CN116716389A

CN116716389A - Primer group for detecting TRPC6 mRNA level, kit, detection method and application thereof in Alzheimer disease diagnosis

Info

Publication number: CN116716389A
Application number: CN202310426626.8A
Authority: CN
Inventors: 郑志强; 闫锦锦
Original assignee: Wuhu Maozhi Biotechnology Co ltd
Current assignee: Wuhu Maozhi Biotechnology Co ltd
Priority date: 2023-04-20
Filing date: 2023-04-20
Publication date: 2023-09-08
Also published as: WO2024217526A1

Abstract

The invention discloses a primer group, a kit, a detection method and application of the primer group in detecting the level of TRPC6 mRNA in Alzheimer disease diagnosis, wherein the primer group corresponds to the 1 st to 2 nd exon regions, the 8 th to 9 th exon regions or the 11 th to 13 th exon regions of TRPC6 gene mRNA. The kit for detecting the TRPC6 mRNA level comprises a PCR amplification primer group of a TRPC6 gene, a PCR amplification primer group of an Actin internal reference gene, a TRPC6 target gene calibrator, an Actin internal reference gene calibrator, a PCR amplification reaction liquid system and reverse transcriptase. The detection method comprises the following steps: and (3) extracting an RNA sample, performing PCR amplification detection, and calculating the concentration ratio of TRPC6 mRNA to the Actin mRNA in the RNA sample by taking the Actin mRNA as an internal reference, wherein the concentration ratio is used as a detection result. The detection primer has high specificity, and the detection method has the characteristics of accuracy, high cost-effective ratio, convenience and the like for diagnosing the Alzheimer's disease, and has great clinical significance.

Description

Primer group for detecting TRPC6 mRNA level, kit, detection method and application thereof in Alzheimer disease diagnosis

Technical Field

The invention belongs to the technical field of biomedical detection, and particularly relates to a primer group, a kit and a detection method for detecting the level of TRPC6 mRNA and application of the primer group and the kit in diagnosis of Alzheimer's disease.

Background

Alzheimer's Disease (AD), senile dementia, is the most common neurodegenerative disease causing dementia in the elderly population. The disease is hidden, and clinical manifestations are mainly progressive decline of cognitive functions and daily life abilities. Depending on doctor's inquiry and neuropsychological tests, subjectivity is strong, clinical experience of doctors and whether neuropsychological tests are normative can influence diagnosis accuracy, and for example, imaging examination MRI and PET-CT detection are expensive, cerebrospinal fluid detection (CSF) is traumatic, so that there is an urgent need for objective, accurate and cost-effective biomarkers for AD diagnosis.

Transient Receptor Potential Channels (TRPC) are non-selective cation channels whose function is primarily to sense a series of physical or chemical signals that, when activated, open to cause the influx of ions of calcium, sodium, etc., thereby allowing the cell and body to respond to the external environment. There are seven members of the TRPC channel family, TRPC1 to TRPC7. The human TRPC6 gene is located on chromosome 11 and contains 13 exons encoding 931 amino acids. TRPC6 also plays an important role in the nervous system: TRPC6 has a protective effect on neurons during cerebral ischemia. Studies show that TRPC6 has a direct relationship with the nervous system, neurons, learning and memory and the generation of AD toxic protein Abeta, and the TRPC6 mRNA content in peripheral blood is a biomarker which has high application value, better specificity and relevance and is suitable for diagnosing AD.

Patent document CN103966309a discloses a method for detecting TRPC6 mRNA levels of peripheral blood cells, wherein total RNA is extracted first, then reverse transcribed, and finally PCR amplification is performed to determine TRPC6 mRNA levels. However, the detection method adopts multi-step reaction, so that systematic errors are easy to accumulate, and on the other hand, the relative expression level of TRPC6 mRNA is measured, and the accuracy of the detection result is reduced, so that the judgment of the Alzheimer disease of a subject is affected. Patent document CN113881765a also discloses a method for detecting TRPC6 expression products for early diagnosis and prognosis evaluation of type 2 diabetes, but in this method, RT-PCR and qPCR processes are separated, systematic errors are easily amplified, and high requirements are placed on the test operation. The development of a method for detecting TRPC6 mRNA level with higher accuracy is of great significance for clinical diagnosis of Alzheimer's disease.

Disclosure of Invention

In view of the above, it is an object of the present invention to provide a kit for detecting TRPC6 mRNA levels.

The technical scheme is as follows:

the kit for detecting the TRPC6 mRNA level is characterized by comprising a PCR amplification primer group of a transient receptor potential channel 6 TRPC6 gene, a PCR amplification primer group of an Actin internal reference gene, a TRPC6 target gene calibrator, an Actin internal reference gene calibrator, a PCR amplification reaction liquid system and reverse transcriptase;

the PCR amplification primer set of the TRPC6 gene comprises at least one of the following primer sets:

an upstream primer TPS-1-F with a nucleotide sequence shown as SEQ ID NO.1, and a downstream primer TPS-1-R with a nucleotide sequence shown as SEQ ID NO. 2;

an upstream primer TPS-3-F with a nucleotide sequence shown as SEQ ID NO.5 and a downstream primer TPS-3-R with a nucleotide sequence shown as SEQ ID NO. 6;

an upstream primer TPS-4-F with a nucleotide sequence shown as SEQ ID NO.7 and a downstream primer TPS-4-R with a nucleotide sequence shown as SEQ ID NO. 8.

In one embodiment, the PCR amplification reaction liquid system comprises DNA polymerase, dNTPs, mgSO ₄ Fluorescent dye.

The second purpose of the invention is to provide a purpose of the kit, which is used for preparing a kit for screening and diagnosing Alzheimer's disease.

In one embodiment, the kit for screening for diagnosis of Alzheimer's disease uses total RNA extract of human peripheral blood cells as a clinical sample.

In one embodiment, the PCR amplification primer set of the TRPC6 gene in the kit has detection accuracy of not less than 85%, sensitivity of not less than 80% and specificity of not less than 80% in clinical sample diagnosis; further accuracy is not lower than 90%, specificity is not lower than 80%, and sensitivity is not lower than 85%.

The third object of the present invention is to provide a method for detecting the level of TRPC6 mRNA in blood.

The technical scheme is as follows:

a method for detecting the level of TRPC6 mRNA in blood, using the kit according to any one of the above, comprising the following steps:

s1, extracting an RNA sample: taking blood of a subject, and extracting total RNA in blood white cells;

s2, PCR amplification detection: performing reverse transcription amplification detection on the RNA sample by adopting two PCR amplification detection systems and using a one-step real-time fluorescent quantitative PCR amplification method to respectively obtain a CT value of TRPC6 mRNA and a CT value of action mRNA in the RNA sample;

performing PCR amplification detection on the TRPC6 target gene calibrator with the serial gradient concentration to obtain CT values of the TRPC6 target gene calibrator with different concentrations;

performing PCR amplification detection on the serial gradient concentration of the Actin reference gene calibrator to obtain CT values of the Actin reference gene calibrators with different concentrations;

s3, data processing: using CT values of TRPC6 target gene calibrators with different concentrations and the concentrations of the TRPC6 target gene calibrators to establish a relationship between the CT values and the number of the concentrations of the TRPC6 target genes;

using CT values of the reference gene calibrator of the Actin with different concentrations and the concentration of the reference gene calibrator of the Actin to establish the relation between CT values and the number of the concentrations of the reference gene of the Actin;

calculating the concentration of TRPC6 mRNA and the concentration of the Actin mRNA in the RNA sample according to the CT value of TRPC6 mRNA and the CT value of the Actin mRNA in the RNA sample based on the CT value-concentration number relation of the TRPC6 target gene and the Actin internal reference gene respectively;

and calculating the concentration ratio of TRPC6 mRNA to Actin mRNA in the RNA sample, and taking the concentration ratio as a detection result.

In one embodiment, the method further includes step S4 of comparing the detection result with a set threshold, and determining that the detection result is an abnormal result when the detection result is lower than the threshold.

In one embodiment, in the step S3, the method for establishing the relationship between the CT value and the concentration number is as follows:

using CT values of TRPC6 target gene calibrators with different concentrations as an abscissa, using the logarithmic value of 2 of the concentration of the TRPC6 target gene calibrators as an ordinate, establishing a CT value-concentration standard curve of the TRPC6 target gene, and calculating a standard curve equation;

and (3) using CT values of the reference gene calibrator with different concentrations as an abscissa, using a logarithmic value of 2 of the reference gene calibrator concentration with the reference gene of the different concentrations as an ordinate, establishing an CT value-concentration standard curve of the reference gene of the different concentrations, and calculating a standard curve equation.

In one embodiment, the reverse transcription amplification detection process in the step S2 is as follows:

(1) Preparing a reaction system, wherein the total reaction system is 20ul, and the reaction system comprises 10ul of reaction mixed solution, 0.4ul of reverse transcriptase, 0.6ul (10 uM) of primer, 9ul of calibrator and 150ng of template RNA;

(2) The reverse transcription condition is that the temperature is kept at 50 ℃ for 5min, and the denaturation condition is that the temperature is kept at 94 ℃ for 30s;

(3) 40 amplification cycles were performed under the following conditions: denaturation, 94 ℃,5s; annealing at 61 ℃ for 15s; extending at 72 ℃ for 15s;

(4) Generating a melting curve, wherein the temperature rise process is to gradually rise from 65 ℃ to 95 ℃ and the temperature rise rate is 5s and 0.5 ℃.

The fourth object of the present invention is to provide a primer set for detecting the level of TRPC6 mRNA.

The technical scheme is as follows:

the primer group for detecting the TRPC6 mRNA level is characterized by comprising an upstream primer and a downstream primer, wherein the upstream primer is a primer TPS-1-F with a nucleotide sequence shown as SEQ ID NO.1, and the downstream primer is a primer TPS-1-R with a nucleotide sequence shown as SEQ ID NO. 2;

or (b)

The upstream primer is a primer TPS-3-F with a nucleotide sequence shown as SEQ ID NO.5, and the downstream primer is a primer TPS-3-R with a nucleotide sequence shown as SEQ ID NO. 6;

or (b)

The upstream primer is a primer TPS-4-F with a nucleotide sequence shown as SEQ ID NO.7, and the downstream primer is a primer TPS-4-R with a nucleotide sequence shown as SEQ ID NO. 8;

or (b)

The upstream primer is a primer TPS-5-F with a nucleotide sequence shown as SEQ ID NO.9, and the downstream primer is a primer TPS-5-R with a nucleotide sequence shown as SEQ ID NO. 10.

The fifth object of the present invention is to provide an application of any one of the above primer sets in preparing a kit for detecting TRPC6 mRNA level in a sample.

Drawings

FIG. 1 is a graph of the results of electrophoresis detection of a blood total RNA sample;

FIG. 2 is a one-step qRT-PCR amplification curve of a TRPC6 calibrator for the kit;

FIG. 3 is a one-step qRT-PCR amplification curve of an action calibrator for the kit;

FIG. 4 is a one-step qRT-PCR amplification curve of RNA detection samples;

fig. 5 is a standard curve of a kit TRPC6 calibrator.

FIG. 6 is a standard curve of the kit action calibrator.

Detailed Description

The invention is further described below with reference to examples and figures.

Example one primer set for detecting mRNA of TRPC6 Gene

Real-time fluorescent quantitative PCR (qRT-PCR) amplification primer sets for detecting the expression level of mRNA of the TRPC6 Gene were designed according to the 1 st to 13 th exon regions of the TRPC6 Gene disclosed by NCBI Gene Bank (NM-004621.6) using primer5.0 software, and each primer set includes an upstream primer and a downstream primer. In this example, the primer sets have five sets corresponding to the 1 st to 2 nd exon regions, 8 th to 9 th exon regions, and 11 th to 13 th exon regions of the TRPC6 gene mRNA, respectively, as shown in Table 1.

TABLE 1 TRPC6 amplification primer nucleotide sequence

Example two kit for detecting mRNA level of TRPC6 Gene

A kit for detecting the level of TRPC6 mRNA comprises a PCR amplification primer group of TRPC6 genes, an Actin internal reference gene amplification primer group, a TRPC6 target gene calibrator, an Actin internal reference gene calibrator, a PCR amplification reaction liquid system and reverse transcriptase. Taking a human detection kit as an example, the method specifically comprises the following steps:

(1) PCR amplification primer set of TRPC6 gene. Including any one or more of the primer sets as in example one, the primer set concentration is 10. Mu.M, 30. Mu.l.

(2) Amplification primer group for action internal reference gene

The primer set for amplifying the internal Gene of the action is designed into a real-time fluorescent quantitative PCR (qRT-PCR) primer set for detecting the expression quantity of mRNA of the Gene of the action by utilizing primer5.0 software according to the sequence of the Gene of the action disclosed by NCBI Gene Bank (XM-032773537.1), and comprises an upstream primer and a downstream primer. In this example, the sequence of the action primer set is shown in Table 2.

TABLE 2 Actin amplification primer nucleotide sequence

(3) TRPC6 target gene calibrator. The solution with larger concentration can be used as mother solution, and can be diluted into gradient concentration solution in time, or prepared gradient concentration solution in advance. In this example, there are five gradient solutions of different concentrations, 54. Mu.l each, T1 (4X 10) ⁵ Copy number/. Mu.l), T2 (1X 10) ⁵ Copy number/. Mu.l), T3 (2.5X10) ⁴ Copy number/. Mu.l), T4 (6.25X10) ³ Copy number/. Mu.l), T5 (1.5625X 10) ³ Copy number/. Mu.l).

(4) An action reference gene calibrator. The solution with larger concentration can be used as mother solution, and can be diluted into gradient concentration solution in time, or prepared gradient concentration solution in advance. In this example, there are five gradient solutions of different concentrations, 54. Mu.l each, A1 (3.2X10 ⁸ Copy number/. Mu.l), A2 (8X 10) ⁷ Copy number/. Mu.l), A3 (2X 10) ⁷ Copy number/. Mu.l), A4（5×10 ⁶ Copy number/. Mu.l), A5 (1.25X10) ⁶ Copy number/. Mu.l).

(5) PCR amplification reaction liquid system. For example, 2X reaction mixture 1.92ml:5Units/μl Taq DNA polymerase (Beijing full gold organism, cat# AP 151-01) 80 μl, 2.5mM dNTP 80 μl, 0.1M MgSO4 100 μl, 1000×SYBR Green I fluorescent dye 40 μl.

(6) Reverse transcriptase. A total of 80. Mu.l, including 4 Units/. Mu.l of One-step enzyme (Beijing full gold organism, cat. No.: AH 101) 40ul,50% glycerol 40. Mu.l.

(7) Blank reference: 1X library dilutions.

As the reagent, a commercially available biomedical reagent can be used.

Example III application of kit for detecting mRNA level of TRPC6 Gene in screening and diagnosing Alzheimer's disease

The kit of the second embodiment is used for detecting the mRNA expression level of the TRPC6 gene in the blood cells of the subject. The process is as follows:

Taking one person as an example, the specific process is as follows:

(1) A sample of 1ml of blood from a subject was collected and total RNA from blood leukocytes was extracted for later use. Total RNA was extracted using the Kangzhi blood RNA extraction kit (cat# CWY 027S) with reference to the kit instructions. Electrophoresis was performed using a 1% Agarose gel, and the results are shown in FIG. 1.

(2) One-step qRT-PCR amplification:

(1) amplification detection

TRPC6 target gene calibrator: performing 5 PCR reactions;

an action reference gene calibrator: performing 5 PCR reactions;

RNA sample: 2 PCR reactions are carried out, namely TRPC6 target gene primer reactions and Actin target gene primer reactions are respectively carried out;

blank reference: 2 PCR reactions were performed, respectively TRPC6 target gene primer reactions and Actin target gene primer reactions.

(2) The total volume of each reaction system was 20ul, including:

10ul of reaction mixture;

reverse transcriptase 0.4ul;

0.6ul of upstream and downstream pooled primers (10 uM); 16.7 ng/. Mu.l RNA sample 9ul;

TRPC6 target gene calibrator 9ul;

and 9ul of an action reference gene calibrator.

(3) One-step qRT-PCR amplification reaction conditions:

reverse transcription is carried out at 50 ℃ for 5min, and denaturation is carried out at 94 ℃ for 30s;

40 amplification cycles under the following conditions: denaturation at 94℃for 5s, annealing at 61℃for 15s, extension at 72℃for 15s;

a melting curve is generated, and the temperature is slowly increased from 65 ℃ to 95 ℃ with the temperature increasing rate of 5s to 0.5 ℃.

The kit detects TRPC6 calibrator amplification curve, action calibrator amplification curve and amplification curve of 1 human sample of a human sample, as shown in figures 2, 3 and 4 respectively. (3) Data processing

Drawing of a Standard Curve

And (3) taking the detection CT values of the TRPC6 target gene calibrator and the action reference gene calibrator as the abscissa, taking the logarithmic value of 2 of the concentration (copy number/. Mu.l) of the TRPC6 target gene calibrator and the action reference gene calibrator as the ordinate, establishing a standard curve, adopting linear fitting to obtain a standard curve equation, and calculating a linear correlation coefficient. . Standard curve equations for TRPC6 and action calibrator are shown in fig. 5 and 6, respectively.

(2) And substituting CT values detected by a human sample into a standard curve equation by taking the CT values as abscissa values to obtain the concentrations of TRPC6 mRNA and action mRNA of the detected RNA sample.

The ratio of TRPC6 mRNA concentration/action mRNA concentration was used as the detection result.

Clinical subject samples (55 cases of AD patients, 55 cases of healthy persons) collected in 110 cases were tested by using the primer set of table 1 according to the above-described method. Statistical analysis was performed on the detection data with GraphPad prism 5.0 to obtain the accuracy, specificity, sensitivity of the detection and the optimal kit threshold line. The detection indexes of the five pairs of specific primer groups are shown in Table 3.

Table 3 contains the results of 5 pairs of PCR amplification primer set kits for testing 110 samples

As can be seen from table 3: any 1 pair of primers and kit in the 5 pairs of PCR amplification primer groups can be effectively used for screening and diagnosing the Alzheimer's disease; when the TRPC6 mRNA concentration/Actin mRNA concentration ratio of the test sample is below the threshold, it is considered to be at risk for alzheimer's disease.

Notably, the primer set of primer TPS-4-F having the nucleotide sequence shown as SEQ ID NO.7 and primer TPS-4-R having the nucleotide sequence shown as SEQ ID NO.8 showed extremely high reliability, 96.5% accuracy, 95% sensitivity and 80% specificity in clinical sample screening diagnosis.

Compared with the TRPC6 mRNA expression level detection method reported in the prior art, the method adopts one-step qRT-PCR amplification detection, the RT-PCR and qPCR of the RNA sample are completed in the same tube, the target gene calibrator and the internal reference gene calibrator are introduced, the concentration of the sample is detected by establishing a standard curve equation, and the data analysis method of concentration ratio is innovatively introduced, so that the method can be reliably used for screening and diagnosing Alzheimer's disease.

The invention has the beneficial effects that: the invention provides a primer group for detecting the expression level of TRPC6 mRNA, a real-time fluorescence quantitative PCR detection kit and a detection method, which can finish reverse transcription and amplification of a target gene in a reaction tube, avoid systematic errors caused by step-by-step transfer operation in the prior art, creatively introduce a target gene calibrator and an action calibrator when detecting the expression level of TRPC6 mRNA, calculate the concentration of TRPC6 mRNA and the concentration of action mRNA in an extracted RNA sample by establishing a relation between the concentration and a fluorescence PCR amplification detection Ct value, and take the ratio of the concentration of the TRPC mRNA and the action mRNA as a risk index for judging that a subject suffers from Alzheimer's disease, and have more accurate and reliable judgment result. The method has the characteristics of accuracy, high cost efficiency, convenience and the like for diagnosing the Alzheimer's disease, and has great clinical significance.

Finally, it should be noted that the above description is only a preferred embodiment of the present invention, and that many similar changes can be made by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A kit for detecting TRPC6 mRNA levels, characterized in that: comprises a PCR amplification primer group of a transient receptor potential channel 6 TRPC6 gene, a PCR amplification primer group of an Actin internal reference gene, a TRPC6 target gene calibrator, an Actin internal reference gene calibrator, a PCR amplification reaction liquid system and reverse transcriptase;

2. The kit for detecting TRPC6 mRNA level according to claim 1, wherein: the PCR amplification reaction liquid system comprises DNA polymerase, dNTPs, mgSO ₄ Fluorescent dye.

3. Use of a kit according to claim 1 or 2 for the preparation of a kit for screening for diagnosis of alzheimer's disease.

4. A method for detecting TRPC6 mRNA levels in blood using the kit according to claim 1 or 2, characterized by the steps of:

5. The method for detecting the level of TRPC6 mRNA in blood according to claim 4, wherein: and S4, comparing the detection result with a set threshold value, and judging as an abnormal result when the detection result is lower than the threshold value.

6. The method for detecting the level of TRPC6 mRNA in blood according to claim 5, wherein: in step S3, the method for establishing the relationship between the CT value and the concentration number is as follows:

7. The method for detecting the level of TRPC6 mRNA in blood according to claim 6, wherein the reverse transcription amplification detection process in step S2 is:

8. A primer set for detecting TRPC6 mRNA levels, characterized in that: the primer TPS-1-F comprises an upstream primer and a downstream primer, wherein the upstream primer is a primer TPS-1-F with a nucleotide sequence shown as SEQ ID NO.1, and the downstream primer is a primer TPS-1-R with a nucleotide sequence shown as SEQ ID NO. 2;

or (b)

9. Use of a primer set according to claim 8 for the preparation of a kit for detecting TRPC6 mRNA levels in a sample.