CN111154868A - Method for detecting tree shrew SLC22A6 gene transcription level by real-time fluorescence quantitative PCR method, primer and kit - Google Patents

Abstract

The invention relates to a method, primers and a kit for detecting the transcriptional level of a tree shrew SLC22A6 gene by a real-time fluorescent quantitative PCR method, belonging to the technical field of molecular biology. The method takes cDNA reverse transcribed from total RNA of a sample as a template toSLC22A6F andSLC22A6r, GAPDH F and GAPDH R are used as specific primers to carry out real-time fluorescence quantitative PCR amplification, and the quantitative determination is carried out according to the change situation of fluorescence in a reaction systemSLC22A6The level of gene transcription. The method can be used for the tree shrewsSLC22A6The quantitative detection is implemented on the change condition of the gene transcription level, and the method has the advantages of simple operation, high repeatability, strong specificity, good sensitivity and easy popularization and application.

Description

Method for detecting tree shrew SLC22A6 gene transcription level by real-time fluorescence quantitative PCR method, primer and kit

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a method, primers and a kit for detecting the transcriptional level of the SLC22A6 gene of tree shrew by using a real-time fluorescent quantitative PCR (RT-qPCR) method.

Background

On chromosome 11SLC22A6The gene-encoded OAT1 protein belongs to the family of organic anion transporters,SLC22A6expressed on the basolateral membrane of proximal tubular epithelial cells.SLC22A6Mainly transports endogenous and exogenous organic anions, and the excretion of uric acid mainly depends on renal tubulesSLC22A6Gene expression of OAT1, OAT1 is a transporter closely related to uric acid secretion and excretion, OAT1 and OAT3 use a tertiary transport mechanism to transport urate from the peritubular space to the proximal tubular epithelial cells, i.e., the first step of urate secretion.SLC22A6Reduced gene expression and reduced renal secretion reduces the accumulation of organic anions, leading to renal inflammation.

Hyperuricemia is a disease caused by an increase in serum uric acid level due to urate deposition or uric acid metabolic disorder, and the incidence rate thereof is increasing in recent years. Research shows that the compound is not only the pathological basis of gout attack, but also has close relation with diseases such as diabetes, hypertension, metabolic disorder syndrome, insulin resistance syndrome and the like, and an animal model has very important function in scientific development as a method for researching disease pathogenesis and pharmacological efficacy. Studies have shown that blood uric acid is an independent risk factor for renal dysfunction, affecting renal function even beyond the amount of urine protein. With the rise of blood uric acid, the probability of renal failure is also greatly increased. On the contrary, the kidney is an important organ for regulating the excretion of uric acid in vivo, and the kidney injury can block the excretion of uric acid, so that the blood uric acid concentration is further increased, therefore, hyperuricemia and renal function damage are interactive, and clinically, the hyperuricemia shows as gouty nephropathy, acute hyperuricemia nephropathy, uric acid calculi and the like. Therefore, the research on the pathogenic factors of the renal injury caused by the rise of blood uric acid is very important for treating the renal injury diseases and the hyperuricemia. Meanwhile, the study on the pathogenesis of hyperuricemia and the establishment of an animal model are particularly important for overcoming the hyperuricemia.

The literature reports toSLC22A6The research of the gene mainly focuses on the structure and the function of the protein, and the research is carried out on the geneSLC22A6The study of gene transcription level is also extremely important, and at present, gene editing technology is adopted to knock outSLC22A6The study of the genes can be further exploredSLC22A6The action mechanism of the gene, and a reliable and efficient gene transcription level detection method is established.

Disclosure of Invention

The invention aims to solve the defects of the prior art and provides a method, a primer and a kit for detecting the transcription level of the tree shrew SLC22A6 gene by using a real-time fluorescent quantitative PCR (RT-qPCR) method. The invention selects the primer with high sensitivity and single product, adopts the primer for detection, is applied to the liver, the kidney and the small intestine of the tree shrew, and can efficiently detect the tree shrewSLC22A6Gene, for the studySLC22A6Further studies of the gene and its expression product OAT1 lay the foundation.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the method for detecting the transcription level of the tree shrew SLC22A6 gene by using the real-time fluorescent quantitative PCR method for the non-diagnosis purpose comprises the following steps:

step (1), respectively taking total RNA extracted from fresh tissues of healthy and to-be-detected tree shrews as templates, and performing reverse transcription to synthesize a first chain of cDNA of the kidney tissues of the tree shrews; the fresh tissues are fresh kidney, liver and small intestine tissues;

step (2), establishing tree shrew GAPDH gene andSLC22A6gene standard curve: diluting the first chain of cDNA of the healthy tree shrew tissue obtained in the step (1) by Easy dilution gradient, respectively taking the undiluted first chain of cDNA and the diluted cDNA as templates, carrying out real-time fluorescence quantitative detection, and respectively taking the undiluted first chain of cDNA and the diluted cDNA as templatesSLC22A6F andSLC22A6r, GAPDH F and GAPDH R are used as specific primers to carry out real-time fluorescent quantitative PCR amplification to respectively obtain healthy tree shrew GAPDH gene,SLC22A6A lysis curve and an amplification curve of the gene;

saidSLC22A6F、SLC22A6The sequences of the primers R, GAPDH F and GAPDH R are as follows:

SLC22A6F：5＇-acagcagaagcagatggtcc-3＇；

SLC22A6R：5＇-ggctctgtaaggccccattt-3＇；

GAPDHF：5＇-agccccatcaccatcttcc-3＇；

GAPDHR：5＇-aatgagccccagccttctc-3＇；

the number of copies Log is then determined as the initial template amount₁₀The logarithm value of (A) is taken as an X axis, and the Ct value is taken as a Y axis to draw, so as to respectively obtain the GAPDH gene,SLC22A6A standard curve of the gene;

step (3), the first chains of the cDNA of the tree shrew tissues to be detected obtained in the step (1) are respectively treated withSLC22A6F andSLC22A6r, GAPDH F and GAPDH R are used as specific primers to carry out real-time fluorescent quantitative PCR amplification, the amplification system and the amplification program are the same as the step (2), and the tree shrew GAPDH gene to be detected, the GAPDH gene to be detected and the GAPDH gene to be detected are respectively obtained,SLC22A6A lysis curve and an amplification curve of the gene;

and (3) calculating according to the standard curve obtained in the step (2) to obtain the tree shrewSLC22A6The level of gene transcription.

Based on the principle of real-time fluorescence quantitative detection, that is, the Cq value of each template has a linear relationship with the logarithm of the initial copy number of the template, the formula is as follows: cq = -1/lg (1+ Ex) × lgX0+ lgN/lg (1+ Ex) (N is the number of cycles of the amplification reaction, X0 is the initial template amount, Ex is the amplification efficiency, and N is the amount of amplification product when the fluorescence amplification signal reaches the threshold intensity.) the higher the initial copy number, the smaller the Cq value. Standard koji can be made by using standard substance with known initial copy numberLine, where the abscissa represents the logarithm of the starting copy number and the ordinate represents the Cq value. Thus, the Cq value of an unknown sample is obtained, i.e., the initial copy number of the sample can be calculated from the standard curve. Therefore, according to the standard curve obtained in the step (2) and the Cq value of each sample, the tree shrew can be obtained by utilizing the self-contained gene expression calculation function in the Bio-Rad CFXManager 3.1 softwareSLC22A6The level of gene transcription.

Further, it is preferable that the first strand of the tree shrew tissue cDNA in step (1) is diluted with Easy dilution gradient at 5-fold, 25-fold, 125-fold, 625-fold.

Further, it is preferable that the real-time fluorescent quantitative PCR amplification system is as follows: 5 mu L of SYBR Premix Ex Taq II (2x), 0.4 mu L of each of upstream and downstream primers, 0.8 mu L of cDNA template, 3.4 mu L of deionized water, and 10 mu L of total primer concentration, wherein the concentrations of the upstream and downstream primers are both 10 mu M;

the real-time fluorescent quantitative PCR amplification program comprises the following steps: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 10s, and annealing at 60 ℃ for 30s, and 40 cycles.

The invention also provides a kit for detecting the SLC22A6 gene transcription level of the tree shrew, and the kit comprisesSLC22A6F、SLC22A6R, GAPDH F and GAPDH R primers.

Further, SYBR Premix Ex Taq II (2X) is preferably also included.

The invention also provides primers for detecting the transcription level of the SLC22A6 gene of the tree shrew, comprising the tree shrewSLC22A6Specific upstream and downstream primers of gene expression level and specific upstream and downstream primers of the tree shrew GAPDH gene serving as an internal reference gene;

wherein, the tree shrewSLC22A6The specific upstream and downstream primer sequences of the gene expression level are as follows:

SLC22A6F：5＇-acagcagaagcagatggtcc-3＇；

SLC22A6R：5＇-ggctctgtaaggccccattt-3＇；

the specific upstream and downstream primer sequences of the tree shrew GAPDH gene as the reference gene are as follows:

GAPDHF：5＇-agccccatcaccatcttcc-3＇；

GAPDHR：5＇-aatgagccccagccttctc-3＇。

the invention also provides application of the kit and the primer on the kit to non-diagnosis-purpose detection of the transcription level of the tree shrew SLC22A6 gene.

When the peak on the dissolution curve is not unique during detection, the pollution exists in the experiment, and the detection needs to be carried out again.

Compared with the prior art, the invention has the beneficial effects that:

the application of the primer sequence disclosed by the invention can realize the alignment of tree shrewsGAPDHGenes andSLC22A6the specificity of the gene is amplified, the specificity of the primer is good, no non-target gene is amplified, and the primer can be used for treating the tree shrewsSLC22A6The quantitative detection is implemented according to the change condition of the gene transcription level, the detection method has high repeatability, high automation and simple operation, can detect only a trace amount of samples, has high flux, and can detect a plurality of samples and a plurality of tissues simultaneously. The invention relates to a method for researching tree shrewsSLC22A6The function and influencing factors of the gene provide an effective tool.

Drawings

FIG. 1 Tree shrewGAPDHAn amplification curve of the gene;

FIG. 2 Tree shrewGAPDHA standard curve of the gene;

FIG. 3 Tree shrewSLC22A6An amplification curve of the gene;

FIG. 4 Tree shrewSLC22A6A standard curve of the gene;

FIG. 5 Tree shrewGAPDHGene expression amplification curve in kidney;

FIG. 6 Tree shrewGAPDHExpression melting peak of gene in kidney;

FIG. 7 Tree shrewSLC22A6Gene expression amplification curve in kidney;

FIG. 8 Tree shrewSLC22A6Expression melting peak of gene in kidney;

FIG. 9 Tree shrewSLC22A6Relative expression of genes in kidney;

FIG. 10 Tree shrewGAPDHGene expression amplification curve in liver;

FIG. 11 TreeShrewGAPDHThe expression melting peak of the gene in the liver;

FIG. 12 Tree shrewSLC22A6Gene expression amplification curve in liver;

FIG. 13 Tree shrewSLC22A6The expression melting peak of the gene in the liver;

FIG. 14 Tree shrewSLC22A6Relative expression of the gene in the liver;

FIG. 15 Tree shrewGAPDHGene expression amplification curve in small intestine;

FIG. 16 Tree shrewGAPDHThe expression melting peak of the gene in the small intestine;

FIG. 17 Tree shrewSLC22A6Gene expression amplification curve in small intestine;

FIG. 18 Tree shrewSLC22A6The expression melting peak of the gene in the small intestine;

FIG. 19 Tree shrewSLC22A6Relative expression of gene expression in the small intestine;

FIG. 20 Tree shrewSLC22A6Gel diagram of amplification products of gene liver and small intestine, wherein M is marker, 1 isSLC22A6The liver amplification product of the gene 2 isSLC22A6Gene small intestine amplification product.

Detailed Description

The present invention will be described in further detail with reference to examples.

It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The materials or equipment used are not indicated by manufacturers, and all are conventional products available by purchase.

The test methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples were all commercially available unless otherwise specified.

1. Experimental animals used in this example: tree shrews, 9 females and 10 males.

2. Grouping and administration of experimental animals: 1 male tree shrew to-be-taken kidney tissue of 19 tree shrews subjected to blank serum detection is taken out to be subjected to standard curve, and the rest 18 tree shrews are randomly divided into a control group, a 30-day administration group and a 120-day administration group, wherein 6 tree shrews are respectively subjected to the standard curve. The weight of each female is 110-135 g, and the weight of each male is 120-150 g. The control group was intraperitoneally injected (iP) daily with 40mg/kg of 1% sodium carboxymethylcellulose solution (CMC-Na, 1g sodium carboxymethylcellulose dissolved in 100ml deionized water) for 120 days, and the administration group was injected with potassium Oxonate (OA) in the same dose iP for 30 days and 120 days, respectively.

3. Experimental methods

3.1 Collection of various tissues of Tree shrew such as kidney, liver and small intestine

Kidney, liver and small intestine tissues were taken and placed in RNA co-solvent (Tripure, Roche) for eachSLC22A6And (4) detecting the transcription level of the gene.

3.2 extraction of Total RNA from tissues of kidney, liver and Small intestine

Taking fresh kidney, liver and small intestine of 19 animals 0.1g respectively, adding 1mL Tripure into homogenizer, homogenizing at room temperature, standing for 5min, transferring to 1.5mL EP tube, standing for 5min, adding 200 μ L-20 deg.C pre-cooled chloroform, shaking with vortex sufficiently, standing for 15min, 4 deg.C, centrifuging at 12000r/min for 25min, sucking 450 μ L of the supernatant into another 1.5mL EP tube, adding pre-cooled isopropanol at-20 ℃ in the same volume, fully mixing and standing for 10min, centrifuging at 12000r/m for 10min at 4 ℃, discarding the supernatant, washing the precipitate with 75% ethanol pre-cooled at1 mL-20 ℃ when the inner wall of the tube is slightly dry, centrifuging at 7500r/m for 5min at 4 ℃, discarding the supernatant, dissolving the precipitate with 30 μ L DEPC water when the inner wall of the tube is slightly dry, bathing in water at 65 ℃ for 10min, and measuring the concentration and absorbance ratio of 1 μ L of the total RNA sample by a Nanodrop-1000 nucleic acid measuring instrument. After the concentration is measured, DEPC water is added to dilute the solution to 1000 ng/mu L, and the solution is put into a refrigerator at the temperature of 80 ℃ below zero for standby.

3.3 Synthesis of cDNA

According to the instructions of the reverse transcription Kit PrimeScript RT gene Kit, 5 XPrimeScript Buffer 6. mu.L, PrimeScript RT Enzyme Mix 1.5. mu.L and Oligo dT Primer were sequentially added to each 30. mu.L system1.5. mu.L, Random 6 mers 1.5. mu.L, total RNA (diluted to 1000 ng/. mu.L) 3. mu.L, RNaseFreedH₂O16.5. mu.L, reverse transcription conditions: 15min at 37 ℃, 5s at 85 ℃ and 10min at 4 ℃.

3.4 design of primers for Gene of interest

According to tree shrews in NCBI gene bankSLC22A6AndGAPDHthe gene sequences are respectively subjected to primer design by using primer design software Pimerexpress 5.0, synthesized by Beijing Baitach company,GAPDHused as an internal reference gene.

3.5 Tree shrew of the embodimentSLC22A6The primer sequences and fragment sizes for quantifying the level of gene expression are shown in Table 1

TABLE 1

3.6 determination of fluorescent quantitative PCR reaction System of target Gene

According to TAKARA biological products PCR reagent (SYBR Premix Ex TaqII), in the Real-Time fluorescence quantitative instrument CFX96 Real-Time System for amplification and data analysis, PCR amplification System as follows: SYBR Premix ExTaq II (2X) 5. mu.L, upstream and downstream primers 0.4. mu.L each, cDNA template 0.8. mu.L, deionized water 3.4. mu.L, total 10. mu.L, and upstream and downstream primer concentrations 10. mu.M each. The real-time fluorescent quantitative PCR amplification program comprises the following steps: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 10s, and annealing at 60 ℃ for 30s, and 40 cycles.

Wherein the nucleotide sequence of the amplified tree shrew SLC22A6 gene fragment is shown as SEQ ID NO. 5; the nucleotide sequence of the amplified fragment of the internal reference gene GAPDH of the tree shrew is shown as SEQ ID NO. 6.

3.7 Tree shrewGAPDHGenes andSLC22A6establishing a gene standard curve: and (3) diluting the first chain of the cDNA of the non-dosed tree shrew, the kidney, the liver and the small intestine which are subjected to reverse transcription and synthesized in the step 3.3 by using Easy dilution gradient, diluting the first chain of the cDNA of the non-dosed tree shrew by 5 times, 25 times, 125 times and 625 times respectively, taking the first chain of the original cDNA and the diluted cDNA as templates, respectively performing real-time fluorescent quantitative detection on 2 parallel samples, and performing real-time fluorescent quantitative PCR according to a fluorescent quantitative PCR system in the step 3.6. Real-time fluorescence detectorThe principle of quantitative detection, i.e., the Cq value of each template is linearly related to the logarithm of the starting copy number of the template, is as follows: cq = -1/lg (1+ Ex) × lgX0+ lgN/lg (1+ Ex) (N is the number of cycles of the amplification reaction, X0 is the initial template amount, Ex is the amplification efficiency, and N is the amount of amplification product when the fluorescence amplification signal reaches the threshold intensity.) the higher the initial copy number, the smaller the Cq value. Based on the principle, the Bio-Rad CFX Manager 3.1 software is utilized to obtainSLC22A6Genes andGAPDHthe gene lysis curve and standard curve in kidney, liver and small intestine.

3.8 analysis of Gene expression differences in samples

Obtained according to step 3.7SLC22A6Genes andGAPDHthe standard curve of the gene and the Cq value of each sample can be used for obtaining the tree shrew by utilizing the self-carried gene expression calculation function in the Bio-Rad CFX Manager 3.1 softwareSLC22A6The level of gene transcription.

4 results

4.1 Tree shrewSLC22A6Melting curve of gene fluorescent quantitative PCR reaction

As can be seen from FIGS. 8, 13 and 16, inSLC22A6The dissolution curve of the fluorescent quantitative PCR reaction of the gene in the amplification process of the kidney, the liver and the small intestine only sees a single peak, and the electrophoresis result of the reaction product only sees a specific expression strip, which indicates that the specificity of the amplified target fragment is good.

4.2 Tree shrewSLC22A6Genes andGAPDHstandard curve of gene

As can be seen from FIGS. 2 and 4, the tree shrewSLC22A6Genes andGAPDHstandard Curve R of Gene²All are close to 1, which indicates that the relative quantification performed by the standard curve is more accurate, and because the fluorescence intensity is stronger, the relative synchronization between the increase of the fluorescence intensity and the amplification of the PCR can be ensured, and the expression of the PCR can be accurately detected, so as toGAPDHChanges in mRNA expression levels were obtained as endogenous controls.

4.3 hyperuricemia Tree shrew kidney, liver and small intestine tissue caused by Potassium OxonateSLC22A6Quantitative detection of changes in mRNA expression levels

Step 2, RNA extracted from the fresh kidney tissue obtained from each group is subjected to reverse transcriptionObtaining cDNA, and obtaining each group through real-time fluorescent quantitative PCR detectionSLC22A6mRNA expression level difference, injecting potassium oxonate with 40mg/kg dose into abdominal cavity,SLC22A6the mRNA expression level in the kidney was 1.4 in the control group, and was not significantly reduced by 1.0 in the 30-day administration group compared to the control group. The up-regulation was evident in the 120-day administration group at 1.6 compared to the control group, as shown in FIG. 9.

RNA extracted from fresh liver tissue obtained from each group is reversely transcribed to obtain cDNA, and each group is detected by real-time fluorescence quantitative PCRSLC22A6mRNA expression level difference, injecting potassium oxonate with 40mg/kg dose into abdominal cavity,SLC22A6the expression level of mRNA in liver is 1.4 in the control group, 0.9 in the 30-day administration group, and is obviously reduced compared with the control group. The dose in the 120-day group was 0.7, and the down-regulation was evident in comparison with the control group, as shown in FIG. 14.

RNA extracted from fresh small intestine tissue obtained from each group is reversely transcribed to obtain cDNA, and each group is detected by real-time fluorescence quantitative PCR to obtainSLC22A6mRNA expression level difference, injecting potassium oxonate with 40mg/kg dose into abdominal cavity,SLC22A6the expression level of mRNA in small intestine is 1.0 in control group and 0.8 in 30 days administration group, and the down regulation is not obvious compared with the control. The dose in the 120-day administration group is 1.7, and the up-regulation is obvious compared with that in the control group; see fig. 19.

4.4 Potassium Oxonate (OA) is a uricase inhibitor which can reduce the decomposition and excretion of uric acid by inhibiting uricase activity, and can cause hyperuricemia in tree shrews, and is used in the above examples to cause hyperuricemia in tree shrewsSLC22A6The expression of mRNA in the kidney is first up-regulated and then down-regulated over time, expression in the liver continues to be down-regulated, and expression in the small intestine is first down-regulated and then up-regulated. The specific mechanism of action has yet to be studied, but the above examples illustrate that the invention can be used inSLC22A6Detection of mRNA expression level for the study of Tree shrewSLC22A6The function and the influencing factors of the gene provide effective tools and provide reliable means for the research of diseases such as hyperuricemia and the like.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

SEQ ID NO.1

acagcagaag cagatggtcc 20

SEQ ID NO.2

ggctctgtaa ggccccattt 20

SEQ ID NO.3

agccccatca ccatcttcc 19

SEQ ID NO.4

aatgagcccc agccttctc 19

SEQ ID NO.5

acagcagaag cagatggtcc cactccaggc ctctgcacaa gagaagactg gactctgaga 60

actgaaatgg ggccttacag agcc 84

SEQ ID NO.6

ggcacagtca aggctgagaa tgggaagctg gtcatcaacg ggaaacccat caccatcttc 60

caggagcgag atcccgctaa catcaaatgg ggtgatgctg gtgctgagta tgtcgtggag 120

tctactggcg tcttcaccac cat 143

Sequence listing

<110> institute of medical science and biology of China academy of medical sciences

<120> method for detecting tree shrew SLC22A6 gene transcription level by real-time fluorescence quantitative PCR method, primer and kit

<160>6

<170>SIPOSequenceListing 1.0

<210>1

<211>20

<212>DNA

<213> Artificial sequence ()

<400>1

acagcagaag cagatggtcc 20

<210>3

<211>20

<212>DNA

<213> Artificial sequence ()

<400>3

ggctctgtaa ggccccattt 20

<210>3

<211>19

<212>DNA

<213> Artificial sequence ()

<400>3

agccccatca ccatcttcc 19

<210>4

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aatgagcccc agccttctc 19

<210>5

<211>84

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<213> Artificial sequence ()

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acagcagaag cagatggtcc cactccaggc ctctgcacaa gagaagactg gactctgaga 60

actgaaatgg ggccttacag agcc 84

<210>6

<211>143

<212>DNA

<213> Artificial sequence ()

<400>6

ggcacagtca aggctgagaa tgggaagctg gtcatcaacg ggaaacccat caccatcttc 60

caggagcgag atcccgctaa catcaaatgg ggtgatgctg gtgctgagta tgtcgtggag 120

tctactggcg tcttcaccac cat 143

Claims (7)

1. The method for detecting the transcription level of the tree shrew SLC22A6 gene by using a non-diagnostic real-time fluorescent quantitative PCR method is characterized by comprising the following steps:

step (1), respectively taking total RNA extracted from fresh tissues of healthy and to-be-detected tree shrews as templates, and performing reverse transcription to synthesize a first chain of cDNA of the kidney tissues of the tree shrews; the fresh tissues are fresh kidney, liver and small intestine tissues;

step (2), establishing tree shrew GAPDH gene andSLC22A6gene standard curve: diluting the first chain of cDNA of the healthy tree shrew tissue obtained in the step (1) by Easy dilution gradient, respectively taking the undiluted first chain of cDNA and the diluted cDNA as templates, carrying out real-time fluorescence quantitative detection, and respectively taking the undiluted first chain of cDNA and the diluted cDNA as templatesSLC22A6F andSLC22A6r, GAPDH F and GAPDH R are used as specific primers to carry out real-time fluorescent quantitative PCR amplification to respectively obtain healthy tree shrew GAPDH gene,SLC22A6A lysis curve and an amplification curve of the gene;

saidSLC22A6F、SLC22A6The sequences of the primers R, GAPDH F and GAPDH R are as follows:

SLC22A6F：5＇-acagcagaagcagatggtcc-3＇；

SLC22A6R：5＇-ggctctgtaaggccccattt-3＇；

GAPDHF：5＇-agccccatcaccatcttcc-3＇；

GAPDHR：5＇-aatgagccccagccttctc-3＇；

the number of copies Log is then determined as the initial template amount₁₀The logarithm value of (A) is taken as an X axis, and the Ct value is taken as a Y axis to draw, so as to respectively obtain the GAPDH gene,SLC22A6A standard curve of the gene;

step (3), the first chains of the cDNA of the tree shrew tissues to be detected obtained in the step (1) are respectively treated withSLC22A6F andSLC22A6r, GAPDH F and GAPDH R are used as specific primers to carry out real-time fluorescent quantitative PCR amplification, the amplification system and the amplification program are the same as the step (2), and the tree shrew GAPDH gene to be detected, the GAPDH gene to be detected and the GAPDH gene to be detected are respectively obtained,SLC22A6A lysis curve and an amplification curve of the gene;

and (3) calculating according to the standard curve obtained in the step (2) to obtain the tree shrewSLC22A6The level of gene transcription.

2. The method for detecting the SLC22A6 gene transcript level of tree shrew by using the real-time fluorescent quantitative PCR method for non-diagnostic purposes according to claim 1, wherein the first strand of the tree shrew tissue cDNA in the step (1) is diluted by Easy dilution gradient, and the dilution gradient is respectively 5 times, 25 times, 125 times and 625 times.

3. The method for detecting the transcriptional level of the tree shrew SLC22A6 gene by the real-time fluorescent quantitative PCR method for non-diagnostic purposes according to claim 1, wherein the method comprises the following steps:

under a real-time fluorescent quantitative PCR amplification system: 5 mu L of SYBR Premix Ex Taq II (2x), 0.4 mu L of each of upstream and downstream primers, 0.8 mu L of cDNA template, 3.4 mu L of deionized water, and 10 mu L of total primer concentration, wherein the concentrations of the upstream and downstream primers are both 10 mu M;

the real-time fluorescent quantitative PCR amplification program comprises the following steps: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 10s, and annealing at 60 ℃ for 30s, and 40 cycles.

4. The kit for detecting the SLC22A6 gene transcription level of the tree shrew is characterized by comprisingSLC22A6F、SLC22A6R, GAPDH F and GAPDH R primers.

5. The kit for detecting the transcription level of the SLC22A6 gene of tree shrew according to claim 4, further comprising SYBR Premix Ex Taq II (2 x).

6. The primer for detecting the transcription level of the SLC22A6 gene of the tree shrew is characterized by comprising the primer for detecting the transcription level of the tree shrew SLC22A6 geneSLC22A6Specific upstream and downstream primers of gene expression level and specific upstream and downstream primers of the tree shrew GAPDH gene serving as an internal reference gene;

wherein, the tree shrewSLC22A6The specific upstream and downstream primer sequences of the gene expression level are as follows:

SLC22A6F：5＇-acagcagaagcagatggtcc-3＇；

SLC22A6R：5＇-ggctctgtaaggccccattt-3＇；

the specific upstream and downstream primer sequences of the tree shrew GAPDH gene as the reference gene are as follows:

GAPDHF：5＇-agccccatcaccatcttcc-3＇；

GAPDHR：5＇-aatgagccccagccttctc-3＇。

7. the kit of claim 4 or 5, and the use of the primers of claim 6 for detecting the transcriptional level of the SLC22a6 gene in tree shrew for non-diagnostic purposes.

Images