, and the stabilizing solution B comprises the following components: 10% glycerol, 20% DMSO.

In the method, the collected bovine blood sample contains an anticoagulant, and the anticoagulant is at least one selected from EDTA, heparin, potassium oxalate, and the like. The concentration of the anticoagulant in the blood sample is 1-2 mg/mL.

Further, the method comprises: adding 5 mu L of the bovine blood sample into a 96-well plate, adding 50 mu L of cell lysate A, and performing shaking lysis at room temperature for 10 min; then adding 50 mu L of stabilizing solution B, standing, taking the supernatant to dilute by 10-20 times and using the supernatant as a template to perform real-time fluorescence quantitative PCR reaction. The collected cow blood sample contains an anticoagulant, and the anticoagulant is EDTA (1-2 mg/ml), heparin (10-12 IU/ml) or potassium oxalate (2 mg/ml).

The detected target gene is located on the No. 6 chromosome of the cow, and the nucleotide sequences of the primers and the probes used in the real-time fluorescent quantitative PCR reaction are respectively as follows (SEQ ID NO: 1-4):

and (3) primer F: 5'-CCAGGATGAACTCCAGGATAAA-3'

And (3) primer R: 5'-CACAGGGGTTTGAGTAAGAGGA-3'

1, probe 1: 5'-CCCATCCATAACAGCCTCCC-3'

And (3) probe 2: 5'-CCCATCCCTAACAGCCTCC-3'

Wherein, the probe 1 and the probe 2 are respectively provided with different fluorescent groups and quenching groups.

Preferably, the PCR reaction system is as follows: 2 × RealFAST Probe No-ROX Mix 2.5 μ L, ROX I0.1 μ L, template 1, 10 μ M primer F, primer R, Probe 1, Probe 2 each 0.2 μ L, and nuclease-free water 0.6 μ L.

RealFAST Probe No-ROX Mix from 2 XT 5 FastqPCR Mix (Probe) kit from New Biotechnology Ltd of Kyoto Ongzhico.

Preferably, the PCR reaction procedure is as follows: 5min at 95 ℃; 10sec at 95 ℃,20 sec at 60 ℃, 40 cycles; storing at 4 ℃.

In a second aspect, the invention provides a kit for rapidly extracting bovine blood DNA for β -casein genotype detection, which comprises the cell lysate A, the stabilizing solution B, and primers and probes shown in SEQ ID NO. 1-4.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the method for rapidly obtaining DNA from the bovine blood for β -casein genotype detection is suitable for large-scale on-site rapid detection requirements of dairy farms/breeding farms, can rapidly distinguish calves, and is favorable for hurdle feeding and core group construction.

The method has low extraction cost, takes about 10min for extracting DNA, only takes about 60min for total detection, and realizes large-scale 96-pore plate quick extraction operation. Compared with the conventional DNA extraction-PCR-electrophoresis-imaging method, the method has obvious advantages: 1) and (3) the speed is high. The blood lysis only needs about 10min, the Taqman typing only needs about 50min, and the total time consumption is reduced by more than 50% compared with the conventional extraction method. 2) The operation is simple. The DNA can be released by cracking at room temperature without additional heating; the cracking system is mixed evenly by vortex without centrifugation. 3) Can be used in batches. And in order to facilitate large-scale field use, the expansion is optimized to 96-pore plate operation. 4) The detection cost is low. The cost of the reagent for blood cracking and Taqman reaction detection is far lower than that of the conventional DNA extraction-PCR-electrophoresis-imaging method. 5) The instrument is less in matching. Only a real-time fluorescent quantitative PCR (qPCR) instrument is utilized, and other complex instrument equipment is not required to be relied on.

Drawings

FIG. 1 shows the results of gene typing detection of β -casein on a 96-well plate sample from a cow in example 1.

FIG. 2 shows the results of the optimization of cell lysate A and stabilizer B in example 2 of the present invention.

FIG. 3 shows the result of β -casein genotyping test on blood samples in which the anticoagulant is EDTA in example 3 of the present invention.

FIG. 4 shows the β -casein genotyping assay results of blood samples with heparin as the anticoagulant in example 3 of the present invention.

FIG. 5 shows the result of β -casein genotyping test on blood samples in which the anticoagulant is potassium oxalate in example 3 of the present invention.

In the figure, Allele X: dominant homozygote, Allele Y: recessive homozygote, Both: heterozygote, NTC: negative control, x: it was not detected.

Detailed Description

Unless otherwise indicated, the examples follow conventional experimental conditions, such as Sambrook et al Molecular Cloning handbook (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or conditions suggested by the manufacturer's instructions example 1 method for rapid extraction of milk cow blood DNA for gene-based detection of β -casein

The method for rapidly extracting the DNA of the milk cow blood to carry out β -casein genotype detection comprises the following steps of rapidly and simply cracking the milk cow blood, diluting a lysate into an amplification template, and carrying out typing and identification by a Taqman probe method.

Adding 5 μ L blood sample (anticoagulant is EDTA, concentration is 1mg/ml) collected from milk cow into 96 deep-well plate, adding 50 μ L cell lysate A, and performing room temperature shaking lysis for 10 min; and then adding 50 mu L of stabilizing solution B, standing, and taking supernatant to dilute by 10-20 times with water as a template to perform Taqman typing detection.

The cell lysate A comprises the following components: 50mM Tris-HCl, 50mM DTT.

The stabilizing solution B comprises the following components: 10% glycerol, 20% DMSO.

The Taqman typing detection system comprises the following reaction systems and procedures:

reaction system:

reagent	System (5. mu.L)/well
		RealFAST Probe No-ROX Mix(2×)	2.5μL
ROX I	0.1μL
		Cow blood lysate dilution sample	1μL
Primer F (10. mu.M)	0.2μL
		Primer R (10. mu.M)	0.2μL
Probe 1 (10. mu.M)	0.2μL
		Probe 2 (10. mu.M)	0.2μL
Nuclease-free water	0.6μL
		Total volume	5μL

Reaction procedure:

RealFAST Probe No-ROX Mix 2 XT 5 FastqPCR Mix (Probe) kit from New Biotechnology GmbH of Beijing Opticaldaceae contains PCR buffer system, PCR intensifier, hot start Taq DNA polymerase and the like, and is used for rapid PCR amplification reaction. The primers and probes used in the Taqman genotyping reaction system are synthesized by Beijing Optimalaceae New Biotechnology Co., Ltd, and have the following sequences:

the detected β -casein gene is located on the cow No. 6 chromosome, and the nucleotide sequences of the primers and the probes used in the real-time fluorescent quantitative PCR reaction are respectively as follows:

and (3) primer F: 5'-CCAGGATGAACTCCAGGATAAA-3'

And (3) primer R: 5'-CACAGGGGTTTGAGTAAGAGGA-3'

1, probe 1: 5'-CCCATCCATAACAGCCTCCC-3'

And (3) probe 2: 5'-CCCATCCCTAACAGCCTCC-3'

The probe 1 and the probe 2 are respectively provided with different fluorescent groups (such as VIC and FAM) and quenching groups (BHQ 1).

The results of the 96-well plate genotyping assay are shown in FIG. 1. The detection rate of 96 samples applying the method is 100%, and compared with DNA obtained by a conventional extraction method, the detection consistency is 100%. The Taqman typing clustering degree shows that the VIC, FAM and heterozygosity clustering are distinguished obviously.