阅读说明：本技术 一种评估慢性心力衰竭预后的引物组、试剂盒及评估慢性心力衰竭预后的方法 (Primer group and kit for assessing chronic heart failure prognosis and method for assessing chronic heart failure prognosis ) 是由 汪道文 孙阳 李诗洋 于 2019-09-19 设计创作，主要内容包括：本发明公开了一种评估慢性心力衰竭预后的引物组、试剂盒及评估慢性心力衰竭预后的方法,属于基因检测领域。所述引物组包括：第一上游引物、第一下游引物、第二上游引物、第二下游引物、第三上游引物、第三下游引物、第四上游引物、第四下游引物、第五上游引物、第五下游引物、第六上游引物、第六下游引物、第七上游引物、第七下游引物、第八上游引物和第八下游引物。引物组扩增的基因分别对应预后敏感的八个风险等位基因,能够保证评估结果的准确性和全面性；该方法通过对目标扩增区域的测序和数据判读,能够准确识别与预后相关的突变,从而判断疾病种类和病因,为临床提供及时可靠的检测报告。(The invention discloses a primer group and a kit for assessing chronic heart failure prognosis and a method for assessing chronic heart failure prognosis, and belongs to the field of gene detection. The primer group comprises: a first upstream primer, a first downstream primer, a second upstream primer, a second downstream primer, a third upstream primer, a third downstream primer, a fourth upstream primer, a fourth downstream primer, a fifth upstream primer, a fifth downstream primer, a sixth upstream primer, a sixth downstream primer, a seventh upstream primer, a seventh downstream primer, an eighth upstream primer, and an eighth downstream primer. The genes amplified by the primer group respectively correspond to eight risk alleles with sensitive prognosis, so that the accuracy and comprehensiveness of an evaluation result can be ensured; the method can accurately identify the mutation related to prognosis by sequencing the target amplification region and judging data, thereby judging the type and cause of diseases and providing a timely and reliable detection report for clinic.)

1. A primer set for assessing prognosis of chronic heart failure, wherein the primer set comprises: the primer pair comprises a first upstream primer, a first downstream primer, a second upstream primer, a second downstream primer, a third upstream primer, a third downstream primer, a fourth upstream primer, a fourth downstream primer, a fifth upstream primer, a fifth downstream primer, a sixth upstream primer, a sixth downstream primer, a seventh upstream primer, a seventh downstream primer, an eighth upstream primer and an eighth downstream primer, wherein the sequence of the first upstream primer is shown as SEQ ID NO. 1 in the sequence table, the sequence of the first downstream primer is shown as SEQ ID NO. 2 in the sequence table, the sequence of the second upstream primer is shown as SEQ ID NO. 3 in the sequence table, the sequence of the second downstream primer is shown as SEQ ID NO. 4 in the sequence table, the sequence of the third upstream primer is shown as SEQ ID NO. 5 in the sequence table, the sequence of the third downstream primer is shown as SEQ ID NO. 6 in the sequence table, the sequence of the fourth upstream primer is shown as SEQ ID NO:7, and the sequence of the fourth downstream primer is shown as SEQ ID NO: as shown in figure 8, the flow of air, the sequence of the fifth upstream primer is shown as SEQ ID NO:9, and the sequence of the fifth downstream primer is shown as SEQ ID NO: as shown in figure 10 of the drawings, the sequence of the sixth upstream primer is shown as SEQ ID NO: as shown in figure 11, the first and second, the sequence of the sixth downstream primer is shown as SEQID NO:12, and the sequence of the seventh upstream primer is shown as SEQ ID NO: as shown in figure 13, the first and second, the sequence of the seventh downstream primer is shown as SEQ ID NO: as shown in figure 14, the first and second, the sequence of the eighth upstream primer is shown as SEQ ID NO: as shown in the drawing 15, the flow rate of the gas, the sequence of the eighth downstream primer is shown as SEQ ID NO: shown at 16.

2. A kit for assessing prognosis of chronic heart failure, the kit comprising: the primer set of claim 1.

3. The kit of claim 2, further comprising: TaKaRa Ex Taq HS, 10 XEx Taq, dNTP mix, Primer Pool 1/2, genomic DNA and sterilized water.

4. A method for assessing prognosis of chronic heart failure using the primer set according to claim 1, wherein the method comprises:

extracting the genome DNA of a sample to be detected;

amplifying the genomic DNA through the primer group to obtain eight amplification products;

respectively carrying out Sanger sequencing on the eight amplification products to obtain eight sequencing products, wherein each sequencing product is a homozygous wild-type sequencing product, a heterozygous-type sequencing product or a homozygous mutant-type sequencing product;

assigning a value to the sequencing product, wherein when the SNP site corresponding to the amplification product is a protective factor, the homozygous wild-type sequencing product is 0, the heterozygous-type sequencing product is-1, and the homozygous mutant-type sequencing product is-2; when the SNP locus corresponding to the amplification product is a risk factor, the homozygous wild-type sequencing product is 0, the heterozygous-type sequencing product is 1, and the homozygous mutant-type sequencing product is 2;

the SNP site corresponding to the amplification product obtained by amplification of the first upstream primer, the first downstream primer, the second upstream primer, the second downstream primer, the fourth upstream primer, the fourth downstream primer, the sixth upstream primer, the sixth downstream primer, the eighth upstream primer and the eighth downstream primer is the risk factor;

the SNP site corresponding to the amplification product obtained by the amplification of the third upstream primer, the third downstream primer, the fifth upstream primer, the fifth downstream primer, the seventh upstream primer and the seventh downstream primer is the protective factor;

and adding the eight sequenced products after assignment, wherein the sum of the added products is X, if X is more than or equal to-3 and less than or equal to 0, the sample to be tested is at low risk, if X is more than or equal to 1 and less than or equal to 3, the sample to be tested is at medium risk, and if X is more than 3, the sample to be tested is at high risk.

5. The method of claim 4, wherein the procedure for amplification is: pre-denaturation at 95 ℃ for 10s and denaturation at 55 ℃ for 30s, and 35 cycles are carried out; annealing at 72 ℃ for 30 s; extension was performed at 10 ℃ for 1 h.

6. The method of claim 4, wherein the test sample is a peripheral blood sample, a body fluid sample, or a tissue organ sample.

Technical Field

The invention relates to the field of gene detection, in particular to a primer group and a kit for evaluating chronic heart failure prognosis and a method for evaluating chronic heart failure prognosis.

Background

Chronic heart failure is the end state of cardiovascular disease resulting from an inability of the heart to meet the body's needs with respect to ejection fraction, the main clinical symptoms being chronic fatigue, shortness of breath, dyspnea, pulmonary congestion and edema. In the general population, women have higher morbidity than men, the prevalence rate is 9/1000, and about 400 thousands of chronic heart failure patients exist in China at present. Although new drugs and auxiliary facilities are currently used for treating chronic heart failure, the prognosis of chronic heart failure is poor, the five-year survival rate is equivalent to that of malignant tumors, and therefore, early prognosis evaluation and active treatment through the evaluation result are very important.

The existing prognosis evaluation technology of chronic heart failure mainly comprises amino terminal natriuretic peptide (NT-proBNP) detection and cardiac pulse pressure detection. NT-proBNP detection reflects the deterioration degree of the chronic heart failure by using the level of a cardiac function marker NT-proBNP, and the accuracy of the detection method is influenced by age and obesity degree although the detection method is widely applied to the evaluation of chronic heart failure. The examination of central arterial pressure can cause trauma to the patient and is generally applicable to critically ill patients with chronic heart failure.

Disclosure of Invention

In order to solve the problems in the prior art, the embodiments of the present invention provide a primer set and a kit for assessing prognosis of chronic heart failure, and a method for assessing prognosis of chronic heart failure. The technical scheme is as follows:

in one aspect, the embodiments of the present invention provide a primer set for assessing prognosis of chronic heart failure, where the primer set includes: the primer pair comprises a first upstream primer, a first downstream primer, a second upstream primer, a second downstream primer, a third upstream primer, a third downstream primer, a fourth upstream primer, a fourth downstream primer, a fifth upstream primer, a fifth downstream primer, a sixth upstream primer, a sixth downstream primer, a seventh upstream primer, a seventh downstream primer, an eighth upstream primer and an eighth downstream primer, wherein the sequence of the first upstream primer is shown as SEQ ID NO. 1 in the sequence table, the sequence of the first downstream primer is shown as SEQ ID NO. 2 in the sequence table, the sequence of the second upstream primer is shown as SEQ ID NO. 3 in the sequence table, the sequence of the second downstream primer is shown as SEQ ID NO. 4 in the sequence table, the sequence of the third upstream primer is shown as SEQ ID NO. 5 in the sequence table, the sequence of the third downstream primer is shown as SEQ ID NO. 6 in the sequence table, the sequence of the fourth upstream primer is shown as SEQ ID NO:7, and the sequence of the fourth downstream primer is shown as SEQ ID NO: as shown in figure 8, the flow of air, the sequence of the fifth upstream primer is shown as SEQ ID NO:9, and the sequence of the fifth downstream primer is shown as SEQ ID NO: as shown in figure 10 of the drawings, the sequence of the sixth upstream primer is shown as SEQ ID NO: as shown in figure 11, the first and second, the sequence of the sixth downstream primer is shown as SEQ ID NO: as shown in figure 12 of the drawings, the sequence of the seventh upstream primer is shown as SEQ ID NO: as shown in figure 13, the first and second, the sequence of the seventh downstream primer is shown as SEQ ID NO: as shown in figure 14, the first and second, the sequence of the eighth upstream primer is shown as SEQ ID NO: as shown in the drawing 15, the flow rate of the gas, the sequence of the eighth downstream primer is shown as SEQ ID NO: shown at 16.

In another aspect, the embodiments of the present invention provide a kit for assessing prognosis of chronic heart failure, the kit including: the primer set is described above.

Specifically, the kit further comprises: TaKaRa Ex Taq HS, 10 XEx Taq, dNTP mix, Primer Pool 1/2, genomic DNA and sterilized water.

In still another aspect, the present invention provides a method for assessing prognosis of chronic heart failure using the primer set, the method including:

extracting the genome DNA of a sample to be detected;

amplifying the genomic DNA through the primer group to obtain eight amplification products;

respectively carrying out Sanger sequencing on the eight amplification products to obtain eight sequencing products, wherein each sequencing product is a homozygous wild-type sequencing product, a heterozygous-type sequencing product or a homozygous mutant-type sequencing product;

assigning a value to the sequencing product, wherein when the SNP site corresponding to the amplification product is a protective factor, the homozygous wild-type sequencing product is 0, the heterozygous-type sequencing product is-1, and the homozygous mutant-type sequencing product is-2; when the SNP locus corresponding to the amplification product is a risk factor, the homozygous wild-type sequencing product is 0, the heterozygous-type sequencing product is 1, and the homozygous mutant-type sequencing product is 2;

the SNP site corresponding to the amplification product obtained by amplification of the first upstream primer, the first downstream primer, the second upstream primer, the second downstream primer, the fourth upstream primer, the fourth downstream primer, the sixth upstream primer, the sixth downstream primer, the eighth upstream primer and the eighth downstream primer is the risk factor;

the SNP site corresponding to the amplification product obtained by the amplification of the third upstream primer, the third downstream primer, the fifth upstream primer, the fifth downstream primer, the seventh upstream primer and the seventh downstream primer is the protective factor;

and adding the eight sequenced products after assignment, wherein the sum of the added products is X, if X is more than or equal to-3 and less than or equal to 0, the sample to be tested is at low risk, if X is more than or equal to 1 and less than or equal to 3, the sample to be tested is at medium risk, and if X is more than 3, the sample to be tested is at high risk.

Specifically, the procedure for amplification is: pre-denaturation at 95 ℃ for 10s and denaturation at 55 ℃ for 30s, and 35 cycles are carried out; annealing at 72 ℃ for 30 s; extension was performed at 10 ℃ for 1 h.

Specifically, the sample to be detected is a peripheral blood sample, a body fluid sample or a tissue organ sample.

The technical scheme provided by the embodiment of the invention has the following beneficial effects: the genes amplified by the primer group respectively correspond to eight risk alleles with sensitive prognosis, so that the accuracy and comprehensiveness of an evaluation result can be ensured, meanwhile, the length of an amplification product obtained by the primer group is about 500bp, and an SNP locus is designed at the middle position of the amplification product, namely about 250bp, so that the 5 'end interference result and the 3' end passivation phenomenon can be effectively prevented, the primer group can respectively and accurately amplify the SNP locus in a sample to be detected, and the amplification product can accurately reflect the prognosis condition of chronic heart failure. The eight amplification primers can be used for sequencing under a PCR amplification mode, so that the sequencing accuracy is further ensured. The method can accurately identify the mutation related to prognosis by sequencing the target amplification region and judging data, thereby judging the type and cause of diseases and providing a timely and reliable detection report for clinic. The detection method designed by the invention can achieve 100% of accuracy through sanger sequencing and typing point mutation, and the outcome of chronic heart failure has obvious heterogeneity and different characteristics of different primary factors, so that the outcome can be caused to be different.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 is a typing diagram of a sequencing product corresponding to an amplification product obtained by a first upstream primer and a first downstream primer provided in the third embodiment of the present invention;

FIG. 2 is a typing diagram of a sequencing product corresponding to an amplification product obtained by a second forward primer and a second backward primer provided in the third embodiment of the present invention;

FIG. 3 is a typing diagram of a sequencing product corresponding to an amplification product obtained by a third upstream primer and a third downstream primer provided in the third embodiment of the present invention;

FIG. 4 is a typing diagram of a sequencing product corresponding to an amplification product obtained by a fourth upstream primer and a fourth downstream primer provided in the third embodiment of the present invention;

FIG. 5 is a typing diagram of a sequencing product corresponding to an amplification product obtained by a fifth upstream primer and a fifth downstream primer provided in the third embodiment of the present invention;

FIG. 6 is a typing diagram of a sequencing product corresponding to an amplification product obtained by a sixth forward primer and a sixth backward primer provided in the third embodiment of the present invention;

FIG. 7 is a typing chart of a sequencing product corresponding to an amplification product obtained by a seventh upstream primer and a seventh downstream primer provided in the third embodiment of the present invention;

FIG. 8 is a typing chart of the sequencing products corresponding to the amplification products obtained by the eighth forward primer and the eighth backward primer provided in the third embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail with reference to the accompanying drawings.